Assembly of Self-Cleaning Electrode Surface for the Development of

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Assembly of Self-cleaning Electrode Surface for the Development of Refreshable Biosensors Xiaoli Zhu, Yaoyao Chen, Chang Feng, Wei Wang, Bing Bo, Ruixin Ren, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05177 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Analytical Chemistry

Assembly of Self-cleaning Electrode Surface for the Development of Refreshable Biosensors Xiaoli Zhu,† Yaoyao Chen,† Chang Feng,‡ Wei Wang,† Bing Bo,§ Ruixin Ren,∥ and Genxi Li*,†,‡ Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China ABSTRACT: Passivation of electrode surface and tedious reconstruction of biosensing architectures have long plagued researchers for the development of electrochemical biosensors. Here, we report a novel self-cleaning electrode by modifying the commonlyused working electrode with superhydrophobic and conductive nanocomposite. Owing to the superhydrophobicity and the chemical stability, the electrode avoids passivation result from both adsorption of molecules and oxidation in air. The high conductivity and the high effective area also allow the achievement of enhanced electrochemical signals. On the basis of comprehensive studies on this novel electrode, we have applied it in the fabrication of refreshable electrochemical biosensors for both electro-active and electro-inactive targets. For both cases, detection of the targets can be well performed, and the self-cleaning electrode can be refreshed by simply washing and applied for successive measurements in a long period.

be achieved. Specifically, superhydrophobic and conductive nanocomposite of polydimethylsiloxane (PDMS) and multiwalled carbon nanotubes (MWCNT) is synthesized and adopted to modify a commonly-used glassy carbon electrode (GCE) so as to fabricate the self-cleaning working electrode. PDMS is well known as a widely-used hydrophobic organic polymer material with favorable cost-efficiency, biocompatibility and stability, while MWCNT have been proven to have excellent conductivity.18-20 Here, the electrode modified with PDMS@MWCNT nanocomposite is expected to have both of the properties: the superhydrophobicity allows the selfcleaning, and the conductivity allows the electron transfer capability.

Electrochemical techniques are powerful tools for the analysis of a variety of targets.1-5 Owing to the high sensitivity, easyoperation and availability for miniaturization, they have received broad focus and developed many applications.6-9 Generally from the point of the targets, the electrochemical biosensing systems can be assigned to two categories. One is for electro-active species; another is for the electro-inactive species. For the former, direct electrochemical response of electro-active targets can be obtained at the working electrode. A great deal of efforts has been made by modifying the working electrode with specific materials, especially nanomaterials, to enhance the obtained signals as well as to reduce the overpotential.10-12 In the meantime, it should be noticed that owing to the unspecific adsorption and electrochemical deposition of the target and interfering molecules, the working electrode tends to passivate, and consequently cannot be applied for another detection.13-15 Thereafter, tedious treatments, or even reconstruction of the sensing layer have to be conducted to regenerate the electrode. As for the other category of electrochemical biosensing system, in which elegant molecular architectures are usually constructed to convert the specific molecular recognition of the electro-inactive targets to readable electrochemical signals, similar problems also exist. Though different strategies, including the use of disposable screen printed electrodes (SPE) and the development of label-free biosensors, have been proposed to address this problem, it is far from resolved.16,17 And, it has been a challenge for the development of electrochemical biosensors with favorable performance, cost-efficiency and usability. In this work, inspired by self-cleaning lotus leaves, we attempt to fabricate a self-cleaning working electrode, on which unspecific adsorption of molecules can be eliminated. And refreshable electrochemical biosensors are thereby expected to

EXPERIMENTAL SECTION Materials. PDMS was purchased from Dow Corning. Biotin-labelled anti-CEA antibody and carcinoembryonic antigen (CEA) were obtained from Abcam. CEA aptamer with a sequence of 5'-ATACCAGCTTATTCAATT-T12-SH-3' was synthesized by Sangon Biotech (Shanghai) Co. Ltd. MWCNT, trimethylsilanol, dopamine, quercetin, horseradish peroxidase (HRP) and some other chemicals were all obtained from Sigma. CEA ELISA kits were purchased from Abcam (kit1) and Thermo Scientific (kit2), respectively. All chemicals were of analytical grade and used without further purification. All solutions were prepared with Milli-Q water (18.2 MΩ.cm−1) from a Milli-Q purification system (Branstead, USA). Preparation of PDMS@MWCNT nanocomposite. PDMS@MWCNT nanocomposite was prepared according to a previous report.21 Briefly, PDMS precursor and MWCNT with a 1:1 weight ratio were fully mixed and added to a 25 mL clean reacting kettle. The kettle was sealed and maintained at 300 °C for 12 hours to allow the formation of PDMS layer on MWCNT. After cooling down to room temperature, the nano-

