Development of an ultra-sensitive hydrogen peroxide sensor for cell culture in a micro-incubator

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1 Development of an ultra-sensitive hydrogen peroxide sensor for cell culture in a micro-incubator Kuan-Chung Fang, Chen-Pin Hsu, Yen-Wen Kang, Jung-Ying Fang, Chih-Cheng Huang, Chia-Hsien Hsu, Yu-Fen Huang, Chih-Chen Chen, Sheng-Shian Li, J. Andrew Yeh, Da-Jeng Yao, and Yu-Lin Wang Abstract Horseradish peroxidase (HRP)-immobilized conducting polymer, polyaniline (PANI), was coated on gold electrodes to characterize hydrogen peroxide in buffer solution by measuring the polymer conductance change, which was resulted from the oxidation of PANI due to the reduction of hydrogen peroxide by HRP. 10 μl of hydrogen peroxide solution was detected with the sensor from 0.1 nm to 1 mm. The detection limit obtained from the experiment is 0.7 nm. And the detectable concentration of H 2O 2 is from 0.7 nm to 1 μm. In the region of detectable concentration, H 2 O 2 was fully consumed by HRP, and therefore the conductance change of the PANI thin film was accumulated by the total amount of H 2O 2, leading to the high sensitivity of the sensor. Above 1 μm, the sensor gradually saturated and some H 2O 2 remained, indicating the inhibition of HRP activity at high concentration of H 2O 2. The saturated Manuscript received August 23, This work was partially supported by National Science Council grant (NSC E MY3) and by the research grant (102N2047E1) at National Tsing Hua University. We thank the technical support from National Nano Device Laboratories (NDL) in Hsinchu and the Center for Nanotechnology, Materials science, and Microsystems (CNMM) at National Tsing Hua University. Kuan-Chung Fang is with Institute of Nanoengineering and Microsystems, kcfang113@gmail.com). Chen-Pin Hsu is with Institute of Nanoengineering and Microsystems, kenneybin@gmail.com). Yen-Wen Kang is with Institute of Nanoengineering and Microsystems, u @oz.nthu.edu.tw). Jung-Ying Fang is with Institute of Nanoengineering and Microsystems, quic123k176@yahoo.com.tw) Chih-Cheng Huang is with Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu, 300, Taiwan, R.O.C. ( s @m99.nthu.edu.tw) Chia-Hsien Hsu is with Division of Medical Engineering, National Health Research Institutes, MiaoLi, Taiwan, R.O.C. ( chsu@nhri.org.tw). Yu-Fen Huang is with Department of Biomedical Engineering and Environmental Science, National Tsing Hua University, Hsinchu 300, Taiwan, R.O.C. ( yufen@mx.nthu.edu.tw). Chih-Chen Chen is with Institute of Nanoengineering and Microsystems, chihchen23@gmail.com). Sheng-Shian Li is with Institute of Nanoengineering and Microsystems, ssli@mx.nthu.edu.tw). J. Andrew Yeh is with Institute of Nanoengineering and Microsystems, jayeh@mx.nthu.edu.tw) Da-Jeng Yao is with Institute of Nanoengineering and Microsystems, djyao@mx.nthu.edu.tw) Yu-Lin Wang is with Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu, 300, Taiwan, R.O.C. ( phone: #62405; ylwang@mx.nthu.edu.tw). sensor could be re-used by washing the sensor with buffer solution, showing that the inhibition of the HRP at high H 2 O 2 concentration could be recovered, and the reaction for the inactive HRP complex was reversible. Near or lower than the detection limit, the signal was dominated by the affinity between HRP and H 2 O 2. There was no response to hydrogen peroxide for the PANI without HRP immobilized, showing the stability of the PANI to hydrogen peroxide and the specificity of the HRP. This electronic hydrogen peroxide sensor with high sensitivity and low detection limit is very promising for applications in low concentration hydrogen peroxide detections, such as for reactive oxygen species (ROS) level relevant to the defects during to the growth of embryo and the surviving rate of In vitro fertilization (IVF). H I. INTRODUCTION ydrogen peroxide attracts a great interest due to its important role in food, pharmaceutical, and clinical applications [1]. Hydrogen peroxide is a by-product in many enzyme-catalytic reactions, such as glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, urate oxidase, aldehyde oxidase, and oxalate oxidase, which are used to detect glucose, lactic acid, cholesterol, ethanol, urea, formaldehyde, and oxalate, respectively. These biomolecules are important markers in many biologically metabolic reactions. Besides, hydrogen peroxide is also an important signaling molecule for many biological reactions [2], [3]. Moreover, hydrogen peroxide is one of the reactive oxygen species (ROS), which is often detected for oxidative stress studies. Oxidative stress is currently an important research topic and is believed to be relevant to tumors, cancers, Parkinsus disease, and aging [4]. Oxidative stress was found to be relevant to the defects during the growth of an embryo [5]. It was also reported that lower ROS level can yield a higher successful rate of In vitro fertilization (IVF) [6]. The concentration of hydrogen peroxide in biological systems could be very low. For example, the intramitochondria H 2 O 2 concentration is estimated and reported as 4.8 nm only [7]. Therefore, it is required to develop highly sensitive H 2 O 2 sensor for these applications. Several methods, including fluorimetry [8], chemiluminescence [9], spectrometry [10], high performance liquid phase (HPLC) chromatography [11], electrochemistry [12] were used to detect H 2 O 2. Fluorimetry, chemiluminescence, and spectrometry require an expensive optical system for high sensitivity detection. High performance liquid phase (HPLC) chromatography is sensitive but also expensive. Instead, electronic biosensors,

