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A flexible electrochemical glucose sensor with composite nanostructured surface of the working electrode
Accepted Manuscript Title: A flexible electrochemical glucose sensor with composite nanostructured surface on the working electrode Author: Zhihua Pu Ridong Wang Jianwei Wu Haixia Yu Kexin Xu Dachao Li PII: DOI: Reference:
S0925-4005(16)30264-7 http://dx.doi.org/doi:10.1016/j.snb.2016.02.115 SNB 19775
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
28-10-2015 20-2-2016 23-2-2016
Please cite this article as: Zhihua Pu, Ridong Wang, Jianwei Wu, Haixia Yu, Kexin Xu, Dachao Li, A flexible electrochemical glucose sensor with composite nanostructured surface on the working electrode, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.02.115 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A flexible electrochemical glucose sensor with composite
nanostructured surface on the working electrode
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Zhihua Pu, 1Ridong Wang, 1Jianwei Wu, 2Haixia Yu, 1Kexin Xu and 1Dachao Li*
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State Key Laboratory of Precision Measuring Technology and Instruments, Tianjin University,
Tianjin, China 2
Tianjin Key Laboratory of Biomedical Detecting Techniques and Instruments, Tianjin University,
Tianjin, China
Tel: +86-22-27403916 *E-mail: [email protected]
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Highlights:
A flexible electrochemical sensor was fabricated into the ISF extraction chip to build a wearable micro device for continuous glucose monitoring.
Composite nanostructured surface with graphene and AuNPs was constructed on the WE to enable accurate measurement of low levels of glucose.
Graphene was directly written onto the tiny-sized WE surface by inkjet printing method without complicated CVD deposition and transferring process.
AuNPs and GOD were electrodeposited onto the WE surface without any protection for the other parts that did not need the modification.
Abstract Detecting hyperglycemia remains a great challenge with regard to continuous glucose monitoring in clinical settings. This paper investigates a novel, flexible three-electrode electrochemical sensor with a composite nanostructured working electrode surface that has been modified by graphene and gold nanoparticles in order to detect low levels of glucose with high accuracy. The sensor electrodes were fabricated on a polyimide substrate using the flexible printed circuit board (PCB) method. Graphene was modified directly onto the working electrode surface via inkjet printing, an emerging method for micro-scale fabrications, to enable glucose detection at low levels. Gold nanoparticles were electrodeposited directly onto the graphene layer to enhance the sensitivity of the sensor. The experimental results demonstrate that the proposed sensor can precisely measure glucose with a linear range of 0~40 mg/dL and a detection limit of 0.3 mg/dL (S/N=3), thereby demonstrating potential for hypoglycemia detection. Moreover, this flexible sensor was suitable for integration within a microfluidic chip, which could be used to transdermally extract and collect ISF, such that a wearable device could be developed for continuous glucose monitoring.
Keywords: electrochemical sensor; flexible substrate; glucose sensing; graphene; gold nanoparticles; inkjet printing
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1. Introduction Continuous blood glucose monitoring holds great significance for the treatment of diabetes. However, it is difficult to continuously monitor glucose in the blood directly. Glucose concentrations of interstitial fluid (ISF) are closely related to blood glucose levels [1] [2] [3]. Thus, research attention has focused on developing a minimally invasive blood glucose measuring method based on ISF analysis, the advantages of which include painless application and the realization of continuous glucose monitoring [4]. Electrochemical biosensors have played a leading role in the advancement of continuous blood glucose monitoring [5]. To date, most continuous glucose monitoring systems based on ISF analysis that have been used in clinical environs have been based on implantable enzyme electrode sensors [6]. However, this methodology is restricted by its invasiveness and the short lifetimes of the electrodes caused by the foreign body reaction in subcutaneous tissue. Furthermore, glucose concentrations cannot be measured accurately due to a signal drift caused by the bioelectricity of the human body [7]. Thus, blood glucose predictions based on glucose measurements in transdermally extracted ISF has become an emerging technology for continuous glucose monitoring. This method can enable continuous glucose monitoring externally, thereby avoiding interferences from internal environments, such as interferences related to the foreign body reaction and the bioelectricity within the human body. Our research group used ultrasound and a vacuum to extract ISF transdermally over an area of 0.07 cm2. Ultrasound was used to create micro pores to increase the permeability of the skin [8], and a vacuum pump was used to enhance ISF convection. It was difficult to collect the extracted ISF, which was scattered on the surface of the skin and whose volume was approximately 1 μL [9]. Therefore, a predefined volume of phosphate buffer was used to dilute the transdermally extracted ISF for easy collection. However, the glucose concentration in the ISF decreased significantly after dilution. Thus, we developed a microfluidic system for the extracting, diluting and collecting ISF [10]. This system featured five polydimethylsiloxane (PDMS) layers and was fabricated using micromolding techniques. This system required integration of a micro-sized glucose sensor into the microchannel of the microfluidic chip. However, it was difficult to fabricate the glucose sensor’s electrodes on the PDMS surface because of their hydrophobicity and poor adhesion to metals [11]. Accordingly, some groups [12] fabricated electrode-based sensors on glass and then bonded the glass to PDMS-based chips to form functional microfluidic devices. Nevertheless, although the PDMS-based microfluidic chip is flexible, the bonded glass makes the whole device rigid. Unfortunately, skin deformation always happens during ISF extraction by vacuum. Although the PDMS-based microfluidic chip can transmute along with the transformation, the rigid whole chip that results from depositing the sensor on glass
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may lead to spillage of the analytical liquid, resulting in inaccurate measurements. Thus, the present paper describes the fabrication of a high-resolution electrochemical sensor on a flexible polyimide (PI) substrate that can be integrated into the microchannel of the ISF extraction chip and that can transmute with chip deformation to accurately measure glucose concentrations in diluted ISF extractions. The glucose concentration in diluted ISF can be as low as approximately 2 mg/dL; therefore, existing commercial glucometers with detection levels ranging from 20 to 500 mg/dL are not applicable. New technologies are being pursued to develop novel glucose sensors. Zimmermann et al. [13] proposed an enzyme-based electrochemical flow-through glucose sensor that could be integrated into a flow channel and reported a detection limit of 20 mg/dL. Huang et al. [14] proposed a capacitive MEMS affinity sensor for continuous glucose monitoring at physiologically relevant concentrations ranging from 30 to 360 mg/dL. Tierney et al. [15] proposed a glucose-sensitive sensor prepared by incorporating 3-phenylboronic acid and dimethylaminopropylacrylamide into a hydrogel covalently linked to the end of an optical fiber. The latter glucose sensor was only capable of measuring physiological glucose levels with a detection limit of 18 mg/dL. Tashkhourian et al. [16] proposed a glucose biosensor based on chromophore (silver nanoparticles) decolorization for the photometric determination of glucose; the linear range of this glucose sensor was 50 to 800 mg/dL, and the limit of detection was 23 mg/dL. Siegrist et al. [17] proposed a continuous glucose sensor that involves binding genetically engineered polypeptide-based fluorescent molecules to improve the accuracy of the sensor; in this case, the linear range spanned from 35 to 586 mg/dL. Although the aforementioned sensors exhibited various merits, their detection ranges only covered the physiological glucose range, meaning that these methods were barely able to measure the low glucose concentrations in diluted ISF with high accuracy. This paper presents a flexible electrochemical sensor, which utilizes a working electrode with a composite nanostructured surface that is capable of measuring glucose levels in diluted ISF with high accuracy. The composite nanostructure significantly improved the performance of the sensor [18]. Graphene was inkjet printed onto the working electrode surface to improve electroactivity and impart a more uniform distribution of electrochemical active sites in order to achieve glucose detection at low levels. Gold nanoparticles (AuNPs) were electrodeposited onto the graphene layer to improve the electron transfer rate between the activity centers of the enzymes and the electrode and, thereby, enhance the sensor’s sensitivity. The two methods directly deposited graphene and AuNPs onto the working electrode surface without requiring protection from modification of any of the other parts of the sensor. Subsequently, in order to obtain glucose-specific detection, glucose oxidase was immobilized via electrochemical polymerization onto the composite nanostructured surface of the working electrode. The resulting glucose sensor can be integrated with the ISF extraction chip to 4
produce a flexible and wearable device. This wearable device makes a flexible connection to the skin, thereby significantly reducing the relative motion between the device and the skin such that a more stable and accurate detection signal is obtained.