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composite was dissolved in acetone with a concentration of 1 mg/mL. Massy sediment was then removed by ultrasonic dispersion and centrifuge filtration (2000 rpm, 10 min). Finally, the nanocomposite was stored at 4 °C and was ready for use. Preparation of self-cleaning electrode. A substrate GCE was first polished to mirror smoothness on a microcloth (Buehler) with gamma micropolish deagglomerated alumina suspension (particle size of ~0.05 µm). Residual alumina powder was removed by sonicating the electrode in ethanol and double-distilled water for 5 min, respectively. After being rinsed with double-distilled water and dried with nitrogen, the electrode was electrochemically activated in 0.5 M H2SO4 using a cyclic voltammetry (-0.3~2 V, 100 mV/s) until a stable cyclic voltammogram was obtained. The activated electrode was then modified with a layer of trimethylsilanol using EDC as a linker. In detail, the surface of the electrode was incubated with 1-Ethyl-3-(3'-dimethylaminopropyl) carbodiimide (EDC) (0.22 M) and N-Hydroxysuccinimide (NHS) (0.22 M) for 30 min, and then with trimethylsilanol (2 mM) in dark for 12 hours. The trimethylsilanol modified electrode was sonicated again in ethanol and double-distilled water for 5 min respectively to remove any residue, and was ready for the casting of PDMS@MWCNT. 10 µL PDMS@MWCNT nanocomposite was dropped onto the electrode, followed by being dried under 55 °C. This "drop-and-dry" step was repeated for four times until the formation of uniform superhydrophobic surface on GCE. Characterization of PDMS@MWCNT and selfcleaning electrode. Transmission electron microscope (TEM) (JEOL2100F, JEOL, Japan), scanning electron microscope (SEM) (JSM4800F, JEOL, Japan), and fourier transform infrared spectrometer (FT-IR) (VERTEX 70, Bruker, Germany) were adopted for the characterization of the morphology and chemical groups of PDMS@MWCNT. To study the superhydrophobicity, PDMS@MWCNT coated glass slide or self-cleaning electrode were adopted. The water contact angles of droplets on the substrates were measured using a theta optical tensiometer (KSV instruments, Sweden). And, the appearance of droplets on glass slide or selfcleaning electrode was recorded using a Cannon EOS 700D camera. Electrochemical impedance spectroscopic (EIS) and Cyclic voltammetric (CV) were measured on a CHI660C electrochemical analyzer (CH Instruments, USA) to study the conductivity of the self-cleaning electrode. A three-electrode system was used, in which, the working electrode is the selfcleaning electrode or GCE, the reference electrode is a saturated calomel electrode and the counter electrode is a platinum electrode. 5 mM [Fe(CN)6]3−/4− with 1 M KNO3 was adopted as the electrolyte, in which the [Fe(CN)6]3−/4− couple worked as an electrochemical indicator. The parameters for EIS were as follows: initial potential, 0.224 V; frequency range, 1 Hz~100 kHz. And the parameters for CV were as follows: potential scan range, -0.1~0.6 V; scan rate, 100 mV/s. Direct detection of electro-active species using the selfcleaning electrode. CV of electro-active species (dopamine and quercetin) was performed either on self-cleaning electrode or bare GCE. Signals of dopamine were obtained in a scan range from -0.2~0.6 V; while signals of quercetin were obtained in a scan range from -0.1~0.6 V. The scan rate was 100 mV/s unless specified. A phosphate-buffered saline (PBS, 10 mM, pH 7.4) was adopted as the electrolyte.

