KR100952330B1 - Multi-type oxygen sensor for measuring cellular respiration level integrated with cell counter and method for fabricating the same - Google Patents

Abstract

PURPOSE: A multi-type oxygen sensor integrated with a cell counter is provided to measure various properties while processing more than two reagents for one cell solution, and to measure multiple samples while the cell activity is high. CONSTITUTION: A multi-type oxygen sensor is laminated with an oxygen sensor(1`) and a cell counter(101`). The oxygen sensor comprises: a first substrate(10); a membrane substrate bonded to the first substrate and defining multiple chambers between the first substrate and the membrane substrate, while including each membrane to selectively transmit oxygen into the chamber; and an electrode formed in each chamber. The cell counter comprises: a second substrate formed with a hole matching each membrane; a cell inlet to insert a cell solution; a channel substrate(120) defining a channel to insert the cell solution on each membrane; and an electrode formed on the channel connected to the membranes.

Description

Multi-type oxygen sensor for measuring cellular respiration level integrated with cell counter and method for fabricating the same}

The present invention can be broadly defined as a microfluidic device and a method for manufacturing the same, and more particularly, an oxygen sensor in the form of a microfluidic device for measuring the oxygen respiration volume of a cell by moving a cell solution, and a method for manufacturing the same. It is about. A "microfluidic device" is a device in which a fluid port, generally composed of an inlet and an outlet, is connected by a microstructure, such as a microchannel or a microchamber, in which a small amount of fluid can move. Such devices can be used as electrochemical sensors for chemical detection and measurement. The device can also be used as an analytical device, such as a lab on a chip for drug discovery and diagnosis.

In the biomedical field, measurement of the oxygen respiration of cells is very important for finding out the cause of metabolic syndrome and developing the therapeutic agent. The method of measuring the oxygen respiration of cells is using electrochemical analysis, and the representative type is Clark-type Clark-type sensor. Many researchers have been working to miniaturize this oxygen sensor using semiconductor processes.

When measuring the oxygen respiration rate of a cell, it is important to know the exact number of cells since the number of cells is an important factor. One of the most commonly used devices for counting cells is a hemocytometer, also known as a hemocytometer, because it is a method of counting cells with the naked eye using a microscope. Consumed. The other is a particle counter invented by Wallace H. Coulter, which has been commercialized under the name Coulter counter, and research into miniaturization using a semiconductor process is being conducted.

Due to the characteristics of the cells, when the cells are left at room temperature, the respiratory activity of the cells decreases rapidly and almost disappears. Therefore, it is important to develop a multi-type sensor to simultaneously measure the respiratory activity of several samples. In addition, it is important to know the number and concentration of cells quickly and accurately for accurate analysis.

However, the conventional oxygen sensor has a disadvantage that it is not possible to measure several samples at the same time because it is made of a single sensor. In addition, since the cell counter and the oxygen sensor are operated as separate elements, it takes a lot of time while moving to the oxygen sensor to measure the respiratory activity of the cell after measuring the number of cells using the cell counter. There is a problem that the respiratory activity of the cell is sharply reduced.

In addition, the existing oxygen sensor in the form of a microfluidic device has a disadvantage in that it is difficult to distinguish the oxygen concentration even if the sensor for the purpose of measuring the oxygen saturation of the blood, or even a sensor developed for measuring the oxygen respiration volume of the cell because the range of the output value is small.

The present invention has been made to solve the above problems, the problem to be solved by the present invention is a multi-type oxygen sensor for measuring the respiratory activity of several samples at the same time and a cell counter that can determine the number of cells for accurate analysis To provide an integrated sensor and its manufacturing method.

The sensor according to the present invention for solving the above technical problem, is connected to a plurality of channels branched from one cell solution inlet for injecting a cell solution one by one is arranged an oxygen sensor for measuring the amount of dissolved oxygen in the cell solution And a cell counter for counting the number of cells is integrated in each of the cell injection port and the plurality of channels.

According to one configuration, the sensor according to the present invention is a stack of the oxygen sensor element and the cell counter element.

Wherein the oxygen sensor element has a first substrate, each membrane bonded to the first substrate to define a plurality of chambers between the first substrate and selectively permeate oxygen on the chamber. A membrane substrate, and electrodes formed on the first substrate in the respective chambers.

