Preparation method of three-electrode Clark dissolved oxygen sensor

Abstract

The invention discloses a preparation method of a three-electrode Clark dissolved oxygen sensor, and belongs to the technical field of microfluidic sensing and electrochemical detection. The invention aims to solve the problems that the service life and integration level of a device are limited by existing liquid electrolyte; oxygen consumption causes local concentration change, so that the measurement accuracy of static and flowing samples is influenced; the gas permeable membrane is easy to have mechanical faults; and real-time and continuous dissolved oxygen monitoring is difficult to realize. The method comprises the following steps: 1, preparing an LTCC substrate and an electrode; 2, preparing and coating a solid proton conduction matrix; 3, preparing an oxygen permeable membrane; and 4, assembling the micro-channel layer. The preparation method is used for preparing the three-electrode Clark dissolved oxygen sensor.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic sensing and electrochemical detection technology. Background Technology

[0002] The accurate measurement of dissolved oxygen delivery and concentration is crucial in environmental monitoring, bioanalysis, bioreactors, and numerous other applications. Oxygen sensing technology is frequently used to monitor cellular metabolic activity and rapidly determine cellular viability. Adequate oxygen supply plays a key role in cell proliferation and differentiation, while abnormal oxygen supply can lead to pathological conditions. Monitoring cellular oxygen consumption and respiration provides important information for metabolism, apoptosis, mitochondrial function, and the toxicological responses of various drugs in key biochemical studies. Furthermore, with the increasing demand for integrated microfluidic devices in biological applications, the development of low-cost, flexible, and easily fabricated oxygen sensors is becoming increasingly urgent.

[0003] Currently, two common dissolved oxygen sensing technologies in microfluidics applications are optical fluorescence sensing and electrochemical sensing. Both exhibit high sensitivity at low oxygen concentrations. Fluorescent oxygen sensing is widely used due to its inherent stability, but its application is limited by complex fabrication processes and expensive lifetime monitoring equipment. Electrochemical sensors are widely used and have various commercial models. The most common implementation is the Clarke-type sensor, which includes a cathode, anode, electrolyte, and gas-permeable membrane. Compared to fluorescence sensing, Clarke-type dissolved oxygen sensors offer advantages such as simple fabrication, low cost, and fast response. However, these sensors also have limitations and inherent design problems. For example, the consumption of dissolved oxygen at the working electrode may alter the local oxygen concentration, requiring mechanical stirring or circulation of the test sample to address this issue. Furthermore, the gas-permeable membrane used to isolate the liquid electrolyte from the sample is typically thin and prone to mechanical failure. Adapting the functionality of such sensors for continuous monitoring, automation, and high-throughput measurement also presents challenges. Summary of the Invention

[0004] This invention aims to address the limitations of existing liquid electrolytes on device lifespan and integration; oxygen consumption leading to localized concentration changes affecting the accuracy of static and flowing sample measurements; the susceptibility of gas-permeable membranes to mechanical failure; and the difficulty in achieving real-time, continuous dissolved oxygen monitoring. Therefore, this invention provides a method for fabricating a three-electrode Clark dissolved oxygen sensor.

[0005] A method for preparing a three-electrode Clark dissolved oxygen sensor, comprising the following steps:

[0006] I. Fabrication of LTCC Substrate and Electrode:

[0007] ① Use two pieces of green ceramic tape as the base material;

[0008] ② Cut grooves, inlet and outlet on the surface of a piece of green porcelain strip to obtain a green porcelain strip with grooves;

[0009] ③ Cut the inlet and outlet on the surface of another green ceramic strip, and prepare the working electrode, counter electrode and reference electrode on the upper surface to obtain a green ceramic strip with electrodes;

[0010] ④ The lower surface of the green ceramic strip with grooves is bonded and laminated with the upper surface of the green ceramic strip with electrodes, then sintered, and finally the reference electrode is chemically oxidized to obtain the LTCC substrate.