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Detection of electro-inactive species using the selfcleaning electrode and MCMA. CEA, a tumor-associated glycosylated protein that has been identified as a critical biomarker for a variety of cancers, was adopted as an electroinactive target.22 For the detection of CEA, a magnetocontrolled moveable architecture (MCMA) strategy was borrowed. Briefly, anti-CEA antibody functionalized magnetic nanoparticles (antibody@MNPs) and horseradish peroxidaselabeled anti-CEA aptamer (HRP-aptamer) were first synthesized. The details for the synthesis processes can be referred to our previous report.23 In order to balance the sensitivity and the negative influence of excess nonconductive MNPs, an optimized amount of 80 µg antibody@MNPs is adopted for a single measurement.23 100 µL of the antibody@MNPs together with 10 µL of HRP-aptamer (2 µM) and 100 µL of different concentrations of CEA were mixed and incubated at room temperature for 1 hour. Then, the MCMA consisted of antibody@MNPs/antigen/HRP-aptamer was separated under a magnetic field and washed twice using PBS (10 mM, pH 7.4). The MCMA was finally redispersed in 100 µL PBS and was ready for electrochemical measurements. A three-electrode system that has been depicted above was adopted. Instead of using common GCE, here a magnetic GCE was adopted, which was inserted with a powerful Nd-FeB magnet. The as-prepared MCMA was injected into the electrolyte (2 mL, 10 mM PBS, pH 7.4) and was allowed to concentrated onto the surface of the self-cleaning electrode under magnetic field within 2 min. After the enzymatic substrates of HRP (20 µM thionine and 6 mM H2O2) was further added, cyclic voltammetric measurements were then performed in a potential range of 0~-0.25 V at a scan rate of 5 mV/s. To refresh the electrode, the electrode was just rinsed with doubledistilled water and was ready for the next measurements. Detection of CEA in real serum samples. Sera from candidates were provided by Shanghai Pulmonary Hospital and Shanghai Tenth People's Hospital. Serum samples were collected from two lung carcinoma patients (No. 1~2), three breast cancer patients (No. 3~5), and five healthy individuals (No. 6~10). The sera were diluted to 2 fold for the measurements of CEA using either ELISA kits or our method shown above. For the measurements using ELISA kits, the detailed processes can be referred to the online instructions of the kits. While for the measurements using our method, the antibody@MNPs and HRP-aptamer together with the diluted sera were mixed together and incubated at room temperature for 1 hour. The following steps were the same as that shown above. The concentrations of CEA in serum samples were determined using the linear regressive curve obtained from standard CEA samples.

RESULTS AND DISCUSSION Fabrication and comprehensive study of self-cleaning electrode. Schematic illustration of the fabrication process is shown in Figure 1A. Comprehensive characterization of the PDMS@MWCNT nanocomposite is first conducted. FT-IR, HR-TEM, and SEM results suggest the successful synthesis of the PDMS@MWCNT nanocomposite (Figure 1B~1D). A thin and uniform layer of PDMS (~1.5 nm) on MWCNT can be observed (Figure S1 in the Supplementary Material).

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Figure 1. Fabrication and characterization of PDMS@MWCNT modified self-cleaning electrode. (A) Schematic presentation of the synthesis of PDMS@MWCNT nanocomposite and the fabrication of self-cleaning electrode. (B) FT-IR spectrum of the PDMS@MWCNT nanocomposite as well as the MWCNT alone. (C) TEM images of MWCNT (left) and PDMS@MWCNT nanocomposite (right). (D) SEM images of MWCNT (left) and PDMS@MWCNT nanocomposite (right). chemical impedances, down) of the self-cleaning electrodes: (D) time-stability, (E) irradiation-stability, (F) reproducibility.

Superhydrophobicity and conductivity of the PDMS@MWCNT nanocomposite are further studied. As is shown in Figure 2A, the PDMS@MWCNT nanocomposite is

undissolved in water, and a drop of water can form a ball appearance on the layer of PDMS@MWCNT coated electrode or glass slide. Superhydrophobicity is employed to describe a surface with a water contact angle higher than 150° and a sliding angle lower than 10°. Our results show that four layers of PDMS@MWCNT (10 µL of 1mg/mL PDMS@MWCNT in acetone for each layer) are optimal to provide a stable superhydrophobic surface with a contact angle of 153 ± 1° and a sliding angle of 6 ± 2°. As for the conductivity, electrochemical response of [Fe(CN)6]3-/4-, a commonly-used electrochemical redox couple, is studied on the PDMS@MWCNT coated electrode.24 As is shown in Figure 2B, EIS results show that the impedance of the self-cleaning electrode is even lower than freshly polished GCE. With the coating of more layers of PDMS@MWCNT onto the electrode, the impedance decreases from ca. 80 ohm to ca. 20 ohm. Cyclic voltammetric results also show that the current response of [Fe(CN)6]3-/4- increases with the layers of PDMS@MWCNT (Figure 2C). The above electrochemical results together suggest that the self-cleaning electrode owns excellent conductivity. Though PDMS is a non-conductive material, here because the PDMS layer on MWCNT is thin enough, electron hopping between the electrolyte and the MWCNT is allowed.21 In addition, the nanostructured surface of PDMS@MWCNT expands the effective electrode area, resulting the increase of the current response of [Fe(CN)6]3-/4- in CV measurements. Because the self-cleaning electrode is expected to be repetitively used, the stability of the superhydrophobicity and conductivity of the self-cleaning electrode is evaluated. In our

