KR101002716B1 - Multi-layer sensing membranes, devices and methods for simultaneous detection of two or more parameters of dissolved oxygen concentration, pH and temperature - Google Patents

Abstract

The present invention relates to a method for producing a multilayer optical sensor film for the simultaneous detection of two or more variables of dissolved oxygen, pH and temperature, and more particularly, on the support resin layer, a sensor layer for temperature detection and a sensor layer for dissolved oxygen detection. And a high-sensitivity optical sensor film including a pH layer sensor layer in a stacked structure, which can be applied to an optical fiber sensor probe as well as a multi-well plate and used as a monitoring micro bioreactor.

Multi-layer, Simultaneous Detection, Optical Sensor Membrane, Sol-Gel, Fluorescent Dye

Description

Multi-layer sensing membranes, devices and methods for simultaneous detection of two or more parameters of dissolved oxygen concentration, pH and temperature }

The present invention relates to a method for producing a multilayer optical sensor film for the simultaneous detection of two or more variables of dissolved oxygen, pH and temperature, and more particularly, on the support resin layer, a sensor layer for temperature detection and a sensor layer for dissolved oxygen detection. And it relates to a method for producing a multilayer optical sensor film comprising a sensor layer for pH detection.

Dissolved oxygen, temperature and pH are important variables in environmental monitoring, marine research, food industry, biotechnology and medicine. Optical detection has advantages over other methods because non-invasive measurements can be made through the windows of bioreactors or reaction vessels. In this case, the sensing material is located on the inner wall of the reaction vessel, and the detection by reflection or fluorescence is performed outside the reaction vessel. In addition, the optical sensor has a merit that does not generate an electromagnetic field because it measures the signal using light instead of the conventional electronic device and also transmits the measurement information to the light. In particular, the development of optical dyes that selectively emit light according to a specific material (dissolved oxygen molecules, carbon dioxide molecules, etc.) or pH change has been used in various sensor fields. Such optical analysis is widely used in various fields because it has sufficient commercial elements such as time and cost savings. In recent years, small multi bioreactors have been actively developed by miniaturizing the incubator and using optical monitoring techniques. Small multiple bioreactors have the advantage of minimizing process development costs by enabling the development of low cost and short time requirements for the optimization of pharmaceutical and biological product production processes.

On the other hand, fluorescent dyes capable of reacting dissolved oxygen, pH and temperature include Rudpp (tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex) and HPTS (8-hydroxypyrene). -1,3,6-trisulfonic acid trisodium salt) and CdSeTe (cadimium selenium tellurium). Rudpp emits 600 nm of fluorescence when incident light of 480 nm is incident, and generates fluorescence in inverse proportion to the concentration of dissolved oxygen. In addition, HPTS has a characteristic of emitting 520 nm of fluorescence when incident light of 410 nm is incident, and shows a tendency of fluorescence intensity to increase with decreasing hydrogen ion concentration. In addition, CdSeTe (cadimium selenium tellurium) is a material used as a fluorescent dye for temperature detection.

Simultaneous measurement of two or more variables of dissolved oxygen, pH and temperature is required in complex cultures and processes. Moreover, knowing the temperature in optical detection of dissolved oxygen is particularly important because quenching by dissolved oxygen is greatly influenced by temperature. Recently, fluorescent materials for simultaneous optical detection of two variables have been reported. For example, Talanta 47 (1998) 1071-1076 describes two wavelengths separated from iron and pH by immobilizing an iron indicator, ferrozine, and a pH indicator, HPTS, in the vicinity of the same thin film. A technique for detecting by attenuated total reflection (ATR) spectroscopy has been reported. Chem. Mater. 2006, 18, 4609-4616] reported a sensor that can simultaneously detect pH and dissolved oxygen through a single-fiber optical sensor using carboxyfluorescein as a pH probe and Rudpp as a dissolved oxygen probe. . However, they have not disclosed any of the sensor films in which Rudpp, HPTS, and CdSeTe fluorescent dyes are made of the respective sensor layers.