[0011] II. Preparation and Coating of Solid Proton Conducting Matrix:

[0012] ① Mix Nafion solution, polyvinylpyrrolidone solution and 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution to obtain PCM solution;

[0013] ② The LTCC substrate was immersed in a mixture of propyl 3-(trimethoxysilyl)methacrylate, ethanol and dilute acetic acid, then rinsed and dried to obtain the pretreated LTCC substrate;

[0014] ③ The PCM solution is drop-coated into the groove of the pretreated LTCC substrate and then dried to obtain a substrate covered with a PCM film;

[0015] III. Preparation of Oxygen Permeation Membrane:

[0016] ① Mix the PDMS matrix with the curing agent, and then add xylene to obtain a xylene-PDMS mixture;

[0017] ② The xylene-PDMS mixture is spin-coated onto the surface of the substrate covered with PCM membrane, and then cured to obtain a substrate covered with oxygen-permeable membrane OPM.

[0018] IV. Assembly of the microchannel layer:

[0019] ① Cut out microchannels in the PDMS block to obtain a PDMS block with microchannels;

[0020] ② Both the PDMS block with microchannels and a piece of transparent glass are treated with O2 plasma. Then, the plasma-treated PDMS microchannel layer is bonded to the transparent glass to obtain the PDMS-glass microchannel layer.

[0021] ③ The substrate covered with oxygen-permeable membrane OPM is saturated with water, then aligned with PDMS-glass microchannel layer and mechanically fixed to obtain a three-electrode Clark dissolved oxygen sensor.

[0022] The beneficial effects of this invention are:

[0023] This invention utilizes LTCC technology to achieve monolithic integration of sensor electrodes, microchannels, and interfaces, resulting in a compact structure, excellent sealing, and high mechanical strength. The use of solid-state Nafion electrolyte instead of traditional liquid electrolyte avoids electrolyte leakage and drying issues, significantly extending sensor lifespan and achieving high integration. The PDMS oxygen-permeable membrane also serves as a protective layer for the Nafion, preventing contamination by cations in the sample. The microchannel design allows the sample to flow continuously through the sensor's working area, effectively reducing the impact of oxygen consumption at the working electrode on measurements and enabling real-time monitoring of dissolved oxygen changes in the flow environment. The sensor exhibits excellent linear response within a dissolved oxygen concentration range of 0 mg / L to 8.1 mg / L, with a correlation coefficient exceeding 99.5%. It exhibits low residual current (<3.5%) and fast response time (average 10.9 seconds). The LTCC process is suitable for rapid prototyping and mass production, with relatively low cost.

[0024] This invention successfully designed, fabricated, and characterized a three-electrode Clark dissolved oxygen sensor. This integrated device enables accurate measurement and real-time detection of dissolved oxygen. The use of a solid electrolyte extends the sensor's lifetime and realizes microfluidic functionality characteristic of Clark-type sensors. The sensor's performance is comparable to existing sensors, while offering advantages such as rapid and low-cost manufacturing and a more flexible platform, making it a potential candidate for applications in bioanalysis and bioreactors. Attached Figure Description

[0025] Figure 1 This is a conceptual diagram of the working principle and a schematic diagram of the reaction mechanism of the three-electrode Clark dissolved oxygen sensor of the present invention;

[0026] Figure 2 This is a schematic diagram of the structure of the three-electrode Clark dissolved oxygen sensor of the present invention;

[0027] Figure 3 The oxygen reduction cyclic voltammetry curve of the three-electrode Clark dissolved oxygen sensor prepared in Example 1 in oxygen-saturated deionized water;

[0028] Figure 4 CA curves of the three-electrode Clark dissolved oxygen sensor prepared in Example 1: (a) CA curves of oxygen-saturated Duchenne phosphate buffer solution (DPBS) at different flow rates and static conditions, and CA curves of oxygen-depleted DPBS under static conditions; (b) CA curves of oxygen-saturated deionized water (DI) and oxygen-saturated DPBS at a certain flow rate and static conditions.

[0029] Figure 5The CA curves of the three-electrode Clark dissolved oxygen sensor prepared in Example 1 at different flow rates are shown in (a) and (b) respectively. (a) CA curves of deionized water with different oxygen concentrations at a flow rate of 0.5 mL / min. (b) Sensor calibration curves obtained from the CA results at different flow rates.