Figure 2. Electrochemical behavior and stability of self-cleaning electrode. (A) Optical photograph of MWCNT and PDMS@MWCNT in tubes filled with water, and water droplets on electrodes casted with MWCNT or PDMS@MWCNT (up); water droplets on coverslips casted with different layers of PDMS@MWCNT (middle); water droplets with different volumes on coverslips casted with 4 layers of PDMS@MWCNT (down). (B) EIS of electrodes casted with different layers of PDMS@MWCNT (up), and the electrochemical impedances vs. the number layers (down). (C) Cyclic voltammograms of [Fe(CN)6]3-/4- on electrodes modified with different layers of PDMS@MWCNT (up), and the corresponding cathodic peak currents vs. the number layers (down). (D-F) Stability of the superhydrophobicity (contact angels, up) and conductivity (electro-

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Figure 3. Detection of dopamine and quercetin performed by self-cleaning electrode or bare GCE. (A) Structures and redox reactions of dopamine and quercetin. (B-C) Cyclic voltammograms of (B) dopamine and (C) quercetinon on GCE and self-cleaning electrode, respectively. (D-G) Cyclic voltammetric response and linear relationship of different concentrations of (D and E) dopamine and (F and G) quercetin on (D and F) GCE or (E and G) self-cleaning electrode.

as control. It is observed that benefited from the high conductivity and effective electrode area of the self-cleaning electrode, the current response of dopamine and quercetin increases 5 ± 2.4 fold in comparison with that on bare GCE (background current subtracted). Some other interesting electrochemical behaviors have also been explored. For example, it is interesting that the ian / ica is closer to 1.0 (dopamine: 1.5 and 3.1 on self-cleaning electrode and bare GCE respectively; quercetin: 1.4 and 2.0 on self-cleaning electrode and bare GCE respectively), suggesting the increase of the reversibility of the redox reaction.30 But as for peak separation (∆E), another important parameter for reversibility, the changes are opposite for dopamine and quercetin (dopamine: decrease from 0.147 V to 0.091 V; quercetin: increase from 0.101 V to 0.142 V). In addition, there are also potential information to be discovered on the diffusion and adsorption of dopamine and quercetin on self-cleaning electrode and GCE respectively (Figure S2 and S3 in Supplementary Material). Because the application of self-cleaning electrode for refreshable electrochemical biosensors is mainly focused in this work, we will discuss the mechanism of the electrochemical behavior of different molecules on this novel electrode elsewhere. Figure 3D~3G shows the electrochemical detection of dopamine and quercetin by using the self-cleaning electrode and GCE respectively. The currents of the anodic peaks are adopted to quantitate the concentration of dopamine and quercetin. Results reveal that both the detection limit and the sensitivity are improved at self-cleaning electrode (dopamine: detection limit lowers from 0.64 µM to 0.25 µM, sensitivity increases from 0.05 A/M to 0.16 A/M; quercetin: detection limit

experiments, the self-cleaning electrode was treated by putting it in air for as long as 90 days to evaluate the time-stability, or by putting it under UV irradiation for as long as 24 hours to evaluate the irradiation-stability, or by polishing and refabricating successively to evaluate the reproducibility. Experimental results show that in all these cases, both the superhydrophobicity and the conductivity are stable (Figure 2D~2F). So, here it can be concluded that the self-cleaning electrode has the capacity to be employed for the following analytical measurements. Application of self-cleaning electrode for the detection of electro-active species. We first adopt the self-cleaning electrode for the electrochemical analysis of electro-active species. Two biomolecules with similar electrochemical behavior but different hydrophilic-hydrophobic property are elaborately selected. One is dopamine, a hydrophilic chemical of the catecholamine and phenethylamine family that plays several important roles (e.g. neurotransmitter) in brain and body.25-27 The other is quercetin, a hydrophobic flavonol that has been used for quercetin supplements and has been promoted for the treatment of a wide spectrum of diseases.28,29 Both dopamine and quercetin have a 1,2-benzenediol group that can be involved in a two-electron two-proton electrochemical redox reaction (Figure 3A). So, it is possible to compare the electrochemical behavior of these two biomolecules with different hydrophilic-hydrophobic property on the self-cleaning electrode and bare GCE. Figure 3B and 3C show the direct CV response of dopamine and quercetin on self-cleaning electrode and bare GCE

