PDMS composite film for on-site electrochemical analysis

Accepted Manuscript A modularized and flexible sensor based on MWCNT/PDMS composite film for on-site electrochemical analysis

Jiamei Xu, Wenqiong Su, Zonglin Li, Wenjia Liu, Shuopeng Liu, Xianting Ding PII: DOI: Reference:

S1572-6657(17)30741-5 doi:10.1016/j.jelechem.2017.10.033 JEAC 3593

To appear in:

Journal of Electroanalytical Chemistry

Received date: Revised date: Accepted date:

21 July 2017 11 October 2017 15 October 2017

Please cite this article as: Jiamei Xu, Wenqiong Su, Zonglin Li, Wenjia Liu, Shuopeng Liu, Xianting Ding , A modularized and flexible sensor based on MWCNT/PDMS composite film for on-site electrochemical analysis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jeac(2017), doi:10.1016/ j.jelechem.2017.10.033

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ACCEPTED MANUSCRIPT A modularized and flexible sensor based on MWCNT/PDMS composite film for on-site electrochemical analysis Jiamei Xu, Wenqiong Su, Zonglin Li, Wenjia Liu, Shuopeng Liu and Xianting Ding*

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Corresponding author: Xianting Ding ([email protected])

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State Key Laboratory of Oncogenes and Related Genes, Institute for Personalized Medicine, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030 China

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Abstract Flexible sensors based on multi-walled carbon nanotubes (MWCNTs) and their composite materials are widely applied to develop strain sensors that generate a signal as a function of the physical or mechanical deformation. However, on-site analysis of the electrochemical active substances with the flexible electrodes during the biological or medical processes remains a great challenge for current electrochemical technologies. Herein, a universal three-electrode module, adaptable of the synchronous electrochemical analysis in practical applications has been developed with the MWCNTs and polydimethylsiloxane (PDMS) flexible film as the working and the counter electrodes and a painted silver-paste line as the pseudo-reference electrode. The MWCNT/PDMS flexible eletrodes capable of being customized into various sizes, shapes and geometries according to the detection environmental requirements, were prepared through a simple molding transfer method and endowed the on-site electrochemical analysis module with a great potential on the curved surface or in the repeatedly bending situation. Finally, in a 24-well plate and a medical drip which were equipped with the developed three-electrode module, the concentrations of dopamine (DA), uric acid (UA) and nicotinamide adenine dinucleotide (NADH) were successfully detected by Differential Pulse Voltammetry (DPV) with robust stability and repeatability. The technique developed in this study offers an accurate and cost-effective on-site electrochemical analysis system that can be modularly adjoined into various biological and medical applications. Keywords: Flexible electrode; Carbon nanotube; PDMS; three-electrode system; On-site Electrochemical analysis