[0030] Figure 6 The response curve and response time of the three-electrode Clark dissolved oxygen sensor prepared in Example 1 were measured by injecting a zero-oxygen solution. Detailed Implementation

[0031] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0032] Specific Implementation Method 1: This implementation method describes a method for preparing a three-electrode Clark dissolved oxygen sensor, which is carried out according to the following steps:

[0033] I. Fabrication of LTCC Substrate and Electrode:

[0034] ① Use two pieces of green ceramic tape as the base material;

[0035] ② Cut grooves, inlet and outlet on the surface of a piece of green porcelain strip to obtain a green porcelain strip with grooves;

[0036] ③ Cut the inlet and outlet on the surface of another green ceramic strip, and prepare the working electrode, counter electrode and reference electrode on the upper surface to obtain a green ceramic strip with electrodes;

[0037] ④ The lower surface of the green ceramic strip with grooves is bonded and laminated with the upper surface of the green ceramic strip with electrodes, then sintered, and finally the reference electrode is chemically oxidized to obtain the LTCC substrate.

[0038] II. Preparation and Coating of Solid Proton Conducting Matrix:

[0039] ① Mix Nafion solution, polyvinylpyrrolidone solution and 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution to obtain PCM solution;

[0040] ② The LTCC substrate was immersed in a mixture of propyl 3-(trimethoxysilyl)methacrylate, ethanol and dilute acetic acid, then rinsed and dried to obtain the pretreated LTCC substrate;

[0041] ③ The PCM solution is drop-coated into the groove of the pretreated LTCC substrate and then dried to obtain a substrate covered with a PCM film;

[0042] III. Preparation of Oxygen Permeation Membrane:

[0043] ① Mix the PDMS matrix with the curing agent, and then add xylene to obtain a xylene-PDMS mixture;

[0044] ② The xylene-PDMS mixture is spin-coated onto the surface of the substrate covered with PCM membrane, and then cured to obtain a substrate covered with oxygen-permeable membrane OPM.

[0045] IV. Assembly of the microchannel layer:

[0046] ① Cut out microchannels in the PDMS block to obtain a PDMS block with microchannels;

[0047] ② Both the PDMS block with microchannels and a piece of transparent glass are treated with O2 plasma. Then, the plasma-treated PDMS microchannel layer is bonded to the transparent glass to obtain the PDMS-glass microchannel layer.

[0048] ③ The substrate covered with oxygen-permeable membrane OPM is saturated with water, then aligned with PDMS-glass microchannel layer and mechanically fixed to obtain a three-electrode Clark dissolved oxygen sensor.

[0049] Figure 1 This diagram illustrates the working principle and reaction mechanism of the three-electrode Clark dissolved oxygen sensor of this invention. Specifically, the three-electrode Clark dissolved oxygen sensor applies a constant negative voltage between the working electrode (WE) and the reference electrode (RE), forcing dissolved oxygen to permeate through the permeable membrane and be reduced and consumed on the surface of the working electrode, simultaneously generating a diffusion current. The magnitude of this current is directly proportional to the diffusion rate of oxygen molecules, which directly reflects the partial pressure or concentration of dissolved oxygen in the water sample. Therefore, the dissolved oxygen value can be accurately calculated by measuring the current. The reference electrode ensures the stability of the working electrode potential, while the counter electrode (CE) forms the current loop. The three-electrode system together ensures the accuracy and stability of the measurement.

[0050] Figure 2 This is a schematic diagram of the three-electrode Clark dissolved oxygen sensor of the present invention; as shown in the figure, the sensor adopts a multi-layer structure, including:

[0051] Low-temperature co-fired ceramic (LTCC) substrate: The substrate is made of low-temperature co-fired ceramic, with microchannels integrated inside and a three-electrode system fabricated on the surface.

[0052] The three-electrode system includes a gold working electrode, a gold counter electrode, and an Ag / AgCl reference electrode.

[0053] Solid proton conduction matrix: composed of Nafion membrane, which acts as a solid electrolyte.

[0054] Oxygen permeation membrane: A thin film made of polydimethylsiloxane (PDMS) covering a solid electrolyte.