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lowers from 1.1 µM to 0.5 µM, sensitivity increases from 0.018 A/M to 0.042 A/M).

potential during different measurements is also smaller than that on bare GCE. As for quercetin (Figure 4C and 4D), similar results can be also observed. Briefly, the RSD of potential and current reaches as high as 12.0% and 29.0% respectively for bare GCE, whereas for self-cleaning electrode, the values are 3.7% and 5.5%, respectively. So, here it can be concluded that owing to the self-cleaning property, the PDMS@MWCNT coated electrode is capable for reuse simply after washing. Application of self-cleaning electrode for the detection of electro-inactive species. For electro-inactive species, elegant molecular architectures are usually constructed onto working electrode surface so as to convert the specific molecular recognition of the electro-inactive targets to readable electrochemical signals. Since we have prepared self-cleaning electrode in this work, and the regeneration of an electrode surface can be easily achieved, we have then attempted to make use of the self-cleaning electrode to fabricate refreshable biosensors for the electro-inactive analytes. CEA, a tumorassociated biomarker is adopted for detection of electroinactive species. The construction of refreshable biosensors based on the self-cleaning electrode combined with a MCMA strategy is illustrated in Figure 5A. Briefly, antibody@magnetic nanoparticles (antibody@MNPs) is synthesized to capture CEA from a complex sample. A HRPaptamer conjugation is subsequently captured onto the surface of the antibody@MNPs through the specific interaction between CEA and aptamer (characterization of the antibody@MNPs and HRP-aptamer is shown in Figure S4 and S5 in the Supplementary Material). Thus, a MCMA consisted of antibody@MNPs/antigen/HRP-aptamer is fabricated, which can be attracted onto the surface of self-cleaning electrode by using a detachable magnetic electrode (Figure 5B) to allow the HRP-mediated electrochemical reactions. The intensity of current response relies directly on the amount of HRP, which is depended on the concentration of the target CEA. So, electrochemical detection of CEA can be achieved. More importantly, the self-cleaning electrode allows the complete desorption of MCMA from the electrode after a measurement barely through washing. Thus successive measurements can be achieved for batch samples. After the obtaining of HRP-dependent electrochemical signals (Figure S6 in the Supplementary Material), detection of CEA using the MCMA was performed. From Figure 5C, it is observed that the current at -0.25 V increases with the concentration of CEA. The result is reasonable because only in the presence of CEA, the HRP-aptamer can be recruited onto the electrode through magnetic controls to give HRPdependent electrochemical signals. Further studies reveal that a linear relationship can be obtained in a concentration range of 0.1~100 mg/mL (Figure 5D), which can be defined as the detection range of our system. The detection limit is calculated to be 0.041 ng/mL (LOD = 3SD / k, LOD: detection limit, SD: the standard deviation of blank sample, k: the slope of the fitting curve), which rives the recently reported methods and the commonly-used ELISA (Table S1). Selectivity of the biosensing system is also investigated. As is shown in Figure 5E, some other proteins that exist abundantly in serum will not interfere with the analysis of CEA. Here, the MCMA not only

Figure 4. Stability of self-cleaning electrode for successive measurements. (A) Cyclic voltammetric response of dopamine using the same bare electrode at an interval of several days (left); corresponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right). (B) Cyclic voltammetric response of dopamine using the same self-cleaning electrode at an interval of several days (left); corresponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right). (C) Cyclic voltammetric response of quercetin using the same bare electrode at an interval of several days (left); corresponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right). (D) Cyclic voltammetric response of quercetin using the same self-cleaning electrode at an interval of several days (left); corresponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right).

On the basis of the above analytical data, multiple detection of a specific concentration of the electro-active targets using the same electrode is conducted in a week to investigate the possibility for refreshable biosensors. During the experiments, the electrodes were simply washed after a measurement and were ready for the next measurement. As is shown in Figure 4A, for the commonly-used bare GCE, the current signal of dopamine differs a lot among different measurements (RSD = 10.5%). While in the case of selfcleaning electrode (Figure 4B), the current signal is steady with a RSD of only 1.4%. Conventionally to achieve such RSD, some complex pretreatments of the electrode instead of simple washing may be required.31 The deviation of the