2. Methods and materials 2.1. Design of the flexible electrochemical glucose sensor Fig. 1(a) displays a schematic diagram of the proposed flexible glucose sensor, which is a threeelectrode electrochemical sensor that includes a working electrode (WE, 1.2×0.2 mm), a reference electrode (RE, 1.2×0.2 mm), and a counter electrode (CE, 1.2×1 mm). The electrodes were fabricated on a flexible PI substrate via a flexible printed circuit board (PCB) process. Then, 5-μm Au was electroplated onto the surface of the electrodes. After first electroplating Ni onto the Cu layer, the Ni in the KAu(CN)2 solution was replaced with Au so that Au replaced Ni in the Cu layer. The corresponding reaction formulas are as follows: i Ni 2 2e Ni
3Ni 2 Au (CN ) 2 2 Au 3Ni
（2-1） 2
（2-2）
Subsequently, silver was electroplated onto the surface of the RE, then the electrodes were immersed in a solution of 50 mM FeCl3 for 50 s, such that an Ag/AgCl RE was obtained (AgNO3, NH3·H2O and FeCl3 were obtained from Tianjin Jiangtian Huagong Co., China). As shown in Fig. 1(b), the working electrode was first modified with graphene via inkjet printing and evaporating the solvent at 120°C for one hour. A uniform distribution of uniformly sized AuNPs was then electrodeposited onto the graphene layer. Finally, glucose oxidase (GOx) was immobilized onto the graphene layer with AuNPs via electrochemical polymerization to enable glucose-specific detection.
During electrochemical glucose sensing, glucose decomposed catalytically via GOx and the H2O2 generated was subsequently oxidized at the electrode surface, producing a measurable current signal. Therefore, H2O2 could be used to characterize the electrodes directly, without the need for immobilized GOx. The chemical reaction formulas are as follows: （2-3）
GOxox glucose H 2O GOxred gluconic acid H 2O2
（2-4）
GOxred GOxox + ne-
（2-5）
H 2O2 O2 2H 2e
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2.2. Modification of graphene on the WE surface The WE surface was modified with graphene to improve electroactivity and to achieve a significantly more uniform distribution of electrochemical active sites in order to enable glucose detection at low levels. Graphene is a novel nanomaterial that, since 2004, has attracted considerable attention due to its excellent properties, including exceptional thermal and mechanical properties, high electrical conductivity, larger surface area, and good biocompatibility [19]. This material has been shown to exhibit superior conductivity [20], and the special concave-convex topography of graphene generates a high number of electrochemical active sites, making it likely to detect weak signals, i.e., glucose at low concentrations. In addition, due to π-stacking interactions between the hexagonal cells of the electrode material and the carbon-based atomic ring structure of graphene, the electrode surface adsorbs biomolecules that contain carbon-based ring structures [21], increasing its capacity for adsorbing GOx molecules. Additionally, the good biocompatibility of graphene increases the bioactive lifetimes of immobilized GOx molecules.
In general, graphene is deposited via chemical vapor deposition, after which it is cut to the desired shape and transferred to the target surface in water [22]. The procedure involved is complex, and it is difficult to acquire graphene with the desired shape when the size is so small. Its tiny size also makes it difficult to transfer graphene onto the WE surface. Thus, we directly deposited the desired shape of graphene onto the WE surface via inkjet printing, an emerging additive manufacturing technique for forming functionalized microstructures [23]. The inkjet printing system was assembled as shown in Fig. 2. This system consisted of a pump, an air pressure control module, a displacement stage, four printheads, a printhead driver, an observation module and a computer. The pump supplied the pressure power for the system. The air pressure control module was used to wash the printheads and maintain the air pressure within them such that successful printing was ensured. The displacement stage was used to fix the printing substrates and to maneuver them as desired. Four printheads could be used to print different materials onto the target substrates. The printhead driver module was used to drive the printheads based on the corresponding printing signals. The observation module included a horizontal camera with a strobe, which enabled us to observe the printing status, as well as a vertical camera that
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enabled us to calibrate the start-point for printing. We used a computer to design the printing routes and supply the printing signals. The accuracy of this inkjet printing system was 20 μm. Graphene modification employed the following procedures. First, we placed the electrodes onto the working stage of the inkjet printer. Then, a desired image, based on the shape and the size of the WE, was drawn by CAD, which was integrated with the controlling software of the printer. Subsequently, the start-point for printing was calibrated using the vertical camera of the printing system. Finally, one layer of graphene ink (Sigma-Aldrich Inc., USA) was printed directly onto the WE surface using the following optimized conditions: 60 V, 500 Hz, and 40°C. The printhead used was purchased from MicroFab, Inc., and the orifice employed was 20 μm. The density of the graphene ink was 0.9375 g/ml, and the graphene was comprised of pieces of reduced graphene oxide (rGO), which is inkjet printable [24]; the rGO pieces were less than 3 μm in diameter. The modification was completed by evaporating the solvent (in the printed graphene ink) at 120°C for one hour. Once spread on the WE surface, the diameter of the printed graphene droplet was approximately 60 μm. 2.3. Modification of the graphene layer with AuNPs AuNPs were used to modify the graphene layer on the WE in order to enhance the sensitivity of the sensor by constructing a composite nanostructured surface that improved the electron transfer rate between GOx activity centers and the electrode. AuNPs are widely used in biosensing because they possess a number of generally inherent, beneficial characteristics, including a large surface-to-volume ratio, good electrocatalytic activity and high chemical reactivity [25]. Their special size renders AuNPs particularly capable of transferring electrons between GOx activity centers and the electrode surface, effectively producing nanowires that enhance the sensitivity of the sensor. The large surface-to-volume ratio of AuNPs assists with GOx immobilization and generates a large number of unsaturated bonds on the electrode surface, which, in turn, make AuNPs more active such that good electrocatalytic activity and high chemical reactivity are achieved. The resulting improvements with regard to electrocatalytic activity and high chemical reactivity facilitate the catalytic reaction of glucose and H2O2 and, therefore, further enhance the sensitivity of the sensor. Both physical and chemical methods have been used to deposit AuNPs on electrode surfaces. With regard to physical methods, evaporation [26] and sputtering [27] are frequently used and dramatically decrease deposition times. However, these methods require high temperatures and protection for the other parts of the system that do not require modification. Furthermore, it is difficult to control the sizes and distributions of AuNPs using these methods. With regard to chemical methods, self-assembly [28] and sol-gel [29] are frequently employed. However, the AuNPs deposited by these methods aggregate easily, significantly affecting the properties of the sensor, and, as with physical methods, 7