[0055] Microchannel layer: Channels made of PDMS for the introduction and flow of sample solution, and sealed by a glass plate.

[0056] The beneficial effects of this embodiment are:

[0057] This embodiment utilizes LTCC technology to achieve monolithic integration of sensor electrodes, microchannels, and interfaces, resulting in a compact structure, excellent sealing, and high mechanical strength. The use of solid-state Nafion electrolyte instead of traditional liquid electrolyte avoids electrolyte leakage and drying issues, significantly extending sensor lifespan and achieving high integration. The PDMS oxygen-permeable membrane also serves as a protective layer for the Nafion, preventing contamination by cations in the sample. The microchannel design allows the sample to flow continuously through the sensor's working area, effectively reducing the impact of oxygen consumption at the working electrode on measurements and enabling real-time monitoring of dissolved oxygen changes in the flow environment. The sensor exhibits good linear response within a dissolved oxygen concentration range of 0 mg / L to 8.1 mg / L, with a correlation coefficient exceeding 99.5%. It exhibits low residual current (<3.5%) and fast response time (average 10.9 seconds). The LTCC process is suitable for rapid prototyping and mass production, with relatively low cost.

[0058] This embodiment successfully designed, fabricated, and characterized a three-electrode Clark dissolved oxygen sensor. This integrated device enables accurate measurement and real-time detection of dissolved oxygen. The use of a solid electrolyte extends the sensor's lifetime and realizes microfluidic functionality characteristic of Clark-type sensors. The sensor's performance is comparable to existing sensors, while offering advantages in rapid, low-cost fabrication and a more flexible platform, making it a potential candidate for applications in bioanalysis and bioreactors.

[0059] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that: the groove mentioned in step 1, ②, is a through hole with a width of 2mm and a length of 15mm; the inlet and outlet mentioned in steps 1, ②, and ③ are both circular through holes with a diameter of 2mm. Everything else is the same as in Specific Implementation Method One.

[0060] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that: in step one ③, gold paste is used to print the working electrode and the counter electrode, and silver paste is used to print the reference electrode; in step one ④, ferric chloride aqueous solution is used to chemically oxidize the reference electrode to form an Ag / AgCl reference electrode. Everything else is the same as in Specific Implementation Method One or Two.

[0061] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that: the lamination described in step one (④) is specifically carried out under conditions of a pressure of 2000 psi to 3000 psi and a temperature of 70°C to 80°C for 5 to 10 minutes; the sintering described in step one (④) is specifically carried out under conditions of a temperature of 750°C to 850°C for 10 to 30 minutes. Everything else is the same as in Specific Implementation Methods One to Three.

[0062] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in the following ways: the concentration of the Nafion solution in step two① is 2 wt.%~5 wt.%; the concentration of the polyvinylpyrrolidone solution in step two① is 1.25 wt.%~2 wt.%; the concentration of the 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution in step two① is 0.04 wt.%~0.1 wt.%; the volume ratio of the Nafion solution to the polyvinylpyrrolidone solution in step two① is 1:(1~2); the volume ratio of the Nafion solution to the 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution in step two① is 1:(1~2). Everything else is the same as in Specific Implementation Methods One to Four.

[0063] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: in step two ②, the concentration of 3-(trimethoxysilyl)methacrylate in the mixture of propyl 3-methacrylate, ethanol, and dilute acetic acid is 20 mmol / L to 40 mmol / L, the concentration of ethanol is 14.5 mol / L to 16.5 mol / L, and the concentration of dilute acetic acid is 26 mmol / L to 46 mmol / L. Everything else is the same as in Specific Implementation Methods One to Five.

[0064] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that: In step two ②, at a temperature of 25℃~35℃, the LTCC substrate is immersed in a mixture of 3-(trimethoxysilyl)methacrylate, ethanol, and dilute acetic acid for 3min~5min, then rinsed with ethanol, and dried at a temperature of 60℃~80℃ for 5min~10min to obtain the pretreated LTCC substrate; in step two ③, the substrate is dried at a temperature of 70℃~80℃ for 1h~3h; the thickness of the PCM film mentioned in step two ③ is 15μm~30μm. Everything else is the same as in Specific Implementation Methods One to Six.