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1. Introduction Recently, flexible electrodes have attracted extensive research interests for their wide applications in flexible supercapacitors or batteries, artificial electronic skin and point of care testing devices [1-3]. Tremendous efforts have been paid toward developing the flexible electrodes of robustness and high conductivity with diverse materials and approaches [4]. Besides the flexible electrodes produced from the self-standing conducting polymers and their composite films which occupy both of conductivity and flexibility without any supporter [5-8], most of the researches have been focused on developing the flexible electrodes through the combination of the conductive materials and the soft substrates like polyethylene terephthalate (PET), polyimide (PI) and polydimethylsiloxane (PDMS) [9-11]. Compared with the graphene of expensiveness, metal nanomaterials lack of mass production and the conducting polymer of the unsatisfied conductivity and life cycles[12-18], multi-wall and single-wall carbon nanotubes (MWCNTs and SWCNTs) have showed their talents as the proper candidates of great potential owning to the extraordinary electrical properties, low-cost mass-production, high chemical stability and prominent biocompatibility [19-28]. As the most popular elastomeric substrate material used for the flexible electrode fabrication, PDMS possessing the distinct essential features (e.g. easy processing, low toxicity, optical transparency, chemical inertness and well mechanical properties [29-31]) is widely utilized to cooperate with CNTs to develop strain sensors which generate an electronic signal as the function of the substrate deformation introduced by pressure, strain, shear, torsion, humidity and temperature [32-39]. However, the electrochemical analysis of chemical or biological substances with flexible sensors remains a great challenge, in particularly, to realize the accurate on-site detection during the biological and medical processes [40, 41]. More recently, there are a few of records reporting the flexible sensors capable of the biological detection by electrochemical techniques. Lee et al. developed a flexible glucose sensor based on a two-electrode configuration which employed a glucose oxidase-modified MWCNT/Au/PDMS composite film as the working electrode and a Pt counter electrode [42]. Compare to the two-electrode system, the three-electrode system is able to offer more accurate and stable electrochemical signal due to the steady potential of the reference electrode during the measurement process [43]. Li et al. ultrasonically dispersed CNT with 2-Propanol as the solvent to prepare the MWCNT/PDMS film on the hydrophobic substrates like glass and PET. Taking the MWCNT/PDMS films as the working, counter and reference electrodes, a three-electrode system was constructed on the surface of a glass slide to detect DNA by Differential Pulse Voltammetry [44]. However, the use of organic solvent 2-Propanol to disperse MWCNT limits its application in biological area. Jin et al. fabricated a flexible electrode with PEDOT-covered MWCNT on a piece of PDMS [45]. In cooperated with a Pt counter electrode and an Ag/AgCl reference electrode, the MWCNT@PEDOT/PDMS working electrode was applied to detect nitric oxide in a PDMS chamber where human umbilical vein endothelial cells had been cultured for 10 hours before the electrochemical analysis. Another flexible sensor was also

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fabricated for the cardiac Troponin-T detection through the e-beam deposition of 25 nm Cr and 125 nm Au on a porous polyimide membrane under a three-electrode-patterned stencil mask [46]. To crosslink antibodies, ZnO nanorods hydrothermally grew up on the working electrode surface covered by a layer of Zn seeds which were prior prepared by sputtering deposition. In this paper, MWCNTs/PDMS electrodes were utilized for electrochemical analysis rather than the widely studied strain sensors. Meanwhile, our three-electrode system is able to offer more accurate and stable electrochemical signal compare to the two-electrode system. And compared with the time-consuming, sophisticated or toxic fabrication procedures involved in the reports mentioned above, this paper describes a low-cost three-electrode electrochemical analysis system developed under a green and facile manufacture process. The cuttable MWCNT/PDMS flexible composite films are fabricated through a simple molding transfer method and are employed as the working and the counter electrodes. A silver-paste line is painted as the pseudo-reference electrode. The three-electrode system can be customized into various sizes and geometries adaptively, easily assembled into modules (as shown in the last two steps of scheme 1) and equipped into pipes, tubes, cell culture plates and/or petri dishes for biological and medical on-site detection. In order to value the performance of the developed on-site three-electrode electrochemical anaysis system in practice, a medical drip and a 24-well plate are equipped with the developed analysis modules to detect several biomarkers of electrochemical activity, such as dopamine (DA) [47-49], uric acid (UA) [50-52], and nicotinamide adenine dinucleotide (NADH) [53-55], by Differential Pulse Voltammetry (DPV) technique.

Scheme 1. The preparation process of the MWCNT/PDMS flexible film and the three-electrode electrochemical analysis module integrated within a medical drip.

2. Experimental 2.1. Reagents and materials MWCNTs of 10-20 μm in length and 50 nm in diameter were purchased from

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XFNANO Materials Tech Co., Ltd (Nanjing, PRC). SYLGARD 184 silicone elastomer was obtained from Dow Corning GmbH (USA). Dopamine (DA) from Target Molecule Corp (PRC), Uric acid (UA) from Acros Organics (USA) and NADH disodium salt from BBI Life Sciences Corporation (Shanghai, PRC) were used to value the performance of the developed three-electrode system. Sodium dodecyl benzene sulfonate (SDBS) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, PRC). Phosphate buffer solution (PBS, 0.01 M, pH 7.4) was purchased from biosharp (PRC). Reagents of analytical grade or the highest commercially available purity were used without further purification.