protection is required for the other parts of the system that do not require modification. In the present case, the flexible PI substrate cannot sustain high temperatures, and the WE surface is the only part of the system requiring modification with AuNPs. Thus, we employed the electrochemical deposition method at room temperature to directly deposit AuNPs onto the graphene layer of the WE without requiring any protection for the other parts of the sensor. This methodology made it easy to control the size as well as the distribution of AuNPs, which significantly affect the performance of the nanoparticles [30]. An electrochemical setup, in which the target electrode served as the WE, a Pt sheet served as the CE, and an Ag/AgCl electrode served as the RE, was dipped into a plating solution consisting of HAuCl4 and 0.5 M Na2SO4 (Shanghai Xinbo Chemistry Technique Co., China) to electrodeposit AuNPs onto the graphene layer. The AuNPs were deposited via constant current pulses that were applied between the WE and CE by an electrochemical workstation (CHI 660E, CHI Instruments Inc., USA). HAuCl4 concentration, deposition current intensity and deposition time violently affected the modification with AuNPs, so several experiments were conducted to characterize sensor performance. H2O2 was utilized to test the AuNPs-modified sensor under different conditions, and the sensitivity and linearity of the system were evaluated in order to optimize the parameters utilized for AuNPs modification. 2.4. Immobilizing GOx onto the WE surface GOx (Sigma-Aldrich Inc., USA) was mixed with 3,4-ethylenedioxythiophene (EDOT) (SigmaAldrich Inc., USA) before immobilized onto the WE surface. The GOx/EDOT solution was prepared by first dissolving PSS (0.1 M) (Sigma-Aldrich Inc., USA) in H2O. Next, EDOT (0.03 M) was added to the mixture as the solution was agitated. Subsequently, the H2O-dissolved GOx (2 mg/mL) was added to the mixture. This solution was used to electrochemically polymerize GOx onto the composite nanostructured surface, on which graphene and AuNPs were constrained, by applying constant current pulses of 2 mA/cm2 between the WE and CE over 1000 s [31]. Immobilization of GOx onto the WE surface was achieved using methods similar to those stated in section 2.3, and protection for the other parts of the system, which did not require GOx deposition, was also not necessary. 3. Results and discussion 3.1. Measurement system The measurement system consisted of an electrochemical workstation to supply the driving potential and to measure the output current of the proposed sensor, a pipette to provide different concentrations
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of the determinand in the reaction tank, a computer to control and receive signals from the workstation. The fabricated electrochemical sensor was characterized by immersing it into electrolyte solutions containing 0.1 M phosphate buffer solution (PBS) (pH=7.4) and different concentrations of glucose or H2O2 (Tianjin Jiangtian Huagong Co., China). A stir bar was not used because the volume of the analytical solution was extremely small (200 μL) such that diffusion quickly reached equilibrium. All experiments were conducted at room temperature. 3.2. Effect of graphene on the flexible electrochemical glucose biosensor Amperometry, which is used frequently in electrochemical measurements, was used for glucose measurements to study the sensitivity and linearity of the fabricated electrochemical sensors with and without graphene after GOx immobilization. An SEM image of the printed graphene on the WE is shown in Fig. 3(a), and Fig. 3(b) shows typical amperometric responses (using an applied potential of -0.2 V) of the three sensor configurations to successive incremental additions of 5 mg/dL glucose [19]. These results demonstrate that the current signals pertaining to low levels of glucose cannot be distinguished by the sensors if graphene is absent, whether or not AuNPs have been deposited; however, low glucose levels can be identified and measured when graphene is present. This indicates that graphene significantly improves the detection of weak glucose signals.
3.3. Enhancements of the electrochemical sensor achieved via AuNPs In order to evaluate the effects of different AuNPs modification conditions on the sensor’s ability to detect H2O2, sensors with AuNPs modification of the WE graphene layer without GOx immobilization were first tested amperometrically. Each parameter was evaluated while holding the other two parameters constant. Fig. 4 shows the typical amperometric responses obtained, using an applied potential of -0.2 V, for various sensor configurations in response to successive increments of 0.2 mM H2O2. As shown in Fig. 4, the electrochemical responses increase as H2O2 concentrations increase, and all of the sensors demonstrate good linearity from 0 to 1.8 mM H2O2. Fig. 4 also shows that the HAuCl4 concentration, the deposition current intensity and the deposition time significantly affect the AuNPs modification obtained, as demonstrated by the substantial sensitivity changes recorded for sensors that were modified with AuNPs under different conditions. As shown in Fig. 4, the optimized AuNPs deposition conditions determined include 4 mM HAuCl4, a deposition current intensity of 10 mA/cm2 and a deposition time of 200 s.
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After optimizing the experimental conditions, AuNPs were electrodeposited onto the graphene layer of the WE surface under these conditions. An SEM image of AuNPs electrodeposited under optimal condition is shown in Fig. 5(a). After this image was obtained, the fabricated sensor was characterized via H2O2 detection before GOx immobilization. As shown in Fig. 5(b), the sensitivities of the sensors toward H2O2 were 667nA/mM and 171nA/mM for sensors with and without AuNPs, respectively, demonstrating that the sensor sensitivity increased by four-times when AuNPs were deposited onto the graphene layer. Thus, AuNPs deposition significantly improved the sensitivity of the sensor.
3.4. Evaluation of the glucose sensor after GOx immobilization Using amperometry, the sensitivity and linearity of the fabricated flexible electrochemical sensor in response to changing glucose concentrations was determined after GOx immobilization. Fig. 6 shows typical amperometric responses of the fabricated sensor to successive increments of 5 mg/dL glucose using an applied potential of -0.2 V. As shown in Fig. 6, the linear range of glucose measurements obtained using the fabricated glucose sensor was 0~40 mg/dL, and the detection limit was 0.3 mg/dL (S/N=3). Physiological blood glucose levels are approximately 30~330 mg/dL; thus physiological ISF glucose levels are approximately 25~300 mg/dL as they are around 80~90% of those in blood [32]; while in our case, ten-fold dilutions of extracted ISF samples enables measurements in the range of 2.5~30 mg/dL. Therefore, the experimental results shown in Fig. 6 demonstrate that the proposed flexible electrochemical sensor can accurately detect glucose in diluted ISF within the physiological range, thereby demonstrating potential for hypoglycemia detection. These experimental results also demonstrate the good repeatability of the sensor.
3.5. Selectivity and response time of the glucose sensor The selectivity of the sensor was also evaluated by adding dopamine (DA), ascorbic acid (AA), uric acid (UA) and sodium chloride (NaCl) to the analytical solutions [33]. As shown in Fig. 7, the experimental results demonstrate that the sensor exhibits good selectivity for glucose, underscoring the potential of the sensor for glucose detection in extracted ISF samples. Immobilized GOx on the electrode surface enables glucose-specific detection, significantly decreasing or eliminating interferences from other species. Additionally, as shown in Fig. 7, the system exhibited a <1s response
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time when the glucose concentration was changed, demonstrating that the sensor reacts quickly to changes in glucose concentrations.