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Figure 5. Detection of CEA based on self-cleaning electrode together with MCMA. (A) Schematic of the fabrication of MCMA (left) and the detection of CEA based on the self-cleaning electrode and the MCMA (right). (B) The appearance of the magnetic GCE. (C) CV of MCMA on self-cleaning electrode in the presence of different concentrations of CEA. (D) Currents at -0.25 V vs. the concentrations of CEA. (E) Currents response of different proteins. Each concentration was fixed at 100 ng/mL. BSA: bovine serum albumin; IgG: immunoglobin G; VEGF: vascular endothelial growth factor.

detection signals can be obtained during the successive detection (RSD < 5%). In addition, if the self-cleaning electrode is shelved for one week and even one month after the successive detection and is then reenabled, no significant passivation of the electrode and no significant deviation of the detection signals is observed. Here, the self-cleaning property and the high stability of the PDMS@MWCNT nanocomposite have enabled the quick refreshing of the electrode and the persistent activity during successive and long-period measurements. Thus in summary, refreshable electrochemical biosensor for the detection of CEA, an electro-inactive target, is successfully fabricated using the self-cleaning electrode coupled with MCMA.

supports electrochemical signals, but also realizes the of CEA from other interferences. During the electrochemical detection, those interferences (control proteins here) have been eliminated, so that good selectivity can be achieved. Finally, we applied this biosensing system for the detection of some clinical serum samples, which is obtained from both healthy individuals and cancer patients. The accuracy and the successive detection ability are mainly focused. As for the accuracy, two commercial ELISA kits from different companies were adopted to confirm the detection results. As is shown in Figure 6 and Table S2, good correlation among the detection results can be obtained. The mean relative deviation of the electrochemical detection results from ELISA results is 4.0%, which is an acceptable value. For the successive detection ability, two experiments were performed. One is that a batch of MCMA that applied for different serum samples was first fabricated. Then, each of these MCMA was attracted onto a self-cleaning electrode to complete a measurement, after which and a subsequent washing another MCMA was ready for the next measurement (Figure S7 in the Supplementary Material). The interval (including the attracting of MCMA, obtaining of CV signals and washing) between two successive measurements is less than 4 min. In fact, the results shown in Figure 6 and Table S2 are obtained under this way. The successful detection of CEA with favorable accuracy using this mode has validated the successive detection ability of our refreshable biosensing system. To further verify this conclusion, successive detection of the same sample was conducted (Figure S8 in the Supplementary Material). Two serum samples from a healthy individual (No. 8) and a cancer patient (No. 2) respectively were adopted. Results show that excellent stability of the

Figure 6. Quantification of CEA in the serum from cancer patients (No. 1-5) and healthy individuals (No. 6-10) by using either commercial ELISA kits or our method based on self-cleaning electrode and MCMA (**p < 0.01).

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Analytical Chemistry This work was supported by the National Natural Science Foundation of China (Grant Nos. 21575088, 21235003) and the Natural Science Foundation of Shanghai (14ZR1416500).

CONCLUSION In summary, we have fabricated a novel self-cleaning electrode by casting superhydrophobic and conductive PDMS@MWCNT nanocomposite onto a GCE, and applied it for refreshable electrochemical biosensors. For electro-active targets, hydrophilic dopamine and hydrophobic quercetin with the same electro-active group are adopted. Results show that successive detection of these two targets with favorable signal stability can be achieved, whereas for the normal GCE, because of the passivation of the electrode, large deviation of the signals is observed during successive detection, making it unable to be reused. For electro-inactive targets, a tumor biomarker CEA is adopted. Usually, an elegant but unrefreshable molecular architecture should be constructed onto the working electrode to detect electro-inactive targets. In our work, the association of the self-cleaning electrode and a MCMA makes the reuse possible. Results show that favorable accuracy of the detection signals can be achieved by using ELISA kits as the reference. Successive detection of clinical serum samples with an interval of less than 4 min is also achieved using a single self-cleaning electrode. In addition, the self-cleaning electrode can be shelved for over one month without any passivation and then reenabled for the measurements. So, the successful application of the self-cleaning electrode for two kinds of refreshable electrochemical biosensors has suggested the great potential for extensive application in the fabrication of electrochemical biosensors with favorable performance, cost-efficiency and usability in the future.