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2.2. Apparatus and measurements The dispersion of MWCNTs was processed in a SB-5200D ultrasonic cleaner (Ningbo Scientz Biotechnology Co., Ltd, PRC). MWCNT film was formed on the surface of a nylon filter membrane (0.22μm pore size) through a set of vacuum filtration devices (Rocker 300, Taiwan, PRC). PDMS was cured in a DHG-9011A electric thermostat oven (Shanghai Jing Hong Experimental Equipment Co., Ltd.). A digital multimeter (Vc9808, Victor, PRC) was used to measure the resistance of the constructed electrodes. The morphology of MWCNT/PDMS film was investigated by a high resolution scanning electron microscope (SEM, KYKY-EM8000F). A set of TS-1B syringe pumps (Longer Precision Pump Co. Ltd, PRC) was in charge of the intermittent injection of DA, UA and PBS into the modified medical drip. All electrochemical experiments were performed with a CHI 660E electrochemical workstation (Shanghai Chen Hua Instrument Company). Double-distilled water (DD water) was obtained from the Milli-Q academic system (Merck Millipore Ltd., USA) and was used in all experiments.

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2.3. Preparation of the MWCNT/PDMS composite film Schematic of the procedures to prepare MWCNT/PDMS flexible film was shown in Scheme 1. First, 30 mg MWCNT and 10 mg SDBS were ultrasonically dispersed in 200 ml DD water for 1 h. The resulting MWCNT suspension was then filtered through a 0.22 μm filter membrane in vacuum. Subsequently the sediment was washed with DD water for several times and was dried for 15 min at 75 °C. The obtained uniform MWCNT film formed on the surface of the filter membrane was cut into required size and shape to adapt to the different analysis environments. The PDMS prepolymer and curing agent were mixed at a weight ratio of 10:1 and were poured into a Petri dish where the customized MWCNT film along with the filter membrane was placed at the bottom. After curing the PDMS at 75 °C for 1.5 h, the customized MWCNT/PDMS composite film was then easily peeled from the Petri dish and so did the filter membrane from the MWCNT/PDMS film. Thus, the MWCNT film on the surface of the filter membrane was successfully transferred to the flexible PDMS substrate. The morphology of the MWCNT/PDMS film was characterized by SEM and its conductivity measurement in the bending or stretch situations was carried out with a rectangular MWCNT/PDMS film (size of 30 mm in length and 10 mm in width)

ACCEPTED MANUSCRIPT clamped by two fixtures connected to a digital multimeter at specific lengths or radians.