3.6. Integration of the glucose sensor into the ISF extraction chip The proposed glucose sensor was integrated into a PDMS-based ISF extraction chip. This flexible chip (Fig. 8(a)) consists of a Venturi to provide driving force for both ISF extraction and fluid manipulation, pneumatic valves to sequentially control the ISF extraction and collection processes, fluid chambers for the storage of extracted and collected ISF, and interconnecting microchannels. The microfluidic chip was fabricated via five PDMS layers using micromolding techniques. As shown in Fig. 8(b), the glucose sensor was bonded onto the microfluidic chip via an Access Port for Glucose Sensor (APGS) connection. The microfluidic chip integrated with the proposed flexible electrochemical glucose sensor can be used to transdermally extract, dilute, and collect ISF, as well as to detect glucose in ISF, enabling development of a flexible and wearable device for continuous glucose monitoring.
4. Conclusions This paper presents a flexible electrochemical sensor with a composite nanostructured WE surface containing graphene and AuNPs. Electrodes were fabricated on the PI substrate using the flexible PCB method. The WE surface was directly modified with graphene via inkjet printing to improve the electroactivity and to obtain a significantly more uniform distribution of electrochemical active sites on the WE electrode surface, thereby enabling glucose detection at low levels. AuNPs were electrochemically deposited directly onto the graphene layer in order to improve the electron transfer rate between GOx activity centers and the electrode such that sensitivity is enhanced. These two methods resulted in direct deposition of graphene and AuNPs onto the working electrode surface without the need for any protection for the other parts of the sensor that did not require modification. We evaluated the factors affecting the performance of AuNPs, including HAuCl4 concentration, deposition current intensity and deposition time, and optimized the conditions for detection. H 2O2 measurement results indicate that a four-time enhancement of the sensor’s sensitivity was obtained by electrodepositing AuNPs onto the graphene layer. The glucose sensor was characterized after immobilizing GOx onto the composite nanostructured surface to detect glucose specifically, and the
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results demonstrate that the proposed sensor could precisely measure glucose with a linear range of 0~40 mg/dL and a detection limit of 0.3 mg/dL (S/N=3), thereby indicating the potential of the proposed electrochemical glucose sensor for hypoglycemia detection. Additionally, the flexible electrochemical sensor was integrated into an ISF extraction microfluidic chip to produce a wearable device that could be utilized for the extraction, dilution, collection and detection of ISF for the purpose of continuous glucose monitoring.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No.61428402 and No.81571766), the Key Projects in the Science & Technology Pillar Program of Tianjin (No.11ZCKFSY01500), the Key Projects of Tianjin Natural Science Foundation Program (No.15JCZDJC36100), the National High Technology Research and Development Program of China (No.2012AA022602), and the 111 Project of China (No.B07014).
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[28] N. Zhou, J. Wang, T. Chen, Z. Yu, G. Li, Enlargement of Gold Nanoparticles on the Surface of a Self-Assembled Monolayer Modified Electrode: A Mode in Biosensor Design, Anal. Chem., 78 (2006) 5227–5230. [29] R. Toledano, D. Mandler, Electrochemical Codeposition of Thin Gold Nanoparticles/Sol−Gel Nanocomposite Films, Chem. Mater., 22 (2010) 3943–3951. [30] J. C. Claussen, J. B. Hengenius, M. M. Wickner, T. S. Fisher, D. M. Umulis, D. M. Porterfield, Effects of Carbon Nanotube-Tethered Nanosphere Density on Amperometric Biosensing: Simulation and Experiment, J. Phys. Chem. C, 115 (2011) 20896-20904. [31] J. C. Claussen, A. Kumar, D. B. Jaroch, M. H. Khawaja, A. B. Hibbard, D. M. Porterfield, T. S. Fisher, Nanostructuring Platinum Nanoparticles on Multilayered Graphene Petal Nanosheets for Electrochemical Biosensing, Adv. Funct. Mater., 22 (2012) 3399–3405. [32] M. Pleiteza, H. V. Lilienfeld-Toalb, W. Mäntelea, Infrared Spectroscopic Analysis of Human Interstitial Fluid in Vitro and in Vivo Using FT-IR Spectroscopy and Pulsed Quantum Cascade Lasers (QCL): Establishing A New Approach to Non Invasive Glucose Measurement, Spectrochim. Acta A., 85 (2012) 61-65. [33] M. Liu, R. Liu, W. Chen, Graphene Wrapped Cu2O Nanocubes: Non-Enzymatic Electrochemical Sensors for the Detection of Glucose and Hydrogen Peroxide with Enhanced Stability, Biosens. Bioelectron., 45 (2013) 206-212.
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Author Biographies
Zhihua Pu received the B.S. degree in Measurement and control technology and instrument specialty from Chongqing University, Chongqing, China, in 2013. And he received the M.S. degree in the college of precision instrument and opto-electronics engineering at Tianjin University, Tianjin, China, in 2015. He is currently pursuing the PhD degree in the college of precision instrument and optoelectronics engineering at Tianjin University, Tianjin, China. His research interests focus on electrochemical glucose sensor. Ridong Wang received the B.S. degree in Measuring and Controlling Technology and Instrument specialty from Tianjin University, Tianjin, China, in 2011. And he received the M.S. degree in the college of precision instrument and opto-electronics engineering at Tianjin University, Tianjin, China, in 2014. His research interest is electrochemical glucose sensor. Jianwei Wu received the B.S. degree in Measuring and Controlling Technology and Instrument specialty from Zhejiang University, Tianjin, China, in 2012. And he received the M.S. degree in the college of precision instrument and opto-electronics engineering at Tianjin University, Tianjin, China, in 2015. His research interest is inkjet printing techniques. Haixia Yu received her doctoral degree in 2011 from Tianjin University, Tianjin, China. Currently, she is an associate professor in the College of Precision Instruments and Optoelectronics Engineering, Tianjin University. Her research interests focus on biomedical microfluidics and biomedical microsensors. Kexin Xu received his B.S. degree in precision instrument engineering from Harbin University of Science and Technology, Harbin, China, in 1982 and his PhD degree in precision instrument engineering from Tianjin University, Tianjin, China, in 1988. He has been a full-time professor and researcher in the College of Precision Instrument and Optoelectronics Engineering, Tianjin University. His research interests include the design theory of acousto-optic tunable filter and spectroscopic technology, rapid detection of milk ingredients, monitoring of air and flue gas composition and noninvasive glucose monitoring. Dachao Li received a B.S. degree in precision instruments from Tianjin University, Tianjin, China, in 1998 and M.S. and Ph.D. degrees in precision instruments and mechanics from Tianjin University, Tianjin, China, in 2001 and 2004, respectively. Li was previously a research associate at the Department of Electrical Engineering and Computer Science, Case Western Reserve University,
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Cleveland, Ohio, USA. Currently, he is a professor and PhD supervisor in the College of Precision Instruments and Optoelectronics Engineering, Tianjin University. His research interests focus on micro-sensors and opto-fluidics.