REFERENCES (1) Nyein, H. Y.; Gao, W.; Shahpar, Z.; Emaminejad, S.; Challa, S.; Chen, K.; Fahad, H. M.; Tai, L. C.; Ota, H.; Davis, R. W.; Javey, A. ACS. Nano. 2016, 10, 7216-7224. (2) Wiedner, E. S.; Bullock, R. M. J. Am. Chem. Soc. 2016, 138, 8309-8318. (3) Chen, H.; Zhang, J.; Gao, Y.; Liu, S.; Koh, K.; Zhu, X.; Yin, Y. Biosens. Bioelectron. 2015, 68, 777-782. (4) Hsieh, K.; Ferguson, B. S.; Eisenstein, M.; Plaxco, K. W.; Soh, H. T. Acc. Chem. Res. 2015, 48, 911-920. (5) Zhao, J.; Zhu, L.; Guo, C.; Gao, T.; Zhu, X.; Li, G. Biosens. Bioelectron. 2013, 49, 329-333. (6) Sage, A. T.; Besant, J. D.; Lam, B.; Sargent, E. H.; Kelley, S. O. Acc. Chem. Res. 2014, 47, 2417-2425. (7) Yu, Y.; Li, H.; Wei, L.; Li, L.; Ding, Y.; Li, G. Anal. Chem. 2016, 88, 3879-3884. (8) Kolliopoulos, A. V.; Kampouris, D. K.; Banks, C. E. Anal. Chem. 2015, 87, 4269-4274. (9) Adkins, J. A.; Henry, C. S. Anal. Chim. Acta. 2015, 891, 247-254. (10) Wang, Y.; Wang, L.; Chen, H.; Hu, X.; Ma, S. ACS. Appl. Mater. Interfaces. 2016, 8, 18173-18281. (11) Hyder, M. N.; Gallant, B. M.; Shah, N. J.; Shao-Horn, Y.; Hammond, P. T. Nano. Lett. 2013, 13, 4610-4619. (12) Gao, T.; Liu, F.; Yang, D.; Yu, Y.; Wang, Z.; Li, G. Anal. Chem. 2015, 87, 5683-5689. (13) Goda, T.; Yamada, E.; Katayama, Y.; Tabata, M.; Matsumoto, A.; Miyahara, Y. Biosens. Bioelectron. 2016, 77, 208-214. (14) Li, C.; Wu, D.; Hu, X.; Xiang, Y.; Shu, Y.; Li, G. Anal. Chem. 2016, 88, 7583-7590. (15) Zhao, T.; Liu, R.; Ding, X.; Zhao, J.; Yu, H.; Wang, L.; Xu, Q.; Wang, X.; Lou, X.; He, M.; Xiao, Y. Anal. Chem. 2015, 87, 77127719. (16) Zhao, Y.; Xu, L.; Li, S.; Chen, Q.; Yang, D.; Chen, L.; Wang, H. Analyst. 2015, 140, 1832-1836. (17) Yang, Y.; Li, C.; Yin, L.; Liu, M.; Wang, Z.; Shu, Y.; Li, G. ACS. Appl. Mater. Interfaces. 2014, 6, 7579-7584. (18) Smith, M. K.; Singh, V.; Kalaitzidou, K.; Cola, B. ACS. Appl. Mater. Interfaces. 2016, 15, 14788-14794. (19) de Givenchy, E. T.; Amigoni, S.; Martin, C.; Andrada, G.; Caillier, L.; Géribaldi, S.; Guittard, F. Langmuir 2009, 25, 6448-6453. (20) Zhao, Y.; Liu, Y.; Xu, Q.; Barahman, M.; Lyons, A. M. ACS. Appl. Mater. Interfaces. 2015, 7, 2632-2640. (21) Park, E. J.; Kim, K. D.; Yoon, H. S.; Jeong, M. G.; Kim, D. H.; Lim, D. C.; Kim, Y. H.; Kim, Y. D. RSC. Adv. 2014, 4, 30368-30374. (22) Grunnet, M.; Sorensen, J. B. Lung Cancer 2012, 76, 138-143. (23) Zhu, X.; Feng, C.; Ye, Z.; Chen, Y.; Li, G. Sci. Rep. 2014, 4, 4169-4175. (24) Rooney, M. B.; Coomber, D. C.; Bond, A. M. Anal. Chem. 2000, 72, 3486-3491. (25) Baldessarini, R. J.; Tarsy, D. Neuron 2016, 89, 695-710. (26) Schultz, W. Nat. Rev. Neurosci. 2016, 17, 183-195. (27) Kalia, L. V.; Lang, A. E. Lancet 2015, 386, 896-912. (28) Marsh, C. A. Nature 1955, 176, 176-177. (29) Yang, F.; Song, L.; Wang, H.; Wang, J.; Xu, Z.; Xing, N. Oncol. Rep. 2015, 33, 2659-2668. (30) Nicholson, R.S. Anal. Chem. 1965, 37, 1351-1355. (31) Huang, D.; Chen, C.; Wu, Y.; Zhang, H.; Sheng, L.; Xu, H.; Liu, Z. Int. J. Electrochem. Sci. 2012, 7, 5510-5520.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: Supplemental figures (Figure S1−S8) and tables (Table S1 and S2). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail addresses: [email protected].