[0065] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in the following ways: the mass ratio of PDMS matrix to curing agent in step three ① is (5~10):1; the viscosity of the xylene-PDMS mixture in step three ① is 100 mPa·s~300 mPa·s; in step three ②, curing is carried out at a temperature of 60℃~80℃ for 2h~5h; and the thickness of the oxygen-permeable membrane OPM in step three ② is 20μm~40μm. Everything else is the same as in Specific Implementation Methods One to Seven.

[0066] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in that: the thickness of the PDMS block mentioned in step four① is 1mm to 2mm; the microchannel mentioned in step four① is a rectangular through-hole with a width of 2.2mm to 4mm and a length of 23mm to 40mm. Everything else is the same as in Specific Implementation Methods One to Eight.

[0067] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Methods One to Nine in that: the O2 plasma treatment described in step four ② is specifically performed as follows: the PDMS block with microchannels and a piece of transparent glass are cleaned with isopropanol and dried with nitrogen, then placed together in the vacuum chamber of a plasma cleaner. The vacuum is evacuated to a pressure below 10 Pa, high-purity oxygen is introduced and the working pressure is maintained at 20 Pa to 40 Pa. Then, under a radio frequency power of 50 W to 80 W, oxygen is excited to generate plasma for 30 to 60 seconds. The water saturation treatment described in step four ② is specifically performed as follows: the substrate covered with an oxygen-permeable membrane (OPM) is immersed in or ultrapure water is dripped onto its surface to ensure the membrane surface is completely covered with water. Then it is left to stand for 10 to 30 minutes, and finally, excess water is poured off. Everything else is the same as in Specific Implementation Methods One to Nine.

[0068] The beneficial effects of the present invention are verified using the following embodiments:

[0069] Example 1:

[0070] A method for preparing a three-electrode Clark dissolved oxygen sensor, comprising the following steps:

[0071] I. Fabrication of LTCC Substrate and Electrode:

[0072] ① Use two pieces of green ceramic tape as the base material;

[0073] ② Cut grooves, inlet and outlet on the surface of a piece of green porcelain strip to obtain a green porcelain strip with grooves;

[0074] The groove is a through hole with a width of 2mm and a length of 15mm; the inlet and outlet are both circular through holes with a diameter of 2mm.

[0075] ③ Cut the inlet and outlet on the surface of another green ceramic strip, and prepare the working electrode, counter electrode and reference electrode on the upper surface to obtain a green ceramic strip with electrodes;

[0076] Both the inlet and outlet are circular through holes with a diameter of 2mm; the working electrode and counter electrode are printed using gold paste, and the reference electrode is printed using silver paste.

[0077] ④ The lower surface of the green ceramic tape with grooves and the upper surface of the green ceramic tape with electrodes are bonded together, and laminated for 10 min under a pressure of 2000 psi and a temperature of 70 °C. Then, sintering is carried out for 30 min at a temperature of 850 °C. Finally, the reference electrode is chemically oxidized using ferric chloride aqueous solution to form an Ag / AgCl reference electrode, thus obtaining the LTCC substrate.

[0078] II. Preparation and Coating of Solid Proton Conducting Matrix:

[0079] ① Mix Nafion solution, polyvinylpyrrolidone solution and 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution to obtain PCM solution;

[0080] The concentration of the Nafion solution is 5 wt.%; the concentration of the polyvinylpyrrolidone solution is 1.25 wt.%; the concentration of the 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution is 0.04 wt.%; the volume ratio of the Nafion solution to the polyvinylpyrrolidone solution is 1:1; the volume ratio of the Nafion solution to the 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution is 1:1.

[0081] ② At a temperature of 25°C, the LTCC substrate was immersed in a mixture of propyl 3-(trimethoxysilyl)methacrylate, ethanol and dilute acetic acid for 3 min, then rinsed with ethanol, and dried at a temperature of 60°C for 5 min to obtain the pretreated LTCC substrate.