Accordingly, the present invention was invented in view of the above problems, and the present invention provides a sensor layer for temperature detection in which the conditions of the sensor are optimized on the support resin layer for simultaneous detection of two or more variables of dissolved oxygen, pH, and temperature. To provide a method of manufacturing a multilayer optical sensor film comprising a sensor layer for detecting dissolved oxygen and a sensor layer for detecting pH.

Another object of the present invention is to provide an optical detection device including the multilayer optical sensor film.

The present invention is a sensor layer for detecting the temperature is fixed CdSeTe (cadimium selenium tellurium) fluorescent dye on the support resin layer, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) Sensor layer for detection of dissolved oxygen and fluorescence of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) An object of the present invention is to provide a method for manufacturing a multilayer optical sensor film including a sensor layer for pH detection in which a dye is immobilized.

The present invention provides an apparatus for simultaneously detecting two or more variables of dissolved oxygen, pH and temperature comprising the optical sensor film.

Hereinafter, the present invention will be described in more detail.

The present invention,

a) preparing a temperature detection sensor layer on which a CdSeTe (cadimium selenium tellurium) fluorescent dye is fixed;

b) preparing a sensor layer for detecting dissolved oxygen having a tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) fluorescent dye fixed on the temperature detecting sensor layer; And

c) 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) fluorescent dye on the prepared sensor layer for detecting dissolved oxygen Preparing a sensor layer for fixed pH detection;

To provide a multi-layer optical sensor film manufacturing method comprising a.

The present invention is characterized by fabricating a sensor film by fixing a sensor layer having a different function on a support layer, unlike the prior art, for simultaneous optical detection of two or more variables of dissolved oxygen concentration, pH and temperature.

The present invention provides for simultaneous optical detection of two or more variables of dissolved oxygen concentration, pH and temperature, CdSeTe, Tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp), respectively. , 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, HPTS) is characterized by using a fluorescent dye.

The fluorescent dye is 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, phenyltrimethoxysilane, vinyltrimethoxysilane, 3- (trimethoxysilyl) propyl methacrylate, methyl Trimethoxysilane, tetramethoxysilane, dimethyldimethoxysilane, tetraethoxysilane, methyltriethoxysilane, dimethyldiethoxysilane, phenyltrimethoxysilane, diphenyldimethoxysilane, phenyltriethoxysilane, di Phenyl diethoxysilane, decyltrimethoxysilane, isobutyltrimethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, glycidoxyoxytrimethoxysilane, mercaptopropyltrimethoxysilane It is preferable to immobilize using one or more selected silane coupling agents, and CdSeTe (cadimium selenium tellurium) and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (8-hydroxypyrene-1,3 , 6-trisulf onic acid trisodium salt (HPTS) fluorescent dyes may be fixed using a sol-gel comprising 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, or phenyltrimethoxysilane. desirable. In addition, the fixation of the tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) fluorescent dye is more preferably fixed using a sol-gel containing methyltrimethoxysilane. desirable.

In the present invention, these three fluorescent dyes can be used without limitation conventional methods known in the art to immobilize on the support, for example, sol-gel (sol-gel) solution, hydrogel (hydrogel) , Cellulose or derivatives thereof, for example alkyl cellulose, in particular C _1-6 alkyl cellulose, more particularly ethyl cellulose, and preferably sol-gel solutions. Immobilization of fluorescent dyes using a sol-gel solution is well known in the art, and may be performed according to a conventionally known technique.

In the present invention, Rudpp and HPTS are used by adsorbing at least one adsorbent and CdSeTe (cadimium selenium tellurium) is used without adsorption. The adsorbent may be used arbitrarily selected from those known in the art, for example, silica gel, organically-modified silicate (eg, Ormosil beads), poly (styrene-block) Poly (styrene-block-vinylpyrrolidone) beads (Poly) can be used. Preferably, Rudpp and HPTS use silica gel. This is because the adsorption of the fluorescent dyes can be used to prevent the shift of the fluorescent peaks and to effectively detect the dyes.

In the present invention, any of conventional ones known in the art may be used as a support for fabricating the multilayer optical sensor film, but it is more preferable to use a transparent polymer such as a quartz glass plate, a glass plate, a polystyrene or a microtiter plate. Do.