Present Addresses † Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China ‡ State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China § Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University, School of Medicine, Shanghai 200433, P. R. China ∥ Department of Oncology, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai 200072, P. R. China

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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Self-cleaning electrode: Inspired from lotus leaf, a working electrode with both conductivity and superhydrophobicity is fabricated using nanocomposite, which allows the application for refreshable electrochemical biosensors.


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Figure 1. Fabrication and characterization of PDMS@MWCNT modified self-cleaning electrode. (A) Schematic presentation of the synthesis of PDMS@MWCNT nanocomposite and the fabrication of selfcleaning electrode. (B) FT-IR spectrum of the PDMS@MWCNT nanocomposite as well as the MWCNT alone. (C) TEM images of MWCNT (left) and PDMS@MWCNT nanocomposite (right). (D) SEM images of MWCNT (left) and PDMS@MWCNT nanocomposite (right). 108x69mm (300 x 300 DPI)



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Figure 2. Electrochemical behavior and stability of self-cleaning electrode. (A) Optical photograph of MWCNT and PDMS@MWCNT in tubes filled with water, and water droplets on electrodes casted with MWCNT or PDMS@MWCNT (up); water droplets on coverslips casted with different layers of PDMS@MWCNT (middle); water droplets with different volumes on coverslips casted with 4 layers of PDMS@MWCNT (down). (B) EIS of electrodes casted with different layers of PDMS@MWCNT (up), and the electrochemical impedances vs. the number layers (down). (C) Cyclic voltammograms of [Fe(CN)6]3-/4- on electrodes modified with different layers of PDMS@MWCNT (up), and the corresponding cathodic peak currents vs. the number layers (down). (D-F) Stability of the superhydrophobicity (contact angels, up) and conductivity (electrochemical impedances, down) of the self-cleaning electrodes: (D) time-stability, (E) irradiationstability, (F) reproducibility. 118x92mm (300 x 300 DPI)


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Figure 3. Detection of dopamine and quercetin performed by self-cleaning electrode or bare GCE. (A) Structures and redox reactions of dopamine and quercetin. (B-C) Cyclic voltammograms of (B) dopamine and (C) quercetinon on GCE and self-cleaning electrode, respectively. (D-G) Cyclic voltammetric response and linear relationship of different concentrations of (D and E) dopamine and (F and G) quercetin on (D and F) GCE or (E and G) self-cleaning electrode. 441x297mm (300 x 300 DPI)



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Figure 4. Stability of self-cleaning electrode for successive meas-urements. (A) Cyclic voltammetric response of dopamine using the same bare electrode at an interval of several days (left); corre-sponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right). (B) Cyclic voltammetric re-sponse of dopamine using the same self-cleaning electrode at an interval of several days (left); corresponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right). (C) Cyclic voltammetric response of quercetin using the same bare electrode at an interval of several days (left); corresponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right). (D) Cyclic voltammetric response of quercetin using the same self-cleaning electrode at an interval of several days (left); corresponding anodic peak potential vs. time (middle); corresponding anodic peak current vs. time (right). 127x172mm (300 x 300 DPI)


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Figure 5. Detection of CEA based on self-cleaning electrode together with MCMA. (A) Schematic of the fabrication of MCMA (left) and the detection of CEA based on the self-cleaning electrode and the MCMA (right). (B) The appearance of the magnetic GCE. (C) CV of MCMA on self-cleaning electrode in the presence of different concentrations of CEA. (D) Currents at -0.25 V vs. the concentrations of CEA. (E) Currents response of different proteins. Each concentration was fixed at 100 ng/mL. BSA: bovine serum albumin; IgG: immunoglobin G; VEGF: vascular endothelial growth factor. 149x75mm (300 x 300 DPI)


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Figure 6. Quantification of CEA in the serum from cancer patients (No. 1-5) and healthy individuals (No. 610) by using either commercial ELISA kits or our method based on self-cleaning electrode and MCMA (**p < 0.01). 57x33mm (300 x 300 DPI)


Assembly of Self-Cleaning Electrode Surface for the Development of

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