The cell counter device includes a second substrate having a hole corresponding to each of the membranes, a hole formed at a position corresponding to the hole of the second substrate, and a cell injection hole for injecting a cell solution. A channel substrate bonded to a second substrate and defining a channel for allowing the cellular solution to flow onto each of the membranes between the second substrate and the cell inlet portion and a connection portion with the membrane, respectively; An electrode.

In the sensor, the hole of the second substrate and the hole of the channel substrate form a reservoir that can contain the cell solution over the membrane.

The sensor manufacturing method according to the present invention for solving the above technical problem, the oxygen sensor device manufacturing step, the cell counter device manufacturing step, and the step of laminating and bonding the oxygen sensor element and the cell counter device.

The manufacturing of the oxygen sensor device may include forming an electrode on a first substrate, bonding the first substrate to define a plurality of chambers between the first substrate and the oxygen on the chamber. Forming a membrane substrate to have each membrane selectively permeable, and bonding the first substrate and the membrane substrate.

The cell counter device manufacturing step includes forming an electrode on a second substrate having a hole corresponding to each membrane, a hole is formed at a position corresponding to the hole of the second substrate, and injecting a cell solution Forming a channel substrate having a cell inlet formed thereon, the channel substrate being bonded on the second substrate to define a channel between the second substrate and the cell solution to be introduced onto the respective membranes; and Bonding the second substrate and the channel substrate.

In the manufacturing method, the electrode may be formed of Ti / Pt, Ti / Au or Ti / Ag, and a passivation layer for passivation between the chamber and the electrode lead is formed on the first substrate on which the electrode is formed. It may further comprise the step.

In addition, the membrane substrate and the channel substrate may use a polydimethylsiloxane (PDMS) substrate, and the first substrate and the second substrate may use one or more materials selected from the group consisting of glass, silicon, metal oxides, and plastic materials.

In this case, the bonding of the first substrate and the membrane substrate and the bonding of the second substrate and the channel substrate may be preferably performed by covalent bonding of -OH groups formed through oxygen plasma treatment. In some embodiments, the method may further include a 3-APTES (silane primer) treatment before the oxygen plasma treatment.

In addition, the method may further include forming a passivation layer to passivate the electrode unnecessarily exposed on the second substrate on which the electrode is formed, wherein the hole of the second substrate and the hole of the channel substrate are formed on the cell. The method may further include aligning the oxygen sensor element with the cell counter element to form a reservoir for containing a solution.

According to the present invention, a sensor is manufactured by fusing a plurality of oxygen sensors and a cell counter. It is composed of a multi-type oxygen sensor, so when two or more reagents are processed for one cell solution, various characteristics can be measured simultaneously. Simultaneous measurement allows multiple samples to be measured when cell activity is high, enabling accurate analysis. By integrating cell counters that can count the number of cells for accurate analysis, the amount of cells can be measured more accurately than with a hemocytometer. Therefore, it may be usefully used as a sensor for measuring oxygen respiration of cells in the biomedical field.

In addition, the cell counter and the oxygen sensor is integrated, but it is formed as a microfluidic device type to be integrated with the microfluidic engineering system as well as other electrochemical sensors. Cells of various sizes can be measured according to the distance between the electrodes and the width and height of the channel.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, embodiments of the present invention may be modified in many different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. The shape and the like of the elements in the drawings are exaggerated in order to emphasize a more clear description, elements denoted by the same reference numerals in the drawings means the same elements.

In order to solve the above-mentioned problems of the prior art, the present invention not only can measure two or more oxygen sensors on one substrate, but also measure several samples at the same time. It implements a multi-form lab-on-a-chip sensor that counts, measures, and compares and analyzes small changes in cellular respiration.

(1)

(One)

n : the number of electrons

F : Faraday's constant

A : surface area of the working electrode

P _m : permeability of the membrane

In addition, based on Equation 1, the surface area A of the working electrode of the oxygen sensor may be increased and the range of the output current may be increased by using a membrane having a high oxygen transmittance (P _m ).

1 is a schematic conceptual view of a sensor according to the present invention, and FIG. 2 is a cross-sectional view taken along line II-II 'of FIG. 1. 3 is an enlarged view of the oxygen sensor portion B in FIG. 2, and FIG. 4 is an enlarged view of the cell counter portion A in FIG. 1.

As shown in Figs. 1 and 2, the sensor according to the present invention is largely divided into two parts, an oxygen sensor element 1 and a cell counter element 101, in particular a cell counter element (on the oxygen sensor element 1). 101 is stacked and integrated in a single chip form.