First, the preparation of a temperature detecting sensor layer in which a CdSeTe (cadimium selenium tellurium) fluorescent dye is fixed on a support is as follows. Hydroxylic acid may be used as the acid catalyst for the condensation reaction of 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane as a sol-gel forming material, acetone as a solvent and sol-gel. For example, 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, acetone, water and 30 to 40% by weight hydrochloric acid may be added from 1 to 5: 1 to 3: 3 to 8: 3 to 15 It is preferable to use a sol-gel solution containing in a volume ratio of 1: 1 because this can increase the immobilization efficiency of the mixed fluorescent dye. Specifically, 1.25 ml of 3-glycidoxypropyltrimethoxysilane, 625 µl of 3-aminopropyltrimethoxysilane, 2.708 ml of acetone, 5.417 ml of water and 400 µl of 35% by weight hydrochloric acid were added for 2 to 5 hours, especially 3 After aging with stirring for a period of time, fluorescent dyes are added thereto and sonicated for 5 to 20 minutes, in particular 10 minutes, to prepare a homogeneous solution. The mixed fluorescent dye solution thus obtained is thinned by using a handle portion which can be rotated to spread the fluorescent dye evenly when coating the mixed fluorescent dye solution thus obtained, and a one-well knife coating to be thinly coated on the support. After coating, the film was dried at room temperature for 12 to 48 hours, in particular for 24 hours, and then dried at 50 to 70 ° C., particularly at 60 ° C., to produce a sensor layer for temperature detection.

In the next step, the dissolved oxygen detection sensor layer having the tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) fluorescent dye fixed on the prepared temperature detection sensor layer was Manufacture. The sensor layer for detecting dissolved oxygen may use methyl trimethoxy silane (MTMS) as a sol-gel forming material, hydrochloric acid as an acid catalyst for condensation of acetone and sol-gel as a solvent. For example, it is preferable to use a sol-gel solution containing methyltrimethoxysilane, acetone, water and 30 to 40% by weight hydrochloric acid in a volume ratio of 200 to 250: 80 to 120: 30 to 50: 1. This is because the immobilization efficiency of the mixed fluorescent dye can be improved. Specifically, a sol-gel solution containing 3.97 ml of methyltrimethoxysilane, 1.84 ml of acetone, 0.72 ml of water and 18 µl of 35% by weight hydrochloric acid can be used.

In a specific example of the present invention, 3.97 ml of methyltrimethoxysilane, 1.84 ml of acetone, 0.72 ml of distilled water and 18 µl of 35 wt% hydrochloric acid are aged by stirring for 2 to 5 hours, especially 3 hours, and then fluorescent dyes are added thereto. And sonicate for 5 to 20 minutes, in particular 10 minutes, to produce a homogeneous solution. The mixed fluorescent dye solution thus obtained was thinly coated using a one-well knife coating, and then dried at room temperature for 12 to 48 hours, especially 24 hours, and then dried at 50 to 70 ° C, especially 60 ° C, to detect dissolved oxygen. Produce a sensor layer.

Finally, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) on the prepared dissolved oxygen detection sensor layer. Sensor layer for pH detection in which the fluorescent dye is fixed is 3-glycidoxypropyltrimethoxysilane as a sol-gel forming material, 3-aminopropyltrimethoxysilane, acetone as a solvent, and an acid for condensation of sol-gel. Hydrochloric acid can be used as a catalyst. For example, 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, acetone, water and 30 to 40% by weight hydrochloric acid may be added from 1 to 5: 1 to 3: 3 to 8: 3 to 15 It is preferable to use a sol-gel solution containing in a volume ratio of 1: 1 because this can increase the immobilization efficiency of the mixed fluorescent dye. Specifically, 1.25 ml of 3-glycidoxypropyltrimethoxysilane, 625, 3-aminopropyltrimethoxysilane 625, 2.708 ml of water, 5.417 ml of water and 400 µl of 35% by weight hydrochloric acid were added for 2 to 5 hours, especially 3 hours. After aging by stirring for a while, fluorescent dyes are added thereto and sonicated for 5 to 20 minutes, in particular for 10 minutes, to prepare a homogeneous solution. The mixed fluorescent dye solution thus obtained is applied to each well of a support, such as a quartz glass plate or a multi-well (eg 24-well) microtiter plate, and then 15 to 30 ° C., especially at room temperature 12 to 48 hours, in particular After drying for 24 hours, it is dried at 50 to 70 ℃, especially 60 ℃ to produce a multilayer optical sensor film.