2 including the electrochemical and resistive sensors, are much cheaper and smaller due to the highly developed microfabrication techniques. Therefore, it is possible to develop a cheap and disposable electronic microsensors with high sensitivity, which can detect hydrogen peroxide in nm or sub-nm. However, from literatures, to the best of our knowledge, many H 2 O 2 sensors can only detect μm of H 2 O 2, and few can detect nm or sub-nm of H 2 O 2 [13], [17]. Currently only three papers published detection limit at sub-nm of H 2 O 2 using electrochemical sensors [14]-[16]. HRP-immobilized PANI was reported to be fabricated as electrochemical sensors [18]-[20] to detect H 2 O 2. PANI provided an efficient surface for the direct electrochemical reduction of HRP. The oxidation of HRP resulted from the reduction of H 2 O 2, leading to the oxidation of PANI, resulting in the decrease of PANI conductivity, have been reported in literature [18]-[20]. Doping PANI to ensure the high conductivity is usually conducted with hydrogen proton by adding acids. However, in most biological environments, the ph is usually near neutral, which causes low conductivity of PANI. Electrosynthesized N-Alkylated PANI has shown good conductivity at neutral ph and was fabricated as microelectrochemical enzyme transistor, which showed detection limit less than 1 ppm (25 μm) of H 2 O 2 [21]. In this study, PANI was spin-coated on Au electrodes on a SiO2/Si substrate, modified with propane sultone (N-Alkylated PANI), and then immobilized with HRP. Different from electrochemical sensors, which usually work in chronometry or amperometry for the detection of H 2 O 2 [21], in our prepared sensors, the conductance change of PANI was measured in real-time to characterize the concentrations of H 2 O 2 in buffer solution. The detection limit was obtained as 0.7 nm and the detectable region is from 0.7 nm to 1 μm. To the best of our knowledge, the detection limit of our sensor is one of the lowest concentrations of H 2 O 2 that has ever been reported [22]. The high sensitivity is attributed to the enzymatic signal amplification by accumulating conductance change of the PANI film. At different concentration of H 2 O 2, the sensor behaviors are found very different. We investigate the sensing mechanism of our sensor and discuss it in detail. Higher than 1 μm of H 2 O 2, the sensor gradually saturated. We discuss why the sensor saturated at high concentration of H 2 O 2 is due to the inhibition of the activity of the HRP enzyme in excess H 2 O 2, as reported in literatures that utilized absorption spectra. Moreover, the inactive HRP on our saturated sensor can be re-activated, indicating that the reaction of the formation of inactive HRP complex is reversible. This finding is very crucial to the understanding of the interaction between HRP and H 2 O 2, and the sensor as well. Besides, the sensor only requires a small sample volume for detection. The developed ultra-sensitive H 2 O 2 sensor cannot only be used for monitoring oxidative stress for IVF, but also can be applied to personal medical use due to its low cost, small size and ease of operation. II. EXPERIMENTS 2.1 Preparation of PANI thin film on substrate Polyaniline emeraldine base was purchased from Sigma-Aldrich. 0.3 g of polyaniline emeraldine base powder was dissolved in 5 ml of dimethyl sulfoxide (DMSO) with stirring for 6 hours. The polyaniline solution was then mixed with the same volume of 0.5 M sulfuric acid for 24 hours. The sulfuric acid can increase the conductivity and the stability of the PANI thin film (Chen and Hwang, 1995). After that, 1.5 μl of the PANI solution was dropped with a micro pipette and spin-coated at 1600 rpm for 30 seconds on a microchip, followed by baking at 60 for 30 minutes in air. The microchip consists of two metal electrodes comprised of 200 Å Ti and 1000 Å Au deposited with an e-beam evaporator on a Si 3 N 4 /Si substrate. The length and the width of the Au electrodes are 500 μm and 100 μm, respectively. The gap between the two metal electrodes is 10 μm. The PANI/Au interface was confirmed to be ohmic by measuring the current-voltage characteristics of the device. 