The oxygen sensor element 1 includes a plurality of oxygen sensors 1 '. The oxygen sensor 1 'is connected to each of a plurality of channels 70 branched from one cell solution inlet 65 for injecting the cell solution, and the amount of dissolved oxygen in the cell solution injected into the cell solution inlet 65. As shown in FIG. 1, a plurality may be arranged on a plane as shown in FIG.

Meanwhile, the cell counter element 101 includes a plurality of cell counters 101 ′, which are integrated into each of the cell inlet 65 and the plurality of channels 70, and the channel 70. The number of cells is counted by measuring impedance change during cell solution movement. Instead of impedance change measurements, cell counters using optical measurements may be integrated.

As shown in more detail in FIG. 3, each oxygen sensor element 1 comprises a chamber 15 containing an internal electrolyte (eg 0.1 M KCl) 20 for electrochemical measurements on the first substrate 10, The three electrodes (working electrode, counter electrode, reference electrode) 30 formed and disposed on the first substrate 10 in the chamber 15, and the membrane 50 capable of selectively permeating oxygen are provided in the chamber ( 15) and a membrane substrate 55 on it. The internal electrolyte 20 is injected into the chamber 15 through an injection hole (shown only in the injection hole 15c of the upper surface in FIG. 1) extending from the upper surface of the sensor to the inside.

The chamber 15 is formed by the bonding of the first substrate 10 and the membrane substrate 55, for which the membrane substrate 55 is defined so as to define the chamber 15 between the first substrate 10 and the first substrate 10. It is machined with the membrane 50 in position. The passivation layer 35 is formed around the three-electrode 30 using a material such as SU-8 or a photocurable epoxy resin. The passivation layer 35 is for passivation between the chamber 15 and the electrode leads.

Reservoir 60, which is the portion that can contain the cell solution, utilizes holes 60 ', 60 "in cell counter 101 integrated over oxygen sensor 1. At this time chamber 15 ), The internal electrolyte 20, the three-electrode 30, the membrane 50 and the leisure bore 60 can be seen to constitute one oxygen sensor (1 '). As shown in FIG. 2, the electrode pads 25 are formed on the first substrate 10.

The first substrate 10 may be one or more materials selected from the group consisting of glass, silicon, metal oxides, and plastic materials. Preferably pyrex glass transparent to light, but is not limited to these examples. The membrane substrate 55 may use a polydimethylsiloxane (PDMS) substrate.

The measuring principle of each oxygen sensor 1 'is that dissolved oxygen in the cell solution passes through the membrane 50 to the internal electrolyte 20, and the transferred oxygen is reduced at the working electrode to which the negative potential is applied, and this reduction current By measuring the concentration of dissolved oxygen.

As shown in FIG. 2, the cell counter element 101 includes an electrode 110 and a channel substrate 120 on the second substrate 100. The passivation layer 115 may be further included around the electrode 110 to passivate the electrode unnecessarily exposed.

The second substrate 100 and the channel substrate 120 are formed with holes 60 ', 60 "to be used as the leisure bore 60 of the oxygen sensor 1' at positions corresponding to the respective membranes 50. As shown in FIG. 1, a cell injection hole 65 is formed in the channel substrate 120, and the processing is performed to define a channel 70 through which fluid flows between the second substrate 100 and the channel substrate 120. Thus, the channel 70 is formed by bonding with the second substrate 100 to allow the cell solution to flow onto each membrane 50.

In addition, like the first substrate 10, the second substrate 100 may be one or more materials selected from the group consisting of glass, silicon, metal oxides, and plastic materials, but is not limited thereto. The channel substrate 120 may also use a PDMS substrate.

The electrode 110 is formed in the cell injection port 65 part and the connection part channel 70 with the membrane 50 and is connected to the electrode pad 105 formed on the second substrate 100 as shown in FIG. 1. do. Accordingly, a cell counter 101 'is implemented, which measures the change in impedance (resistance) when the cell passes through the cell inlet 65 channel and the channel 70 leading to the oxygen sensor to count the number of cells.