The structure of the multilayer is preferably a temperature detecting sensor layer, a dissolved oxygen detecting sensor layer, and a pH detecting sensor layer, but the positional change between the sensor layers is not particularly limited.

The multilayer optical sensor film according to the present invention may be applied to an optical fiber probe and used as an optical fiber sensor, and the multi-well microtiter plate coated with the optical sensor film may be usefully used as a multichannel micro bioreactor for monitoring.

In the multilayer optical sensor film according to the present invention, since each sensor layer constitutes a sensor film, fluorescent dyes in the sensor film do not interfere with each other, so that a wide measurement range can be achieved even when simultaneous detection of two or more variables of dissolved oxygen, pH, and temperature is performed. It has the advantage of high accuracy and precision.

The multilayer optical sensor film according to the present invention can be applied to a fiber optic sensor probe and mounted in a bioreactor, which can be usefully used for on-line monitoring of dissolved oxygen, pH and temperature in a reactor. It can be monitored and useful in the process.

In addition, by applying to a multi-well microtiter plate to manufacture a multi-channel multi-well micro bioreactor for monitoring, non-invasive method can optimize the various conditions on the sensor membrane, thereby optimizing the production process cost Savings and time savings can minimize process development costs.

Hereinafter, the present invention will be described in detail by way of example, but it is only for better understanding of the present invention and does not limit the scope of the present invention in any way.

Preparation Example 1 Preparation of Tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) Complex (Rudpp) Immobilized Sol-gel (MTMS sol-gel)

MTMS was used to form the sol-gel, 99% acetone was used as the solvent, and 35% HCl was used as the catalyst. MTMS: Acetone: Water: HCl = 3.97 mL: 1.84 mL: 0.72 mL: 18 After mixing at a ratio of μl and vigorously stirring for 3 hours to form a sol-gel.

Preparation Example 2 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) immobilized sol-gel (GA sol-gel ) Produce

GPTMS and APTMS were used to form the sol-gel, 99% acetone was used as the solvent, and 35% HCl was used as the catalyst. GPTMS: APTMS: Acetone: Water: HCl = 1.25 mL: 625 μL: 2.708 mL: 5.417 mL: 400 μL was mixed at vigorous stirring for 3 hours to form a sol-gel.

Preparation Example 3 Preparation of CdSeTe Immobilized Sol-Gel (GA sol-gel)

GPTMS and APTMS were used to form the sol-gel, 99% acetone was used as the solvent, and 35% HCl was used as the catalyst. GPTMS: APTMS: Acetone: Water: HCl = 1.25 mL: 625 μL: 2.708 mL: 5.417 mL: 400 μL was mixed at vigorous stirring for 3 hours to form a sol-gel.

Comparative Example 1 Preparation of Optical Sensor Film for Detection of Dissolved Oxygen and pH

In order to prepare a sensor film for detecting dissolved oxygen and pH, a sensor layer solution was prepared by mixing Rudpp 5mg / mL in the MTMS sol-gel solution obtained in Preparation Example 1. At this time, Rudpp was used by adsorbing on silica gel. That is, 0.6 g of Rudpp was added to 12 ml of chloroform (CHCl ₃ ), 2 g of silica gel (4 μm, Dongyang Steel Chemical) was added and stirred for 24 hours, which was filtered through a filter paper and washed three times with tertiary distilled water. After drying it was used. 20 μl of Rudpp sol-gel solution prepared on the polystyrene surface was dropped and then coated with a one-well knife coating to allow a thin coating on the support, followed by drying in air for a day, and then smoothing the surface of the sensor layer. In order to dry for one day at 60 ℃ to form a sensor layer for detecting dissolved oxygen. After preparing a sensor layer solution by mixing 5.2 mg / mL of HPTS in the GA sol-gel solution prepared in Preparation Example 2, 20 μl of HPTS sol-gel solution was dropped on a completely dried oxygen detection membrane, followed by a coater (one After coating a thin layer using a -well knife coating, the layer was dried for one day in air and dried at 60 ° C for one day to smooth the surface of the sensor layer, thereby completing a multilayer optical detection layer for detecting dissolved oxygen and pH.