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2.4. Fabrication of the three-electrode analysis system Several types of the three-electrode analysis system were set up according to the application requirements. Generally, two pieces of the MWCNT/PDMS film were involved as the working and the counter electrodes, respectively. Electrical contact was achieved by bonding a copper wire with the MWCNT/PDMS surface and was enhanced by applying silver paste between the electrode and the wire. A straight line of 1 cm in length was painted with silver paste between the two pieces of MWCNT/PDMS electrodes, as a pseudo reference electrode. Before use, the three-electrode analysis system was activated in 1 M KCl aqueous solution by cyclic voltammetry (CV) in the range between -1 V and 1 V at a scan rate of 0.05 V/s for several cycles until the CV curves achieved a stable state. Afterwards, the developed three-electrode analysis module attached on the inner wall of a 24-well plate (Fig. S4 A and B) was used to determine the different concentrations of a series of NADH/PBS solutions by DPV from 0.2 V to 0.9 V. DPV parameters were set at an increase voltage of 0.005 V, a amplitude of 0.05 V, and a pulse period of 0.3 s. It cost 42 s for each detection of NADH. Furthermore, a medical drip with an open window of 5 mm × 15 mm was designed to integrate with the developed analysis module in order to simulate an electrochemical detection during a medical infusion process (Fig. S4 C, D and E). Firstly, 1 μM DA/PBS solution was pumped into the equipped medical drip at an injection rate of 1 ml/min while the electrochemical analysis module was kept in liquid by controlling the medical drip regulator. After 5 min, the DPV measurement was conducted from 0 V to 0.6 V at an increase voltage of 0.005 V and a pulse period of 0.3 s to record the concentration of the DA/PBS solution. Accordingly, the response time was 36 s for the once detection of DA or UA. A series of DA/PBS solutions from 10 μM, 20 μM, 30 μM, 40 μM to 50 μM and the UA/PBS solutions of 1 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM and 500 μM were sequentially assayed following the same procedure. The DPV curves were summarized to explore the performance of the electrochemical analysis module. Finally, taking PBS as the carrying solution, 1 mM DA/PBS and 1 mM UA/PBS were simultaneously injected into the equipped medical drip at their own speed (Table 1) for 2 min. When the analytical system reach a steady state, the electrochemical detection of DA and UA was performed by DPV from 0 V to 0.6 V. After each test, the medical drip was washed by injecting PBS for 10 min. Table 1. The injection rates in the simultaneous detection of DA and UA Test a Test b Test c PBS 1 ml/min 1 ml/min 1 ml/min 1 mM DA/PBS 10 μl/min 50 μl/min 100 μl/min 1 mM UA/PBS 50 μl/min 100 μl/min 200 μl/min 3. Results and discussion

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3.1. Characteristics of the MWCNT/PDMS flexible electrode The morphology of the MWCNT/PDMS flexible electrode was visualized by SEM. Fig. 1 A and B showed the SEM images of the cross section and the top-view of the developed composite film, indicating the reticular structure of MWCNTs which was firmly adhered to the surface of the PDMS matrix. The thickness of the MWCNT layer was approximately 80 μm. Some irregular protrusions were observed because of a small amount of PDMS penetrating into the MWCNTs network and molding into the filtration membrane pores. The well-exposed MWCNT net structure on the flexible PDMS substrate could offer an excellent ability of electron transfer under the stretching or compressing situations.

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Fig. 1. SEM images of (A) the cross section and (B) the surface of the MWCNT/PDMS composite film.

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Stretch and bending tests of the MWCNT/PDMS flexible electrode (30 mm × 10 mm) was performed as shown in Fig. S1 and S2 in the Supporting Information. The resistance at the bending radius of 16 mm, 26 mm and 42 mm were nearly invariable (Fig. 2A). However, when stretched into 3.5 cm, 4 cm, 4.5 cm in length, the resistance of the developed flexible electrode exhibited a linear relationship to the deformation ratio, as shown in Fig. 2B. The stable network structure of the conductive layer in the composite film insured the high conductivity and proper mechanical stability of the fabricated flexible electrode, suggesting a great application potential of the MWCNT/PDMS flexible electrode. The newly developed three-electrode analysis module set-up in a 24-well plate or a medical drip have to be subjected in 1 M KCl aqueous solution by cycling the potential from -1 V to 1 V at the scan rate of 0.05 V/s to activate the effective area of the three electrodes. As shown in Fig. 2C, the CV curves obtained from the three-electrode analysis module in a 24-well plate were changeless after 10 cycles, suggesting the stable attachment of the three-electrode analysis module to the curved inner wall and the steady working state of the three electrodes, which would benefit to enhance the electron transfer between electrodes and to assist the silver paste reference electrode in offering a steady electrode potential. The electron transfer ability of the newly developed sensor was further

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investigated in an aqueous solution containing 2 mM K3Fe(CN)6 and 0.1 M KCl by CV from -0.3 V to 0.6 V at various scan rates. As Fig. 2D presented, the reversible redox peaks indicated that the constructed three-electrode system was very stable and the pseudo reference electrode which was made of the conductive silver paste can also achieve the requirements of practical detections compared with the commercial Ag/AgCl reference electrodes. In electrochemistry, redox processes can be either surface controlled or diffusion controlled. For surface confined process, peak current magnitude should be linear with scan rate, while for diffusion controlled process, redox peak magnitude is linear with square root of scan rate. Therefore, a well-defined linear relationship (inset of Fig. 2D) between the redox peak current and the scan rate was observed as expected for the surface control process rather than the diffusion control dominating on the working electrode surface.