2.2 Sultonation and HRP-immobilization of polyaniline thin film The PANI-coated device was held at -0.2 V for 10 minutes followed by being washed with DI water and then immersed into a 1.2 M of NaOH solution for 20 minutes. The device was then dried with nitrogen gas. Propane sultone was dropped on the PANI film and allowed to wait for 8 hours. The device was then washed with DI water to remove the excess propanesultone. Horseradish peroxidase and 1,4-diaminobenzene were purchased from Sigma-Aldrich. The HRP enzyme was prepared in 120 units cm-3 in a citrate phosphate buffer solution (ph=5.5), with 25 mm of 1,4-diaminobenzene. The prepared device was then placed in the HRP enzyme solution for 20 minutes, followed by being applied with 0.4 V for 4 minutes. The sensor was then washed with DI water and ready for H 2 O 2 detection. Figure 1(a) and Figure 1(b) show the schematics of the H 2 O 2 sensor and the photograph of a packaged sensor, respectively. 2.3 Sensor measurements Fig. 1. (a) Schematic of the hydrogen peroxide sensor. HRP-immobilized PANI was spin-coated on Au electrodes (b) plan-view photograph of a packaged hydrogen peroxide sensor.

3 The current of the sensor was measured at a dc bias of 0.1 V at room temperature using an Agilent B1500 parameter analyzer with the polyaniline layer exposed. Different concentrations of hydrogen peroxide prepared in citrate phosphate buffer solutions (ph=5.5) were directly dropped on fresh sensors and the currents were measured. The concentrations of H 2 O 2, including 0.1 nm, 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 1 μm, 10 μm, 100 μm and 1 mm, were tested with fresh sensors. The detection limit is also experimentally confirmed by testing H 2 O 2 concentrations, including 0.1 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, and 1 nm again. The ratio of conductance change (decrease) is found to be proportional to the target concentrations of H 2 O 2. A specificity test was also conducted under different H 2 O 2 concentrations for sensors without HRP immobilization at the same ph and dc bias. III. RESULTS AND DISCUSSION 3.1 Overall sensing capability and range time detection of 10 μl of 0.1 nm, 1 nm, 10 nm, 100 nm, 1 μm, 10 μm, 100 μm, and 1 mm H 2 O 2, respectively, at constant bias of 100 mv with fresh sensors. The interval time of the current measurement is 1 minute. When 10 μl of 0.1 nm H 2 O 2 was dropped onto the surface of the PANI, there was no significant current change. In sharp contrast, when 10 μl of 1 nm H 2 O 2 was dropped on a fresh sensor, a sharp current change was observed as the system reached a steady state. The current keeps decreasing as the concentration of H 2 O 2 increases until 1 μm. Beyond 1 μm, the decreasing rate of the current does not increase significantly. To find out the detection limit, 10 μl of 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, and 0.9 nm H 2 O 2 were further tested with fresh sensors. Figure 5(a), (b), (c), (d) show the real-time measurement at 0.1 V for 10 μl of 0.6 nm, 0.7 nm, 0.8 nm, and 0.9 nm H 2 O 2, respectively. The average percentage of conductance changes and the error bars (standard deviation) from 5 measurements on fresh sensors for H 2 O 2 concentration ranging from 0.1 nm to 1 nm and from 0.1 nm to 1 mm were shown in Figure 6(a) and 6(b), respectively. From Figure 6(a), it is obvious that below 0.6 nm, the percentage of conductance change is not significant. However, at 0.7 nm, the percentage of conductance changes a lot. To the best of our knowledge, the detection limit of our sensor (0.7 nm) is one of the lowest concentrations of H 2 O 2 that have ever been reported (Chen et al., 2013; Ahammad, 2013). From Figure 6(a) and 6(b), the observed detectable concentration of H 2 O 2 for this sensor is from 0.7 nm to 1 μm. When higher than 1 μm, the sensor showed gradually saturation. The specificity was tested with sensors without HRP immobilized under different concentrations of hydrogen peroxide at ph 5.5 with or without 1,4-diaminobenzene, as shown in Figure 7(a) and 7(b), respectively, indicating no significant current change, which demonstrate that hydrogen peroxide do not react with PANI and the specificity of the sensors only depends on HRP. Fig. 2. The current of devices with different waiting time (1 hr, 10 hrs, and 24 hrs) for the mixture of PANI solution and sulfuric acid. The current of the PANI thin films were measured at 0.1 V as baked, 1 day, 3 days, and 7 days after baking. Figure 2 shows the current for devices with different waiting time (1 hr, 10 hrs, and 24 hrs) for the mixture of PANI solution and sulfuric acid, which was described in the Experimental 2.1. The current of the PANI thin films were measured at 0.1 V as baked, 1 day, 3 days, and 7 days after baking. It is obvious that the one with 24-hour waiting time gives the highest current level and the best stability. The PANI thin film with 48-hour waiting time was also measured (data not shown here), but it showed a decreased current level, compared with that of the one with 24-hour waiting time. We intend to prepare PANI film with high conductivity, because the high conductivity indicates the high carrier concentration, which may lead to better sensitivity. Figure 3(a), 3(b), 3(c), and 3(d), and Figure 4(a), 4(b), 4(c), and 4(d) show the real Fig. 3. Real time detection of 10 μl of (a) 0.1 nm, (b) 1 nm, (c) 10 nm, and (d) 100 nm H 2O 2, respectively, at constant bias of 100 mv with fresh sensors..