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Figure captions
Fig. 1. Schematic diagram of (a) Proposed flexible glucose sensor and (b) Structure of the working electrode
Fig. 2. Schematic diagram of the inkjet printing system Fig. 3. (a) SEM image of the printed graphene and (b) Effect of graphene on the flexible glucose sensors Fig. 4. Characterizations of AuNPs-modified electrodes under various conditions: (a) Concentrations of AuCl4, (b) Current intensity; (c) Deposition time
Fig. 5. (a) SEM image of electrodeposited AuNPs and (b) Characterization of the AuNPs-modified electrode under optimized conditions via H2O2 (n=6) Fig. 6. Amperometric measurements in glucose solutions via the proposed flexible electrochemical sensor (n=6) Fig. 7. Selectivity and response time of the glucose sensor (the addition of different analytes in 0.1 M PBS: 0.2 mM Glucose, 0.1 mM Dopamine (DA), 0.1 mM Ascorbic acid (AA), 0.1 mM Uric acid (UA) and 0.1 mM NaCl) Fig. 8. (a) Photo of the integrated microfluidic system and (b) Schematic diagram of the integrating structure
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S0925-4005(16)30264-7 http://dx.doi.org/doi:10.1016/j.snb.2016.02.115 SNB 19775
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
28-10-2015 20-2-2016 23-2-2016
Please cite this article as: Zhihua Pu, Ridong Wang, Jianwei Wu, Haixia Yu, Kexin Xu, Dachao Li, A flexible electrochemical glucose sensor with composite nanostructured surface on the working electrode, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.02.115 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A flexible electrochemical glucose sensor with composite
nanostructured surface on the working electrode
1
Zhihua Pu, 1Ridong Wang, 1Jianwei Wu, 2Haixia Yu, 1Kexin Xu and 1Dachao Li*
1
State Key Laboratory of Precision Measuring Technology and Instruments, Tianjin University,
Tianjin, China 2
Tianjin Key Laboratory of Biomedical Detecting Techniques and Instruments, Tianjin University,
Tianjin, China
Tel: +86-22-27403916 *E-mail: [email protected]
1
Highlights:
A flexible electrochemical sensor was fabricated into the ISF extraction chip to build a wearable micro device for continuous glucose monitoring.
Composite nanostructured surface with graphene and AuNPs was constructed on the WE to enable accurate measurement of low levels of glucose.
Graphene was directly written onto the tiny-sized WE surface by inkjet printing method without complicated CVD deposition and transferring process.
AuNPs and GOD were electrodeposited onto the WE surface without any protection for the other parts that did not need the modification.
Abstract Detecting hyperglycemia remains a great challenge with regard to continuous glucose monitoring in clinical settings. This paper investigates a novel, flexible three-electrode electrochemical sensor with a composite nanostructured working electrode surface that has been modified by graphene and gold nanoparticles in order to detect low levels of glucose with high accuracy. The sensor electrodes were fabricated on a polyimide substrate using the flexible printed circuit board (PCB) method. Graphene was modified directly onto the working electrode surface via inkjet printing, an emerging method for micro-scale fabrications, to enable glucose detection at low levels. Gold nanoparticles were electrodeposited directly onto the graphene layer to enhance the sensitivity of the sensor. The experimental results demonstrate that the proposed sensor can precisely measure glucose with a linear range of 0~40 mg/dL and a detection limit of 0.3 mg/dL (S/N=3), thereby demonstrating potential for hypoglycemia detection. Moreover, this flexible sensor was suitable for integration within a microfluidic chip, which could be used to transdermally extract and collect ISF, such that a wearable device could be developed for continuous glucose monitoring.
Keywords: electrochemical sensor; flexible substrate; glucose sensing; graphene; gold nanoparticles; inkjet printing
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1. Introduction Continuous blood glucose monitoring holds great significance for the treatment of diabetes. However, it is difficult to continuously monitor glucose in the blood directly. Glucose concentrations of interstitial fluid (ISF) are closely related to blood glucose levels [1] [2] [3]. Thus, research attention has focused on developing a minimally invasive blood glucose measuring method based on ISF analysis, the advantages of which include painless application and the realization of continuous glucose monitoring [4]. Electrochemical biosensors have played a leading role in the advancement of continuous blood glucose monitoring [5]. To date, most continuous glucose monitoring systems based on ISF analysis that have been used in clinical environs have been based on implantable enzyme electrode sensors [6]. However, this methodology is restricted by its invasiveness and the short lifetimes of the electrodes caused by the foreign body reaction in subcutaneous tissue. Furthermore, glucose concentrations cannot be measured accurately due to a signal drift caused by the bioelectricity of the human body [7]. Thus, blood glucose predictions based on glucose measurements in transdermally extracted ISF has become an emerging technology for continuous glucose monitoring. This method can enable continuous glucose monitoring externally, thereby avoiding interferences from internal environments, such as interferences related to the foreign body reaction and the bioelectricity within the human body. Our research group used ultrasound and a vacuum to extract ISF transdermally over an area of 0.07 cm2. Ultrasound was used to create micro pores to increase the permeability of the skin [8], and a vacuum pump was used to enhance ISF convection. It was difficult to collect the extracted ISF, which was scattered on the surface of the skin and whose volume was approximately 1 μL [9]. Therefore, a predefined volume of phosphate buffer was used to dilute the transdermally extracted ISF for easy collection. However, the glucose concentration in the ISF decreased significantly after dilution. Thus, we developed a microfluidic system for the extracting, diluting and collecting ISF [10]. This system featured five polydimethylsiloxane (PDMS) layers and was fabricated using micromolding techniques. This system required integration of a micro-sized glucose sensor into the microchannel of the microfluidic chip. However, it was difficult to fabricate the glucose sensor’s electrodes on the PDMS surface because of their hydrophobicity and poor adhesion to metals [11]. Accordingly, some groups [12] fabricated electrode-based sensors on glass and then bonded the glass to PDMS-based chips to form functional microfluidic devices. Nevertheless, although the PDMS-based microfluidic chip is flexible, the bonded glass makes the whole device rigid. Unfortunately, skin deformation always happens during ISF extraction by vacuum. Although the PDMS-based microfluidic chip can transmute along with the transformation, the rigid whole chip that results from depositing the sensor on glass
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may lead to spillage of the analytical liquid, resulting in inaccurate measurements. Thus, the present paper describes the fabrication of a high-resolution electrochemical sensor on a flexible polyimide (PI) substrate that can be integrated into the microchannel of the ISF extraction chip and that can transmute with chip deformation to accurately measure glucose concentrations in diluted ISF extractions. The glucose concentration in diluted ISF can be as low as approximately 2 mg/dL; therefore, existing commercial glucometers with detection levels ranging from 20 to 500 mg/dL are not applicable. New technologies are being pursued to develop novel glucose sensors. Zimmermann et al. [13] proposed an enzyme-based electrochemical flow-through glucose sensor that could be integrated into a flow channel and reported a detection limit of 20 mg/dL. Huang et al. [14] proposed a capacitive MEMS affinity sensor for continuous glucose monitoring at physiologically relevant concentrations ranging from 30 to 360 mg/dL. Tierney et al. [15] proposed a glucose-sensitive sensor prepared by incorporating 3-phenylboronic acid and dimethylaminopropylacrylamide into a hydrogel covalently linked to the end of an optical fiber. The latter glucose sensor was only capable of measuring physiological glucose levels with a detection limit of 18 mg/dL. Tashkhourian et al. [16] proposed a glucose biosensor based on chromophore (silver nanoparticles) decolorization for the photometric determination of glucose; the linear range of this glucose sensor was 50 to 800 mg/dL, and the limit of detection was 23 mg/dL. Siegrist et al. [17] proposed a continuous glucose sensor that involves binding genetically engineered polypeptide-based fluorescent molecules to improve the accuracy of the sensor; in this case, the linear range spanned from 35 to 586 mg/dL. Although the aforementioned sensors exhibited various merits, their detection ranges only covered the physiological glucose range, meaning that these methods were barely able to measure the low glucose concentrations in diluted ISF with high accuracy. This paper presents a flexible electrochemical sensor, which utilizes a working electrode with a composite nanostructured surface that is capable of measuring glucose levels in diluted ISF with high accuracy. The composite nanostructure significantly improved the performance of the sensor [18]. Graphene was inkjet printed onto the working electrode surface to improve electroactivity and impart a more uniform distribution of electrochemical active sites in order to achieve glucose detection at low levels. Gold nanoparticles (AuNPs) were electrodeposited onto the graphene layer to improve the electron transfer rate between the activity centers of the enzymes and the electrode and, thereby, enhance the sensor’s sensitivity. The two methods directly deposited graphene and AuNPs onto the working electrode surface without requiring protection from modification of any of the other parts of the sensor. Subsequently, in order to obtain glucose-specific detection, glucose oxidase was immobilized via electrochemical polymerization onto the composite nanostructured surface of the working electrode. The resulting glucose sensor can be integrated with the ISF extraction chip to 4
produce a flexible and wearable device. This wearable device makes a flexible connection to the skin, thereby significantly reducing the relative motion between the device and the skin such that a more stable and accurate detection signal is obtained.