Fig. 2. The relationships between the resistance of the developed MWCNT/PDMS flexible electrode vs. (A) the bending radius and (B) the stretch ratio (data were presented as the value of mean ± s.d., for n = 4 independent tests). Cyclic voltammograms of the three-electrode analysis system (fabricated as shown in Fig. S4) (C) in 1 M KCl for 20 cycles from -1 V to 1 V at the scan rate of 0.05 V/s and (D) in an aqueous solution containing 2 mM K3Fe(CN)6 and 0.1 M KCl at different scan rates (inset: the linearity between the redox peak current and the scan rate, suggesting the surface control process of electron transfer occurred on the working electrode surface).

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3.2. Performance of the three-electrode analysis module As mentioned above, a 24-well cell culture plate integrated with the fabricated flexible sensor on the inner wall was firstly activated by CV in 1 M KCl for 20 cycles. Then a series of PBS containing different concentrations of NADH was added into the detection chamber successively to value the performance of the developed three-electrode analysis module. The obtained DPV curves were shown in Fig. 3A. The oxidation peak of NADH was located around 0.54 V and the peak current exhibited the linear increasement with the NADH concentration from 10 μM to 1 mM in the function of INADH (μA) = 6.46 + 0.065×CNADH (μM ) with R2 of 0.998.

Fig. 3. DPV data obtained from the three-electrode analytical module in 0.01 M PBS containing different concentrations of (A) NADH (a-f: 10 μM, 100 μM, 300 μM, 500 μM, 700 μM, 1 mM), (B) DA (a-g: 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100μM) and (C) UA (a-h: 1 μM, 10 μM, 50μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM); inset plots: calibration curves (data were presented as mean ± s.d., for n = 3 independent tests). (D) DPV curves of the three-electrode analytical module when simultaneously detecting DA and UA in 0.01 M PBS.

The individual electrochemical behaviors of DA and UA were investigated in a medical drip equipped with the developed three-electrode analysis module described above. As shown in Fig. 3 B and C, the oxidation peak potentials of DA and UA were

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placed at 0.16 V and 0.35 V respectively. The peak current both of the analytes were enhanced linearly with their increased concentrations. The linear regression equation of DA was calibrated as IDA (μA) = 7.447 + 0.273 ×CDA (μM) (R2 = 0.995) in the range of 1 μM to 50 μM. So as UA was IUA (μA) = 1.685 + 0.156 ×CUA (μM) (R2 = 0.999) from 1 μM to 500 μM. It is reported that the normal concentration of dopamine and uric acid in human serum is around 0.02 mM and 0.32 mM, respectively, which can be successfully detected with the developed three-electrode analysis system [56-57]. Finally, the simultaneous detection of DA and UA with the developed three-electrode analysis module was also performed with the equipped medical drip. The DPV peak current presented in Fig. 3D was summarized in Table 2. Two well-defined voltammetric peaks at 0.16 V and 0.37 V were assigned to the oxidation peaks of DA and UA, respectively. The separations of the peak potentials was 210 mV, demonstrating that the developed electrochemical analytical module could offer an excellent selective detection of the analytes. According to the standard equations established above, the concentrations of DA and UA in Test a (Fig. 3D, curve a) were calculated to be 9.91 μM and 49.44 μM from the values of their peak current. After adjusting the injection rates of the analysts in Test b and Test c, the DA and UA concentrations of curve b and curve c were converted to be 44.99 μM and 91.47 μM, 73.45 μM and 162.04 μM, respectively. According to the injection rates in the simultaneous detection of DA and UA in Table 1, the calculated concentration of DA in Test a, b, and c is 9.43 μM, 43.48 μM, 76.92 μM and the calculated concentration of UA in Test a, b, and c is 47.17 μM, 86.96 μM, 153.85 μM, respectively. Compared to the calculated concentration, the detected concentration of DA and UA showed no obvious change (less than 5%), indicating that the co-existence of DA and UA can be successfully detected by the three-electrode module equipped in the medical drip.