As shown in FIG. 4, two or more channels 70 are divided and divided into the cell inlet 65, and each channel 70 is connected to each oxygen sensor 1 ′. When the sample cell solution is placed in the cell inlet 65, cells randomly flow through the channel 70 to each oxygen sensor 1 ′, and the amount of cells flowing into each channel 70 is measured by the cell counter 101. It is measured by '). By injecting different reagents into the leisure bore 60 of each oxygen sensor 1 ′, the oxygen respiration volume of the cells can be measured under different conditions, so that various measurements can be performed simultaneously before cell activity drops. The electrodes 110 of the cell counter 101 'have the same size and have the same spacing. In order to measure cells of various sizes, it is necessary to consider the size of the channel 70 and the area of the electrode suitable for the size of the channel 70. The movement of the fluid depends on the pressure pushed at the cell inlet 65. The sensor thus configured acts as a sensor in the lab on a chip concept.

As described above, the sensor according to the present invention is manufactured by bonding the oxygen sensor element 1 and the cell counter element 101 and then bonding them. The flow chart is as shown in FIG. 5, and FIGS. 6 to 11. Is a perspective view or a sectional view of an oxygen sensor element fabrication process, FIGS. 12 to 16 are perspective views or a sectional view of a cell counter element fabrication process, and FIGS. 17 and 18 are a perspective view and a sectional view of the sensor according to the present invention.

Referring to FIGS. 5 to 18, a sensor manufacturing method according to the present invention will be described.

Oxygen sensor element fabrication process (step S of FIG. 5)

The first substrate 10, for example, a pyrex glass 7740 substrate is cleaned (step S1 of FIG. 5). 6 is a perspective view of the first substrate 10. The washing method may be ultrasonic cleaning using acetone (5 minutes), ultrasonic cleaning using methanol (5 minutes) and deionized water washing (5 minutes).

Next, Ti / Au deposition and patterning are performed to fabricate an electrochemical electrode and, if necessary, a temperature sensor in the oxygen sensor 1 ′ (step S2 of FIG. 5).

E-beam vacuum evaporation or thermal evaporation (E-beam / thermal evaporation) equipment can be used for the deposition, and the thickness of Ti / Au can be 500Å / 2500Å. After Ti / Au deposition, by forming a photoresist pattern, wet etching the Ti / Au through the photoresist pattern, and removing the photoresist pattern, as shown in FIG. The counter electrode 31 and the working electrode 32 are formed among the 30, and the temperature sensor 34 is also formed to surround the 3-electrode 30. At this time, the surface area A of the working electrode 32 may be increased based on Equation 1 to increase the range of the output current.

Next, Ti / Ag deposition and patterning are performed to manufacture a reference electrode among the three electrodes 30 (step S3 of FIG. 5).

Electron beam vacuum deposition equipment can also be used for Ti / Ag deposition, and the thickness of Ti / Ag can be set to 500 mW / 2500 mW. After Ti / Ag deposition, a photoresist pattern is formed, the Ti / Ag is wet-etched through this, and the photoresist pattern is removed, thereby forming the reference electrode 33 as shown in FIG. 8.

Through the above process, the passivation layer 35 is formed on the first substrate 10 on which the 3-electrode 30 is formed (step S4 of FIG. 5). This is for the passivation between the electrode lead and the chamber 15 containing the internal electrolyte. 9 is a perspective view of the substrate in a passivated state. In particular, in this embodiment, since the working electrode 32 is patterned to have five blocks, the passivation layer 35 of this portion is also formed into five strips.

The passivation layer 35 may be formed by any method known in the art. For example, MICROCHEM, SU-8, a negative photoresist material commercially available from the United States, is spin-coated and then exposed to areas requiring passivation to deform the (chemical) bonds of the exposed areas and develop them.

Next, bonding of the first substrate 10 and the membrane substrate 55 is performed (step S5 of FIG. 5).

In this embodiment, a PDMS substrate is used as the membrane substrate 55. First, the PDMS membrane mold is patterned to form the membrane 50 at a predetermined position. At this time, the mold is patterned so that an injection hole 15a through which the electrolyte solution can be injected into the chamber 15 may be formed. The membrane 50 is manufactured using this PDMS membrane mold.

The first substrate 10 on which the three-electrode 30 is patterned and the membrane substrate 55 on which the membrane 50 is formed are subjected to oxygen plasma treatment and then bonded. FIG. 10 is a perspective view after bonding the first substrate 10 and the membrane substrate 55 and FIG. 11 is a cross-sectional view taken along line XI-XI ′ of FIG. 10. PDMS is simple because the adhesion process by inducing Si-O-Si covalent bond from -OH group formed through oxygen plasma treatment. In addition, since PDMS has a high oxygen transmission rate (P _m ), the range of the output current in Equation 1 can be increased.