2. Methods and materials 2.1. Design of the flexible electrochemical glucose sensor Fig. 1(a) displays a schematic diagram of the proposed flexible glucose sensor, which is a threeelectrode electrochemical sensor that includes a working electrode (WE, 1.2×0.2 mm), a reference electrode (RE, 1.2×0.2 mm), and a counter electrode (CE, 1.2×1 mm). The electrodes were fabricated on a flexible PI substrate via a flexible printed circuit board (PCB) process. Then, 5-μm Au was electroplated onto the surface of the electrodes. After first electroplating Ni onto the Cu layer, the Ni in the KAu(CN)2 solution was replaced with Au so that Au replaced Ni in the Cu layer. The corresponding reaction formulas are as follows: i Ni 2 2e Ni
3Ni 2 Au (CN ) 2 2 Au 3Ni
（2-1） 2
（2-2）
Subsequently, silver was electroplated onto the surface of the RE, then the electrodes were immersed in a solution of 50 mM FeCl3 for 50 s, such that an Ag/AgCl RE was obtained (AgNO3, NH3·H2O and FeCl3 were obtained from Tianjin Jiangtian Huagong Co., China). As shown in Fig. 1(b), the working electrode was first modified with graphene via inkjet printing and evaporating the solvent at 120°C for one hour. A uniform distribution of uniformly sized AuNPs was then electrodeposited onto the graphene layer. Finally, glucose oxidase (GOx) was immobilized onto the graphene layer with AuNPs via electrochemical polymerization to enable glucose-specific detection.
During electrochemical glucose sensing, glucose decomposed catalytically via GOx and the H2O2 generated was subsequently oxidized at the electrode surface, producing a measurable current signal. Therefore, H2O2 could be used to characterize the electrodes directly, without the need for immobilized GOx. The chemical reaction formulas are as follows: （2-3）
GOxox glucose H 2O GOxred gluconic acid H 2O2
（2-4）
GOxred GOxox + ne-
（2-5）
H 2O2 O2 2H 2e
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2.2. Modification of graphene on the WE surface The WE surface was modified with graphene to improve electroactivity and to achieve a significantly more uniform distribution of electrochemical active sites in order to enable glucose detection at low levels. Graphene is a novel nanomaterial that, since 2004, has attracted considerable attention due to its excellent properties, including exceptional thermal and mechanical properties, high electrical conductivity, larger surface area, and good biocompatibility [19]. This material has been shown to exhibit superior conductivity [20], and the special concave-convex topography of graphene generates a high number of electrochemical active sites, making it likely to detect weak signals, i.e., glucose at low concentrations. In addition, due to π-stacking interactions between the hexagonal cells of the electrode material and the carbon-based atomic ring structure of graphene, the electrode surface adsorbs biomolecules that contain carbon-based ring structures [21], increasing its capacity for adsorbing GOx molecules. Additionally, the good biocompatibility of graphene increases the bioactive lifetimes of immobilized GOx molecules.
In general, graphene is deposited via chemical vapor deposition, after which it is cut to the desired shape and transferred to the target surface in water [22]. The procedure involved is complex, and it is difficult to acquire graphene with the desired shape when the size is so small. Its tiny size also makes it difficult to transfer graphene onto the WE surface. Thus, we directly deposited the desired shape of graphene onto the WE surface via inkjet printing, an emerging additive manufacturing technique for forming functionalized microstructures [23]. The inkjet printing system was assembled as shown in Fig. 2. This system consisted of a pump, an air pressure control module, a displacement stage, four printheads, a printhead driver, an observation module and a computer. The pump supplied the pressure power for the system. The air pressure control module was used to wash the printheads and maintain the air pressure within them such that successful printing was ensured. The displacement stage was used to fix the printing substrates and to maneuver them as desired. Four printheads could be used to print different materials onto the target substrates. The printhead driver module was used to drive the printheads based on the corresponding printing signals. The observation module included a horizontal camera with a strobe, which enabled us to observe the printing status, as well as a vertical camera that
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enabled us to calibrate the start-point for printing. We used a computer to design the printing routes and supply the printing signals. The accuracy of this inkjet printing system was 20 μm. Graphene modification employed the following procedures. First, we placed the electrodes onto the working stage of the inkjet printer. Then, a desired image, based on the shape and the size of the WE, was drawn by CAD, which was integrated with the controlling software of the printer. Subsequently, the start-point for printing was calibrated using the vertical camera of the printing system. Finally, one layer of graphene ink (Sigma-Aldrich Inc., USA) was printed directly onto the WE surface using the following optimized conditions: 60 V, 500 Hz, and 40°C. The printhead used was purchased from MicroFab, Inc., and the orifice employed was 20 μm. The density of the graphene ink was 0.9375 g/ml, and the graphene was comprised of pieces of reduced graphene oxide (rGO), which is inkjet printable [24]; the rGO pieces were less than 3 μm in diameter. The modification was completed by evaporating the solvent (in the printed graphene ink) at 120°C for one hour. Once spread on the WE surface, the diameter of the printed graphene droplet was approximately 60 μm. 2.3. Modification of the graphene layer with AuNPs AuNPs were used to modify the graphene layer on the WE in order to enhance the sensitivity of the sensor by constructing a composite nanostructured surface that improved the electron transfer rate between GOx activity centers and the electrode. AuNPs are widely used in biosensing because they possess a number of generally inherent, beneficial characteristics, including a large surface-to-volume ratio, good electrocatalytic activity and high chemical reactivity [25]. Their special size renders AuNPs particularly capable of transferring electrons between GOx activity centers and the electrode surface, effectively producing nanowires that enhance the sensitivity of the sensor. The large surface-to-volume ratio of AuNPs assists with GOx immobilization and generates a large number of unsaturated bonds on the electrode surface, which, in turn, make AuNPs more active such that good electrocatalytic activity and high chemical reactivity are achieved. The resulting improvements with regard to electrocatalytic activity and high chemical reactivity facilitate the catalytic reaction of glucose and H2O2 and, therefore, further enhance the sensitivity of the sensor. Both physical and chemical methods have been used to deposit AuNPs on electrode surfaces. With regard to physical methods, evaporation [26] and sputtering [27] are frequently used and dramatically decrease deposition times. However, these methods require high temperatures and protection for the other parts of the system that do not require modification. Furthermore, it is difficult to control the sizes and distributions of AuNPs using these methods. With regard to chemical methods, self-assembly [28] and sol-gel [29] are frequently employed. However, the AuNPs deposited by these methods aggregate easily, significantly affecting the properties of the sensor, and, as with physical methods, 7