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Table 2. The determined concentrations of DA and UA when simultaneously detected in the equipped medical drip. Test a Test b Test c IDA (μA) 10.15 19.73 27.50 CDA (μM) /detected 9.91 44.99 73.45 CDA (μM) /calculated 9.43 43.48 76.92 IUA (μA) 9.40 15.95 26.96 CUA (μM) /detected 49.44 91.47 162.04 CUA (μM) /calculated 47.17 86.96 153.85 3.3. Selectivity and stability of the three-electrode analysis module Ascorbic acid (AA) is the most important interferences in the DA electrochemical detection from biological samples. Therefore, we further investigated the selective detection of DA coexisted with AA in PBS by using the MWCNT/PDMS electrode. As Fig. S5 showed, only a minor change of the sensor peak current was detected for DA in the presence of AA. Our results indicate that two well-defined and individual peaks at 0.1 V and 0.17 V were observed for the oxidation of AA and DA,

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respectively, which demonstrated MWCNT/PDMS sensor can be successfully used for electrochemical analysis of DA in the co-existence of AA. The long-term stability of the three-electrode analysis module was assessed by the electrochemical determination of a series of NADH/PBS solutions of the defined concentrations at the interval of every 3 days. The three-electrode analysis module set up in a 24-well plate was stored in PBS solution at room temperature between the two adjacent measurements. As the DPV oxidation peak current summarized in Fig. 4, the original sensitivity of 64.50 μA/mM (R2=0.998) was turned into 64.15 μA/mM (R2=0.999) after 6 days and to 64.31 μA/mM (R2=0.998) after 15 days which retained 99.7% of the initial sensitivity. After 35 days, the response sensitivity of the three-electrode analysis module was 61.42 μA/mM (R2=0.997), retaining 95.22% of its initial value (data not show). The minimal decrease of the detection sensitivity showed the excellent stability and a great potential of the presented three-electrode system in practical applications. Another application of the electrochemical analytical module as a flexible sensing tag that can directly attach to human skin, is under researching, as shown in Fig. S6 in Supporting Information.

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Fig. 4. (A) Stability test of the presented three-electrode module evaluated in a series of NADH/PBS solutions at the interval of every 3 days and (B) the histogram of their sensitivity.

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4. Conclusions A novel flexible and cuttable MWCNT/PDMS electrode has been prepared through a green molding transfer method and a universal three-electrode analysis module adapting to on-site electrochemical analysis has been proposed with the MWCNT/PDMS flexible electrodes. The individual and/or simultaneous electrochemical determinations of DA, UA and NADH were successfully realized in a cell culture plate and an infusion drip integrated with the three-electrode analysis module. The three-electrode analysis module showed a fast response to the analysts in a broad linear detection range with satisfactory stability and repeatability. The facile fabricating method, versatile process-ability, low cost, as well as the superior conductivity, are of benefit to the proposed on-site electrochemical analysis in adjoining into many biological and medical applications.

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Acknowledgements This work was supported by the State Key Laboratory of Robotics Open Grant (2016-006) and the China Science and Technology Innovation Zone (17-163-15-XJ-002-002-09).

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ACCEPTED MANUSCRIPT Highlights

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MWCNT/PDMS flexible electrodes are prepared through a solution-based green method.

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A three-electrode analytical module is developed with the MWCNT/PDMS flexible electrodes.

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On-site electrochemical detection of NADH, dopamine and uric acid is achieved with the

The developed technique offers an accurate and cost-effective on-site electrochemical

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analysis system that can be modularly adjoined into various biological and medical

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applications.

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analytical module-equipped cell culture plates and medical drips.