Through the above process, the production of the oxygen sensor element 1 is completed.

Cell counter device fabrication process (step T of Fig. 5)

The second substrate 100, for example an acrylic substrate, is cleaned (step T1 in FIG. 5). The second substrate 100 has holes 60 ', as shown in perspective view in FIG. It also has an injection hole 15b corresponding to the injection hole 15a through which the electrolyte solution can be injected into the chamber 15. The hole 60 'and the injection hole 15b are formed using a laser processing technique or a drill. The washing method may use ultrasonic washing with methanol (1 min) and deionized water washing (5 min).

Next, Ti / Au deposition and patterning are performed (step T2 in FIG. 5).

Electron beam vacuum deposition equipment can be used for the deposition, and the thickness of Ti / Au may be 500 kW / 2500 kW. After Ti / Au deposition, a photoresist pattern is formed, the wet etching of Ti / Au is performed, and the photoresist pattern is removed, thereby forming the electrode pad 105 and the electrode 110 as shown in FIG. 13.

The passivation layer 115 such as polyimide is applied onto the second substrate 100 on which the electrode 110 is patterned (step T3 of FIG. 5). This is to passivate the electrodes unnecessarily exposed in the cell counter. 14 is a perspective view of the substrate in a passivated state. The white portion O opened in a circle is a part of the electrode 110 to be used.

Next, the second substrate 100 and the channel substrate 120 are bonded (step T4 in FIG. 5).

In this embodiment, a PDMS substrate is used as the channel substrate 120. First, the PDMS mold is patterned. Using this PDMS mold, there is a hole 60 "at a position corresponding to the hole 60 'of the second substrate 100, an injection hole 15c corresponding to the injection hole 15b, and a channel through which the cell solution can flow. A PDMS channel substrate 120 having the 70 formed thereon is fabricated, after 3-APTES (silane primer) treatment on the patterned second substrate 100, the second substrate 100 and the channel substrate ( Bonding is performed after oxygen plasma treatment 120. Fig. 15 is a perspective view after bonding the second substrate 100 and the channel substrate 120, and Fig. 16 is a cross-sectional view taken along line XVI-XVI 'of Fig. 15.

When bonding PDMS to PDMS, glass, and silicon, it is possible to reliably bond only by oxygen plasma treatment, but not to other types of polymers (acrylic) except PDMS, or have weak adhesive strength. Therefore, when the acrylic substrate is first surface-treated with 3-ATPES and then oxygen plasma treatment, -OH groups can be formed, thus enabling a firm bonding between the PDMS and the acrylic substrate.

Through the above process, the cell counter device 101 is manufactured.

Oxygen sensor element and cell counter element bonding process (step U of FIG. 5)

Oxygen plasma treatment is then performed to bond the oxygen sensor element 1 prepared through steps S1 to S5 and the cell counter element 101 manufactured through steps T1 to T4, followed by 3-APTES treatment. FIG. 17 is a perspective view of the oxygen sensor device and the cell counter device bonded together, and FIG. 18 is a cross-sectional view taken along line XVIII-XVIII ′ of FIG. 17.

And arranging the reservoirs 60 and the holes 60 ', 60 "corresponding to each other in the oxygen sensor element 1 and the cell counter element 101 before bonding. In the method of the present invention, the oxygen sensor Aligning the element 1 and the cell counter element 101 at a position corresponding to each other may be performed manually or by using an aligner.

Since the use of the sensor after filling the internal electrolyte 20 in the chamber 15 through the injection holes (15a, 15b, 15c), while injecting the cell solution into the cell solution inlet (65) while moving to each leisure boa (60) The number of cells moved to each leisure bore 60 through the cell counter 101 'is measured, and each oxygen sensor 1' is reacted with a cell solution by putting different kinds of reagents into each leisure boa 60. Simultaneously measure the oxygen respiration rate of cells.

In the above, the present invention has been described in detail with reference to preferred embodiments, but the present invention is not limited to the above embodiments, and various modifications are possible by those skilled in the art within the technical idea of the present invention. Is obvious. Embodiments of the invention have been considered in all respects as illustrative and not restrictive, which include the scope of the invention as indicated by the appended claims rather than the detailed description therein, the equivalents of the claims and all modifications within the means. I want to.