protection is required for the other parts of the system that do not require modification. In the present case, the flexible PI substrate cannot sustain high temperatures, and the WE surface is the only part of the system requiring modification with AuNPs. Thus, we employed the electrochemical deposition method at room temperature to directly deposit AuNPs onto the graphene layer of the WE without requiring any protection for the other parts of the sensor. This methodology made it easy to control the size as well as the distribution of AuNPs, which significantly affect the performance of the nanoparticles [30]. An electrochemical setup, in which the target electrode served as the WE, a Pt sheet served as the CE, and an Ag/AgCl electrode served as the RE, was dipped into a plating solution consisting of HAuCl4 and 0.5 M Na2SO4 (Shanghai Xinbo Chemistry Technique Co., China) to electrodeposit AuNPs onto the graphene layer. The AuNPs were deposited via constant current pulses that were applied between the WE and CE by an electrochemical workstation (CHI 660E, CHI Instruments Inc., USA). HAuCl4 concentration, deposition current intensity and deposition time violently affected the modification with AuNPs, so several experiments were conducted to characterize sensor performance. H2O2 was utilized to test the AuNPs-modified sensor under different conditions, and the sensitivity and linearity of the system were evaluated in order to optimize the parameters utilized for AuNPs modification. 2.4. Immobilizing GOx onto the WE surface GOx (Sigma-Aldrich Inc., USA) was mixed with 3,4-ethylenedioxythiophene (EDOT) (SigmaAldrich Inc., USA) before immobilized onto the WE surface. The GOx/EDOT solution was prepared by first dissolving PSS (0.1 M) (Sigma-Aldrich Inc., USA) in H2O. Next, EDOT (0.03 M) was added to the mixture as the solution was agitated. Subsequently, the H2O-dissolved GOx (2 mg/mL) was added to the mixture. This solution was used to electrochemically polymerize GOx onto the composite nanostructured surface, on which graphene and AuNPs were constrained, by applying constant current pulses of 2 mA/cm2 between the WE and CE over 1000 s [31]. Immobilization of GOx onto the WE surface was achieved using methods similar to those stated in section 2.3, and protection for the other parts of the system, which did not require GOx deposition, was also not necessary. 3. Results and discussion 3.1. Measurement system The measurement system consisted of an electrochemical workstation to supply the driving potential and to measure the output current of the proposed sensor, a pipette to provide different concentrations
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of the determinand in the reaction tank, a computer to control and receive signals from the workstation. The fabricated electrochemical sensor was characterized by immersing it into electrolyte solutions containing 0.1 M phosphate buffer solution (PBS) (pH=7.4) and different concentrations of glucose or H2O2 (Tianjin Jiangtian Huagong Co., China). A stir bar was not used because the volume of the analytical solution was extremely small (200 μL) such that diffusion quickly reached equilibrium. All experiments were conducted at room temperature. 3.2. Effect of graphene on the flexible electrochemical glucose biosensor Amperometry, which is used frequently in electrochemical measurements, was used for glucose measurements to study the sensitivity and linearity of the fabricated electrochemical sensors with and without graphene after GOx immobilization. An SEM image of the printed graphene on the WE is shown in Fig. 3(a), and Fig. 3(b) shows typical amperometric responses (using an applied potential of -0.2 V) of the three sensor configurations to successive incremental additions of 5 mg/dL glucose [19]. These results demonstrate that the current signals pertaining to low levels of glucose cannot be distinguished by the sensors if graphene is absent, whether or not AuNPs have been deposited; however, low glucose levels can be identified and measured when graphene is present. This indicates that graphene significantly improves the detection of weak glucose signals.
3.3. Enhancements of the electrochemical sensor achieved via AuNPs In order to evaluate the effects of different AuNPs modification conditions on the sensor’s ability to detect H2O2, sensors with AuNPs modification of the WE graphene layer without GOx immobilization were first tested amperometrically. Each parameter was evaluated while holding the other two parameters constant. Fig. 4 shows the typical amperometric responses obtained, using an applied potential of -0.2 V, for various sensor configurations in response to successive increments of 0.2 mM H2O2. As shown in Fig. 4, the electrochemical responses increase as H2O2 concentrations increase, and all of the sensors demonstrate good linearity from 0 to 1.8 mM H2O2. Fig. 4 also shows that the HAuCl4 concentration, the deposition current intensity and the deposition time significantly affect the AuNPs modification obtained, as demonstrated by the substantial sensitivity changes recorded for sensors that were modified with AuNPs under different conditions. As shown in Fig. 4, the optimized AuNPs deposition conditions determined include 4 mM HAuCl4, a deposition current intensity of 10 mA/cm2 and a deposition time of 200 s.
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After optimizing the experimental conditions, AuNPs were electrodeposited onto the graphene layer of the WE surface under these conditions. An SEM image of AuNPs electrodeposited under optimal condition is shown in Fig. 5(a). After this image was obtained, the fabricated sensor was characterized via H2O2 detection before GOx immobilization. As shown in Fig. 5(b), the sensitivities of the sensors toward H2O2 were 667nA/mM and 171nA/mM for sensors with and without AuNPs, respectively, demonstrating that the sensor sensitivity increased by four-times when AuNPs were deposited onto the graphene layer. Thus, AuNPs deposition significantly improved the sensitivity of the sensor.
3.4. Evaluation of the glucose sensor after GOx immobilization Using amperometry, the sensitivity and linearity of the fabricated flexible electrochemical sensor in response to changing glucose concentrations was determined after GOx immobilization. Fig. 6 shows typical amperometric responses of the fabricated sensor to successive increments of 5 mg/dL glucose using an applied potential of -0.2 V. As shown in Fig. 6, the linear range of glucose measurements obtained using the fabricated glucose sensor was 0~40 mg/dL, and the detection limit was 0.3 mg/dL (S/N=3). Physiological blood glucose levels are approximately 30~330 mg/dL; thus physiological ISF glucose levels are approximately 25~300 mg/dL as they are around 80~90% of those in blood [32]; while in our case, ten-fold dilutions of extracted ISF samples enables measurements in the range of 2.5~30 mg/dL. Therefore, the experimental results shown in Fig. 6 demonstrate that the proposed flexible electrochemical sensor can accurately detect glucose in diluted ISF within the physiological range, thereby demonstrating potential for hypoglycemia detection. These experimental results also demonstrate the good repeatability of the sensor.