Comparative Example 2 Preparation of Composite Optical Sensor Membrane for Detection of Dissolved Oxygen, pH and Temperature

In order to prepare dissolved oxygen and a sensor film for pH and temperature detection, Rudpp 5 mg / mL was mixed with the MTMS sol-gel solution obtained in Preparation Example 1 to prepare a sensor membrane solution. In order to prepare a sensor layer for detecting dissolved oxygen and pH, a sensor layer solution was prepared by mixing Rudpp 5mg / mL in the MTMS sol-gel solution obtained in Preparation Example 1. At this time, Rudpp was used by adsorbing on silica gel. That is, 0.6 g of Rudpp was added to 12 ml of chloroform (CHCl ₃ ), 2 g of silica gel (4 μm, Dongyang Steel Chemical) was added and stirred for 24 hours, which was filtered through a filter paper and washed three times with tertiary distilled water. After drying it was used. 20 μl of Rudpp sol-gel solution prepared on the polystyrene surface was dropped and then coated with a one-well knife coating to allow a thin coating on the support, followed by drying in air for a day, and then smoothing the surface of the sensor layer. In order to dry for one day at 60 ℃ to form a sensor layer for detecting dissolved oxygen. After preparing a sensor layer solution by mixing 5.2 mg / mL of HPTS adsorbed on silica gel and 2 mg / mL of CdSeTe in the GA sol-gel solution prepared in Preparation Examples 2 and 3, the HPTS sol- on the completely dried dissolved oxygen detection layer. 20 μl of the gel solution is then thinly coated using a one-well knife coating on a support, followed by one day drying in air and one day at 60 ° C. to smooth the surface of the sensor layer. A composite optical sensor film for detecting dissolved oxygen, pH and temperature was completed. The CdSeTe was heated to 150 ° C. by adding 32 mg of CdO, 1.5 mg of Hexa, and 3.0 g of TOPO under vacuum, and then heated at 320 ° C. until nitrogen was completely dissolved in a reaction vessel. Se and Te dissolved in TOP (Trioctylphosphine) were added to the reaction vessel and reacted at 320 ° C. for 18 seconds to use the synthesized CdSeTe for temperature detection.

Example 1 Preparation of Multi-layer Optical Sensor Film for Dissolved Oxygen, pH and Temperature Detection

In order to prepare a multilayer optical sensor film for detecting dissolved oxygen, pH and temperature, a sensor layer solution was prepared by mixing CdSeTe 1 mg / mL with the GA sol-gel solution obtained in Preparation Example 3. 20 μl of CdSeTe sol-gel solution was dropped on the polystyrene surface, and then thinly coated using a one-well knife coating to allow a thin coating on the support. It dried at 60 degreeC for one day, and formed the sensor film for temperature detection. 20μl of a sensor layer solution prepared by mixing Rudpp 5mg / mL in MTMS sol-gel on a completely dried temperature detection sensor layer was thinned by using a one-well knife coating to make a thin coating on a support. After coating and drying in the air for one day, it was dried for one day at 60 ℃ to smooth the surface of the sensor layer. In the same way, 20 μl of a solution layer of HPTS 5.2mg / mL mixed with a GA sol-gel solution was applied, and then thinly coated using a one-well knife coating. In order to smooth the surface, it was dried at 60 ° C. for one day to complete the multilayer optical sensor film for detecting dissolved oxygen, pH and temperature.

Experimental Example 1 Confirmation of Simultaneous Detection of Dissolved Oxygen, pH and Temperature of Two or More Variables

The detection possibility of the multilayer optical sensor film having a laminated structure of the dissolved oxygen detection film and the pH detection film was investigated. The fluorescence intensity measured at the excitation wavelength of 480 nm and the emission wavelength of 600 nm at the dissolved oxygen concentrations of 100% and 0% using tertiary distilled water and an aqueous sodium sulfate solution (40 g / L) is shown in FIG. 2.