[0082] In the mixture of propyl 3-(trimethoxysilyl)methacrylate, ethanol, and dilute acetic acid, the concentration of propyl 3-(trimethoxysilyl)methacrylate is 20 mmol / L, the concentration of ethanol is 16.5 mol / L, and the concentration of dilute acetic acid is 46 mmol / L.

[0083] ③ The PCM solution is drop-coated into the grooves on the surface of the pretreated LTCC substrate, and then dried at 70°C for 1 hour to obtain a substrate covered with PCM film.

[0084] The thickness of the PCM film is 15 μm;

[0085] III. Preparation of Oxygen Permeation Membrane:

[0086] ① Mix the PDMS matrix with the curing agent, and then add xylene to obtain a xylene-PDMS mixture;

[0087] The mass ratio of the PDMS matrix to the curing agent (dibutyltin dilaurate) is 10:1; the viscosity of the xylene-PDMS mixture in step 3① is 100 mPa·s;

[0088] ② The xylene-PDMS mixture was spin-coated onto the surface of the substrate covered with PCM film, and then cured at 60℃ for 2 hours to obtain a substrate covered with oxygen-permeable OPM film.

[0089] The thickness of the oxygen-permeable membrane OPM is 20 μm;

[0090] IV. Assembly of the microchannel layer:

[0091] ① Cut out microchannels in the PDMS block to obtain a PDMS block with microchannels;

[0092] The thickness of the PDMS block is 1 mm; the microchannel is a rectangular through-hole with a width of 2.2 mm and a length of 23 mm.

[0093] ② The PDMS block with microchannels and a piece of transparent glass are both treated with O2 plasma. Then the plasma-treated PDMS microchannel layer is bonded to the glass sheet to obtain the PDMS-glass microchannel layer.

[0094] The O2 plasma treatment is carried out in the following steps: the PDMS block with microchannels and a piece of transparent glass are cleaned with isopropanol and dried with nitrogen. They are then placed together in the vacuum chamber of a plasma cleaner. The vacuum is evacuated to a pressure below 10Pa. High-purity oxygen is introduced and the working pressure is maintained at 20Pa. Then, under the condition of 50W radio frequency power, oxygen is excited to generate plasma for 30s. Finally, the treated PDMS microchannel layer is aligned with the transparent glass and tightly bonded to obtain the PDMS-glass microchannel layer.

[0095] ③ The substrate covered with oxygen-permeable membrane OPM is saturated with water, then aligned with PDMS-glass microchannel layer and mechanically fixed to obtain a three-electrode Clark dissolved oxygen sensor.

[0096] The water saturation treatment is carried out in the following steps: ultrapure water is dropped onto the surface of the substrate covered with an oxygen-permeable membrane (OPM) to ensure that the membrane surface is completely covered with water, then it is left to stand for 10 minutes, and finally the excess water on the surface is poured off.

[0097] 1. Cyclic Voltmeter-Ampere Test:

[0098] Cyclic voltammetry was performed in oxygen-saturated deionized water at a scan rate of 100 mV / s over a potential range of 0 V to -1.0 V. Figure 3 The oxygen reduction cyclic voltammetry curves of the three-electrode Clark dissolved oxygen sensor prepared in Example 1 are shown in oxygen-saturated deionized water. The results show that a plateau current begins to appear at an applied potential of approximately -0.8 V. The diffusion control region of oxygen reduction was observed in the range of -0.8 V to -1.0 V, with no other competing reactions observed. Therefore, -0.75 V was chosen as the applied potential for subsequent chronoamperometry.

[0099] 2. Influence of oxygen sensing, flow rate, and medium:

[0100] The dynamic range of the sensor was evaluated using the chronoamperometry method. Figure 4 The CA curves for the three-electrode Clark dissolved oxygen sensor prepared in Example 1 are shown in Figures (a) and (b) respectively: (a) CA curves for oxygen-saturated Duchenne phosphate buffer solution (DPBS) and static oxygen-depleted DPBS at different flow rates and under static conditions; (b) CA curves for oxygen-saturated deionized water (DI) and oxygen-saturated DPBS at a certain flow rate and under static conditions. Chronoamperometry (CA) was used to determine the dynamic behavior of the device. Measurement results were obtained by applying a step potential change from -0V to -0.75V and measuring the current response for 1 minute. Figure (a) shows that under static conditions, the average current detected in oxygen-depleted DPBS is -4.9 ± 1.7 nA (mean ± standard deviation). Compared to the oxygen-depleted solution, the absolute current value of oxygen-saturated DPBS is significantly increased, and the higher the flow rate, the higher the absolute current response. When the solution is not flowing, after applying a potential step to oxygen-saturated DPBS, the absolute current value gradually decreases, and the average current of the CA curve is approximately 306.5 ± 5.3 nA from around 55 s to 58 s, and continues to rise. However, the current values ​​measured under flow conditions reached a steady state. In the CA curves, the times when the current reached the plateau were 10s, 11s, and 14s at flow rates of 0.2, 0.5, and 1.0 mL / min, respectively, and the average currents at steady state were -418.9±1.8nA, -469.3±2.2nA, and -502.7±2.4nA, respectively. As shown in (b), the CA curves measured in oxygen-saturated deionized water (DI) and oxygen-saturated DPBS showed similar trends, indicating that the positive and negative ions in DPBS had no effect on the measurement results. This is attributed to the protective effect of the PDMS membrane on the Nafion electrolyte.

[0101] 3. Calibration curve:

[0102] Dissolved oxygen concentration was adjusted by bubbling N2 gas into deionized water and magnetically stirring. Solutions of different concentrations were introduced into the fabricated three-electrode Clark dissolved oxygen sensor at different flow rates. Figure 5The CA curves of the three-electrode Clark dissolved oxygen sensor prepared in Example 1 are shown at different flow rates. (a) is the CA curve of deionized water with different oxygen concentrations at a flow rate of 0.5 mL / min, and (b) is the sensor calibration curve obtained from the CA results at different flow rates. All calibration curves show good linearity, with correlation coefficients all higher than 99.5%. The absolute value of the slope coefficient increases with increasing flow rate. The residual current is less than 3.5% of the current measured under saturated oxygen conditions.

[0103] 4. Response time:

[0104] The response time was measured by injecting a 0.1 M Na2SO3 solution (oxygen scavenger) into an air-filled microchannel. Figure 6 The response curve and response time of the three-electrode Clark dissolved oxygen sensor prepared in Example 1 were measured by injecting a zero-oxygen solution. The time required for the current to drop by 90% when the sensor changes from an oxygen-saturated state to an oxygen-depleted state is defined as the 90% response time, and the measured average response time is 10.9 ± 0.6 s.

Claims

1. A method for preparing a three-electrode Clark dissolved oxygen sensor, characterized in that... It is done in the following steps: I. Fabrication of LTCC Substrate and Electrode: ① Use two pieces of green ceramic tape as the base material; ② Cut grooves, inlet and outlet on the surface of a piece of green porcelain strip to obtain a green porcelain strip with grooves; ③ Cut the inlet and outlet on the surface of another green ceramic strip, and prepare the working electrode, counter electrode and reference electrode on the upper surface to obtain a green ceramic strip with electrodes; ④ The lower surface of the green ceramic strip with grooves is bonded and laminated with the upper surface of the green ceramic strip with electrodes, then sintered, and finally the reference electrode is chemically oxidized to obtain the LTCC substrate. II. Preparation and Coating of Solid Proton Conducting Matrix: ① Mix Nafion solution, polyvinylpyrrolidone solution and 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution to obtain PCM solution; ② The LTCC substrate was immersed in a mixture of propyl 3-(trimethoxysilyl)methacrylate, ethanol and dilute acetic acid, then rinsed and dried to obtain the pretreated LTCC substrate; ③ The PCM solution is drop-coated into the groove of the pretreated LTCC substrate and then dried to obtain a substrate covered with a PCM film; III. Preparation of Oxygen Permeation Membrane: ① Mix the PDMS matrix with the curing agent, and then add xylene to obtain a xylene-PDMS mixture; ② The xylene-PDMS mixture is spin-coated onto the surface of the substrate covered with PCM membrane, and then cured to obtain a substrate covered with oxygen-permeable membrane OPM. IV. Assembly of the microchannel layer: ① Cut out microchannels in the PDMS block to obtain a PDMS block with microchannels; ② Both the PDMS block with microchannels and a piece of transparent glass are treated with O2 plasma. Then, the plasma-treated PDMS microchannel layer is bonded to the transparent glass to obtain the PDMS-glass microchannel layer. ③ The substrate covered with oxygen-permeable membrane OPM is saturated with water, then aligned with PDMS-glass microchannel layer and mechanically fixed to obtain a three-electrode Clark dissolved oxygen sensor.

2. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... The groove mentioned in step 1② is a through hole with a width of 2mm and a length of 15mm; the inlet and outlet mentioned in steps 1② and 3 are both circular through holes with a diameter of 2mm.

3. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... In step 1, ③, gold paste is used to print the working electrode and the counter electrode, and silver paste is used to print the reference electrode; in step 1, ④, ferric chloride aqueous solution is used to chemically oxidize the reference electrode to form an Ag / AgCl reference electrode.

4. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... The lamination described in step 1, ④ is specifically carried out under a pressure of 2000 psi to 3000 psi and a temperature of 70°C to 80°C for 5 to 10 minutes; the sintering described in step 1, ④ is specifically carried out under a temperature of 750°C to 850°C for 10 to 30 minutes.

5. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... The concentration of the Nafion solution mentioned in step 2① is 2 wt.%~5 wt.%; the concentration of the polyvinylpyrrolidone solution mentioned in step 2① is 1.25 wt.%~2 wt.%; the concentration of the 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution mentioned in step 2① is 0.04 wt.%~0.1 wt.%; the volume ratio of the Nafion solution to the polyvinylpyrrolidone solution mentioned in step 2① is 1:(1~2); the volume ratio of the Nafion solution to the 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone solution mentioned in step 2① is 1:(1~2).

6. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... In step 2②, the concentration of propyl 3-(trimethoxysilyl)methacrylate, ethanol, and dilute acetic acid in the mixture is 20 mmol / L to 40 mmol / L, the concentration of ethanol is 14.5 mol / L to 16.5 mol / L, and the concentration of dilute acetic acid is 26 mmol / L to 46 mmol / L.

7. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... Step 2②: Immerse the LTCC substrate in a mixture of propyl 3-(trimethoxysilyl)methacrylate, ethanol, and dilute acetic acid for 3-5 minutes at a temperature of 25℃~35℃, then rinse with ethanol and dry at a temperature of 60℃~80℃ for 5-10 minutes to obtain the pretreated LTCC substrate; Step 2③: Dry at a temperature of 70℃~80℃ for 1-3 hours; The thickness of the PCM film mentioned in Step 2③ is 15μm~30μm.

8. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... The mass ratio of PDMS matrix to curing agent in step 3① is (5~10):1; the viscosity of the xylene-PDMS mixture in step 3① is 100mPa·s~300mPa·s; in step 3②, the curing time is 2h~5h at a temperature of 60℃~80℃; the thickness of the oxygen-permeable membrane OPM in step 3② is 20μm~40μm.

9. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... The thickness of the PDMS block mentioned in step 4① is 1mm~2mm; the microchannel mentioned in step 4① is a rectangular through hole with a width of 2.2mm~4mm and a length of 23mm~40mm.

10. The method for preparing a three-electrode Clark dissolved oxygen sensor according to claim 1, characterized in that... The O2 plasma treatment described in step 4② is carried out as follows: the PDMS block with microchannels and a piece of transparent glass are cleaned with isopropanol and dried with nitrogen, and then placed together in the vacuum chamber of the plasma cleaner. The vacuum is evacuated to a pressure below 10Pa, high-purity oxygen is introduced and the working pressure is maintained at 20Pa~40Pa. Then, under the condition of radio frequency power of 50W~80W, oxygen is excited to generate plasma for 30s~60s. The water saturation treatment described in step 4② is carried out as follows: the substrate covered with oxygen-permeable membrane OPM is immersed in or ultrapure water is dropped onto its surface to ensure that the membrane surface is completely covered with water. Then it is left to stand for 10min~30min, and finally the excess water on the surface is poured off.

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