1 is a plan conceptual view of a sensor according to the present invention.

FIG. 2 is a cross-sectional view taken along line II-II 'of FIG. 1.

3 is an enlarged view of the oxygen sensor part B in FIG. 2.

4 is an enlarged view of the cell counter portion A in FIG. 1.

5 is a flow chart of sensor fabrication according to the present invention.

6 to 11 is a perspective view or a cross-sectional view of the oxygen sensor device manufacturing process according to the present invention.

12 to 16 are a perspective view or a cross-sectional view of the cell counter device manufacturing process according to the present invention.

17 and 18 are perspective and sectional views of the sensor according to the present invention.

<Explanation of symbols for the main parts of the drawings>

1 ... oxygen sensor element 1 '... oxygen sensor

10 ... 1st substrate 15 ... chamber

15a, 15b, 15c ... inlet 20 ... internal electrolyte

25 ... electrode pad 30 ... 3-electrode

31 ... relative electrode 32 ... working electrode

33.Reference electrode 34 ... Temperature sensor

35 Passivation layer 50 Membrane

55 membrane membrane 60 ...

60 ', 60 "... hole 65 ... cell solution inlet

70 ... channel 100 ... second substrate

101 ... cell counter element 101 '... cell counter

105 ... electrode pad 110 ... electrode

115 ... passivation layer 120 ... channel substrate

Claims (12)

An oxygen sensor for measuring the amount of dissolved oxygen in the cell solution is arranged one by one to a plurality of channels branched from one cell solution inlet for injecting a cell solution. Sensors are integrated in each of the channels of the. A first substrate; A membrane substrate having respective membranes bonded on the first substrate to define a plurality of chambers between the first substrate and selectively permeate oxygen on the chamber; And An oxygen sensor element including; an electrode formed on the first substrate in each chamber; A second substrate having a hole corresponding to each of the membranes; A hole is formed at a position corresponding to the hole of the second substrate, and a cell injection hole for injecting a cell solution is formed, and the cell solution is bonded on the second substrate to form the cell solution therebetween. A channel substrate defining a channel to be introduced onto each membrane; And And a cell counter element including electrodes formed on the cell injection port portion and the connection portion channel with the membrane, respectively. The sensor of claim 2, wherein the aperture of the second substrate and the aperture of the channel substrate form a reservoir to contain the cell solution over the membrane. Forming an electrode on the first substrate; Forming a membrane substrate bonded to the first substrate to define a plurality of chambers over the electrode with the first substrate and having respective membranes capable of selectively permeating oxygen thereon; And Fabricating an oxygen sensor element comprising bonding the first substrate and the membrane substrate; Forming an electrode on a second substrate having a hole corresponding to each of the membranes; A hole is formed at a position corresponding to the hole of the second substrate, and a cell injection hole for injecting a cell solution is formed, and the cell solution is bonded on the second substrate to form the cell solution therebetween. Forming a channel substrate to define a channel that can be introduced onto each membrane; And Fabricating a cell counter device comprising bonding the second substrate and the channel substrate; Stacking and bonding the oxygen sensor element and the cell counter element. The method of claim 4, wherein the electrode is formed of Ti / Pt, Ti / Au, or Ti / Ag. The method of claim 4, further comprising forming a passivation layer for passivation between the chamber and the electrode lead on the first substrate on which the electrode is formed. The method of claim 4, wherein the membrane substrate and the channel substrate use a polydimethylsiloxane (PDMS) substrate. 8. The method of claim 4 or 7, wherein the first substrate and the second substrate use one or more materials selected from the group consisting of glass, silicon, metal oxides and plastic materials. The method of claim 8, wherein the bonding of the first substrate and the membrane substrate, and the bonding of the second substrate and the channel substrate, comprise Si—O—Si covalent bonds from an —OH group formed by oxygen plasma treatment. Sensor manufacturing method characterized in that the adhesive by inducing. 10. The method of claim 9, further comprising a 3-APTES (silane primer) treatment prior to the oxygen plasma treatment. 5. The method of claim 4, further comprising forming a passivation layer to passivate the electrode unnecessarily exposed on the second substrate on which the electrode is formed. 5. The method of claim 4, further comprising aligning the oxygen sensor element and the cell counter element such that the aperture of the second substrate and the aperture of the channel substrate form a reservoir that can contain a cell solution over the membrane. Sensor manufacturing method characterized in that.

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