4 3.2 Reuse of the sensor Fig. 4. Real time detection of 10 μl of (a) 1 μm, (b) 10 μm, (c) 100 μm, and (d) 1 mm H 2O 2, respectively, at constant bias of 100 mv with fresh sensors.. Fig. 5. Real time measurement of 10 μl of (a) 0.6 nm, (b) 0.7 nm, (c) 0.8 nm, and (d) 0.9 nm H 2O 2, respectively, at 0.1 V. Fig. 6. The average percentage of conductance changes and the error bars (standard deviation) from 5 measurements on fresh sensors for H 2O 2 concentration ranging from (a) 0.1 nm to 1 nm and from (b) 0.1 nm to 1 mm, respectively. Fig. 7. The current of sultonated PANI without HRP immobilization under different target concentrations of hydrogen peroxide at ph 5.5 (a) with or (b) without 1,4-diaminobenzene, respectively. We are interested in whether the used sensor can be reused again without any regeneration in need. If the H 2 O 2 solution is removed from the sensor, ideally, the sensor should be able to be reused because the PANI still has good conductivity, indicating the considerable carrier concentration left in the polymer available for further redox reactions (oxidation of the PANI and reduction of H 2 O 2 and HRP). However, the HRP has to be active or re-activated for reuse by capturing electrons from an electron donor, the PANI, after being oxidized by H 2 O 2. Therefore, it is proposed that the conjugated system of the PANI can efficiently allow electrons away from HRP to be transferred to the oxidized site near the HRP. Therefore, a sensor was reused to detect H 2 O 2 for three times. Figure 8(a) shows the real-time detection of 1 nm hydrogen peroxide (current level versus time) with a fresh sensor for the 1 st, the 2 nd, and the 3 rd measurements, respectively. Before any measurement, the sensor was washed with buffer solution and dried with N 2 gas. The result shows that the reused sensor has a reduced current level, but still can sense hydrogen peroxide as low concentration as 1 nm. Practically, if the sensor needs to be reused, the calibration curve may need to be collected for the 2 nd or the 3 rd measurement, respectively. However, the cost of this microsensor is not high, so it can be used just once and disposed. The calibration curves shown in Figure 6(a) and 6(b) are only prepared based on the first measurement on fresh sensors. Similarly, Figure 8(b), 8(c), and 8(d) show real-time detection 10 μl of 100 nm, 1 μm, and 5 μm of H 2 O 2, respectively, for 3 measurements on each sensor. IV. CONCLUSIONS In summary, Horseradish peroxidase (HRP)-immobilized conducting polymer, polyaniline (PANI), was used to detect hydrogen peroxide in buffer solution by measuring the polymer conductance change. The low detection limit (0.7 nm) and the high sensitivity are attributed to the enzymatic signal amplification by accumulated conductance change of PANI. The detectable H 2 O 2 concentration is from 0.7 nm to 1 μm. Beyond 1 μm H 2 O 2, the sensor gradually saturated. The saturation of the sensor is due to the inhibition of HRP activity by excess H 2 O 2. However, the saturated sensor can be re-used by washing with buffer solution to re-activate HRP. It is therefore suggested that the reaction of the formation of inactive HRP (verdohemoprotein) can be reversed, resulting to re-activating. In the detectable region, because H 2 O 2 was fully consumed, the signal is determined by the total amount of H 2 O 2. In the low concentration region (below or near detection limit), the concentration of H 2 O 2 dominates the signal, which implies that the affinity between the H 2 O 2 and the HRP plays an important role. This ultra-sensitive disposed H 2 O 2 microsensor is able to be utilized in detecting low H 2 O 2

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