As can be seen in FIG. 2, the fluorescence intensity was low when the dissolved oxygen concentration was high, and the fluorescence intensity was high when the dissolved oxygen concentration was low. In addition, in order to investigate the change in fluorescence characteristics in accordance with pH in detail, the fluorescence intensity was measured with phosphate buffer solution (pH 3 to pH 9) of different pH. In FIG. 2, the measured fluorescence intensity increased with increasing pH, and it was confirmed that simultaneous detection of dissolved oxygen concentration and pH was possible with a multilayer sensor membrane composed of 5 mg / mL Rudpp and 5.2 mg / mL HPTS.

Figure 1 shows a cross section of a multilayer optical sensor film immobilized in the sol-gel solution of the present invention.

2 is a graph showing the results of simultaneous detection of dissolved oxygen and pH of a multilayer optical sensor film according to the present invention.

Figure 3 is a graph showing the results of real-time monitoring of the simultaneous detection of dissolved oxygen and pH in the monitoring microbioreactor fabricated on a 24-well microtiter plate of the multilayer optical sensor film according to the present invention.

* Explanation of symbols for the main parts of the drawings

10: sensor layer for pH detection

20: sensor layer for detecting dissolved oxygen

30: sensor layer for temperature detection

40: support resin layer

Claims (9)

delete In the manufacturing method of the multilayer optical sensor film, a) preparing a temperature detection sensor layer on which a CdSeTe (cadimium selenium tellurium) fluorescent dye is fixed; b) preparing a sensor layer for detecting dissolved oxygen having a tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) fluorescent dye fixed on the temperature detecting sensor layer; And c) 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) fluorescent dye on the prepared sensor layer for detecting dissolved oxygen A method for manufacturing a multilayer optical sensor film, the method comprising: manufacturing a fixed pH detection sensor layer. 3. The method of claim 2, CdSeTe (cadimium selenium tellurium) in step a) and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt in step c), HPTS) fluorescent dyes are 3-glycidoxypropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, phenyltrimethoxysilane, vinyltrimethoxysilane, 3- (trimethoxysilyl) propyl methacrylate A method of manufacturing a multilayer optical sensor film, characterized in that the immobilization is carried out using at least one silane coupling agent selected from the group consisting of: 3. The method of claim 2, The tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) fluorescent dye of step b) is methyltrimethoxysilane, tetramethoxysilane, dimethyldimethoxysilane, tetra Ethoxysilane, methyltriethoxysilane, dimethyldiethoxysilane, phenyltrimethoxysilane, diphenyldimethoxysilane, phenyltriethoxysilane, diphenyldiethoxysilane, decyltrimethoxysilane, isobutyltrimethoxy Immobilized by using at least one silane coupling agent selected from the group consisting of silane, vinyltrimethoxysilane, vinyltriethoxysilane, glycidoxyoxypropyltrimethoxysilane and mercaptopropyltrimethoxysilane. Method for producing a multilayer optical sensor film. The method according to claim 3 or 4, The immobilization method of manufacturing a multilayer optical sensor film, characterized in that the immobilization using a sol-gel (hydro-gel), hydrogel (hydrogel), cellulose or a derivative thereof. 3. The method of claim 2, The support is a method of manufacturing an optical sensor film, characterized in that the quartz glass plate, glass plate, transparent polymer or multi-well microtiter plate. CdSeTe (cadimium selenium tellurium) fluorescent dye fixed temperature sensor layer; A sensor layer for detecting dissolved oxygen to which a tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) fluorescent dye is fixed; And 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, HPTS) is a sensor layer for detecting the pH of the fluorescent dye fixed; comprising Multilayer optical sensor film. The method of claim 7, wherein The multilayer optical sensor film is a sensor layer for detecting the temperature of the CdSeTe (cadimium selenium tellurium) fluorescent dye is fixed on the support; A sensor layer for detecting dissolved oxygen having a tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex (Rudpp) fluorescent dye fixed on the temperature detecting sensor layer; And PH of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) fluorescent dye fixed on the sensor film for detecting dissolved oxygen Detection sensor layer; multilayer optical sensor film comprising a. An apparatus for simultaneously detecting two or more variables of dissolved oxygen, pH and temperature comprising an optical sensor film according to claim 7.

Images