3.5. Selectivity and response time of the glucose sensor The selectivity of the sensor was also evaluated by adding dopamine (DA), ascorbic acid (AA), uric acid (UA) and sodium chloride (NaCl) to the analytical solutions [33]. As shown in Fig. 7, the experimental results demonstrate that the sensor exhibits good selectivity for glucose, underscoring the potential of the sensor for glucose detection in extracted ISF samples. Immobilized GOx on the electrode surface enables glucose-specific detection, significantly decreasing or eliminating interferences from other species. Additionally, as shown in Fig. 7, the system exhibited a <1s response
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time when the glucose concentration was changed, demonstrating that the sensor reacts quickly to changes in glucose concentrations.
3.6. Integration of the glucose sensor into the ISF extraction chip The proposed glucose sensor was integrated into a PDMS-based ISF extraction chip. This flexible chip (Fig. 8(a)) consists of a Venturi to provide driving force for both ISF extraction and fluid manipulation, pneumatic valves to sequentially control the ISF extraction and collection processes, fluid chambers for the storage of extracted and collected ISF, and interconnecting microchannels. The microfluidic chip was fabricated via five PDMS layers using micromolding techniques. As shown in Fig. 8(b), the glucose sensor was bonded onto the microfluidic chip via an Access Port for Glucose Sensor (APGS) connection. The microfluidic chip integrated with the proposed flexible electrochemical glucose sensor can be used to transdermally extract, dilute, and collect ISF, as well as to detect glucose in ISF, enabling development of a flexible and wearable device for continuous glucose monitoring.
4. Conclusions This paper presents a flexible electrochemical sensor with a composite nanostructured WE surface containing graphene and AuNPs. Electrodes were fabricated on the PI substrate using the flexible PCB method. The WE surface was directly modified with graphene via inkjet printing to improve the electroactivity and to obtain a significantly more uniform distribution of electrochemical active sites on the WE electrode surface, thereby enabling glucose detection at low levels. AuNPs were electrochemically deposited directly onto the graphene layer in order to improve the electron transfer rate between GOx activity centers and the electrode such that sensitivity is enhanced. These two methods resulted in direct deposition of graphene and AuNPs onto the working electrode surface without the need for any protection for the other parts of the sensor that did not require modification. We evaluated the factors affecting the performance of AuNPs, including HAuCl4 concentration, deposition current intensity and deposition time, and optimized the conditions for detection. H 2O2 measurement results indicate that a four-time enhancement of the sensor’s sensitivity was obtained by electrodepositing AuNPs onto the graphene layer. The glucose sensor was characterized after immobilizing GOx onto the composite nanostructured surface to detect glucose specifically, and the
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results demonstrate that the proposed sensor could precisely measure glucose with a linear range of 0~40 mg/dL and a detection limit of 0.3 mg/dL (S/N=3), thereby indicating the potential of the proposed electrochemical glucose sensor for hypoglycemia detection. Additionally, the flexible electrochemical sensor was integrated into an ISF extraction microfluidic chip to produce a wearable device that could be utilized for the extraction, dilution, collection and detection of ISF for the purpose of continuous glucose monitoring.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No.61428402 and No.81571766), the Key Projects in the Science & Technology Pillar Program of Tianjin (No.11ZCKFSY01500), the Key Projects of Tianjin Natural Science Foundation Program (No.15JCZDJC36100), the National High Technology Research and Development Program of China (No.2012AA022602), and the 111 Project of China (No.B07014).
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Author Biographies
Zhihua Pu received the B.S. degree in Measurement and control technology and instrument specialty from Chongqing University, Chongqing, China, in 2013. And he received the M.S. degree in the college of precision instrument and opto-electronics engineering at Tianjin University, Tianjin, China, in 2015. He is currently pursuing the PhD degree in the college of precision instrument and optoelectronics engineering at Tianjin University, Tianjin, China. His research interests focus on electrochemical glucose sensor. Ridong Wang received the B.S. degree in Measuring and Controlling Technology and Instrument specialty from Tianjin University, Tianjin, China, in 2011. And he received the M.S. degree in the college of precision instrument and opto-electronics engineering at Tianjin University, Tianjin, China, in 2014. His research interest is electrochemical glucose sensor. Jianwei Wu received the B.S. degree in Measuring and Controlling Technology and Instrument specialty from Zhejiang University, Tianjin, China, in 2012. And he received the M.S. degree in the college of precision instrument and opto-electronics engineering at Tianjin University, Tianjin, China, in 2015. His research interest is inkjet printing techniques. Haixia Yu received her doctoral degree in 2011 from Tianjin University, Tianjin, China. Currently, she is an associate professor in the College of Precision Instruments and Optoelectronics Engineering, Tianjin University. Her research interests focus on biomedical microfluidics and biomedical microsensors. Kexin Xu received his B.S. degree in precision instrument engineering from Harbin University of Science and Technology, Harbin, China, in 1982 and his PhD degree in precision instrument engineering from Tianjin University, Tianjin, China, in 1988. He has been a full-time professor and researcher in the College of Precision Instrument and Optoelectronics Engineering, Tianjin University. His research interests include the design theory of acousto-optic tunable filter and spectroscopic technology, rapid detection of milk ingredients, monitoring of air and flue gas composition and noninvasive glucose monitoring. Dachao Li received a B.S. degree in precision instruments from Tianjin University, Tianjin, China, in 1998 and M.S. and Ph.D. degrees in precision instruments and mechanics from Tianjin University, Tianjin, China, in 2001 and 2004, respectively. Li was previously a research associate at the Department of Electrical Engineering and Computer Science, Case Western Reserve University,
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Cleveland, Ohio, USA. Currently, he is a professor and PhD supervisor in the College of Precision Instruments and Optoelectronics Engineering, Tianjin University. His research interests focus on micro-sensors and opto-fluidics.
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Figure captions
Fig. 1. Schematic diagram of (a) Proposed flexible glucose sensor and (b) Structure of the working electrode
Fig. 2. Schematic diagram of the inkjet printing system Fig. 3. (a) SEM image of the printed graphene and (b) Effect of graphene on the flexible glucose sensors Fig. 4. Characterizations of AuNPs-modified electrodes under various conditions: (a) Concentrations of AuCl4, (b) Current intensity; (c) Deposition time
Fig. 5. (a) SEM image of electrodeposited AuNPs and (b) Characterization of the AuNPs-modified electrode under optimized conditions via H2O2 (n=6) Fig. 6. Amperometric measurements in glucose solutions via the proposed flexible electrochemical sensor (n=6) Fig. 7. Selectivity and response time of the glucose sensor (the addition of different analytes in 0.1 M PBS: 0.2 mM Glucose, 0.1 mM Dopamine (DA), 0.1 mM Ascorbic acid (AA), 0.1 mM Uric acid (UA) and 0.1 mM NaCl) Fig. 8. (a) Photo of the integrated microfluidic system and (b) Schematic diagram of the integrating structure
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