KR100845425B1 - Optical sensor probe and detection method using the same - Google Patents

Abstract

The present invention a) a sensor membrane is fixed a fluorescent dye on the surface; b) light emitting diodes emitting light of a constant wavelength; c) a photodiode for detecting emitted light; d) an optical fiber connecting the sensor film and the light emitting diode and the photodiode, or the sensor film and the photodiode, which pass light emitted by the light emitting diode or the fluorescent dye; e) an excitation filter positioned in front of the light emitting diode, which selectively transmits only excitation light which can be sensitive by a fluorescent dye among light emitted from the light emitting diode; And f) divergence filters located in front of the photodiode, which selectively transmit only fluorescence emitted by the fluorescent dye, including optical sensor probes and using them in-situ in a bioreactor. Method for detecting dissolved oxygen, dissolved carbon dioxide, pH, and the like.

Optical fiber, light emitting diode, sensor probe, in-situ, fluorescent dye

Description

Optical sensor probes and detection methods using the same}

1 is a view showing the structure of an optical sensor probe for detecting fluorescence using two optical fibers;

2 is a diagram showing the structure of an optical sensor probe for detecting fluorescence using one strand of optical fiber;

3 to 5 show the main body of the optical sensor probe;

6 is a view showing a sensor film prepared by spin coating a fluorescent dye on a quartz plate using a sol-gel method;

7 is a view showing a sensor film produced by coating a fluorescent dye on a quartz plate using silicon.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to optical sensor probes and a detection method using the same, and more particularly, using a fluorescent dye, a light emitting diode (LED) and an optical fiber for detecting dissolved oxygen, dissolved carbon dioxide or pH. The present invention relates to an optical sensor probe manufactured by using the same, and a method for detecting dissolved oxygen, dissolved carbon dioxide, or pH using the same.

In bioprocessing, dissolved oxygen, dissolved carbon dioxide and pH are closely related to microbial growth. Therefore, in recent years, technology development for monitoring dissolved oxygen, dissolved carbon dioxide or pH in the incubator has been actively made. In particular, the development of optical fiber carbon dioxide sensor, pH and dissolved oxygen sensor using optical detection technology. Optical analysis and online monitoring technology are simpler to construct and produce no electromagnetic noise than conventional electrochemical systems, and are particularly compact and easy to manufacture. Recently, a sol-gel technique in which a fluorescent dye capable of reacting dissolved oxygen, pH and carbon dioxide is immobilized has been studied. The sol-gel technology has the advantages of being simple, strong and resistant to other environmental factors compared to other immobilization techniques. In addition, because of the transparent crystals, the applicability of the optical detection system is excellent.

Fluorescent dyes capable of reacting dissolved oxygen, pH and carbon dioxide include ruthenium complex, HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt), and fluoresceamine (fluoreceinamine). Ruthenium-based complexes have fluorescence at specific wavelengths, typically RuDPP (tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) complex) has an excitation light of 470 nm. It has a characteristic of emitting 580 nm fluorescence when it is incident, and generates fluorescence in inverse proportion to the concentration of oxygen. In addition, HPTS is used as a fluorescent dye for detecting carbon dioxide, and fluorine amine is a substance used as a fluorescent dye for pH detection.

However, the optical fiber sensor developed so far is a plastic material, which cannot be applied to a bioreactor in-situ because it is impossible to sterilize at high temperature and pressure, and detects only one of dissolved oxygen, carbon dioxide, and pH. It could be monitored.

The inventors of the present invention can develop an optical sensor probe using a fluorescent dye that can be applied to a bioreactor in an in-situ manner and capable of detecting various analytes such as dissolved oxygen, carbon dioxide, and pH by being capable of high temperature and high pressure sterilization. We have been conducting continuous research. As a result, it was confirmed that the above object can be achieved by manufacturing an optical sensor probe using dissolved oxygen, dissolved carbon dioxide or a fluorescent dye for detecting pH, a light emitting diode, and an optical fiber, and have completed the present invention.

Accordingly, an object of the present invention is not only can be applied to the bioreactor in-situ method at high temperature and high pressure sterilization, but also can detect a variety of analytes such as dissolved oxygen, carbon dioxide, pH, etc. To provide a sensor probe.

Another object of the present invention is to provide a method for detecting various analytes in-situ in a bioreactor using the optical sensor probe.

The first aspect of the present invention

a) a sensor film in which a fluorescent dye is immobilized on the surface;

b) light emitting diodes emitting light of a constant wavelength;

c) a photodiode for detecting emitted light;

d) an optical fiber connecting the sensor film and the light emitting diode and the photodiode, or the sensor film and the photodiode, which pass light emitted by the light emitting diode or the fluorescent dye;

e) an excitation filter positioned in front of the light emitting diode, which selectively transmits only excitation light which can be sensitive by a fluorescent dye among light emitted from the light emitting diode; And

f) an emission filter located in front of the photodiode, which selectively transmits only fluorescence emitted by the fluorescent dye:

It relates to an optical sensor probe, including.

In the optical sensor probe according to the present invention, the optical fiber passes through the light emitted from the light emitting diode and connects the first optical fiber connecting the sensor film and the light emitting diode, and the light emitted from the fluorescent dye to connect the sensor film and the photodiode. It may be made of a second optical fiber, or may be made of one strand of optical fiber that passes the light emitted from the light emitting diode and the light emitted from the fluorescent dye and connects the sensor film and the photodiode. In the case of using one strand of optical fiber, the light emitting diode and the photodiode are positioned at right angles, and a dichroic filter which reflects the excitation light at 45 ° and transmits the incident light at 180 ° can be positioned in the middle. have. In addition, the fluorescent dye may be selected from the group consisting of ruthenium complex, HPTS and fluoresceamine, and may be immobilized on the surface of the sensor film using a sol-gel method or silicon. The optical sensor probe according to the present invention preferably further includes an amplifier for amplifying a signal coming into the outer container and / or photodiode of stainless steel, and the sensor film and the optical fiber may be detachable from the optical sensor probe. desirable.

The second aspect of the present invention relates to a method for detecting dissolved oxygen, dissolved carbon dioxide or pH in-situ in a bioreactor using the optical sensor probe.

Hereinafter, the present invention will be described in detail.

The optical sensor probe according to the invention comprises the following components:

a) a sensor film in which a fluorescent dye is immobilized on the surface;

b) light emitting diodes emitting light of a constant wavelength;

c) an excitation filter for selectively transmitting only excitation light that can be sensitive to the fluorescent dye among the light emitted from the light emitting diode;

d) a photodiode for detecting emitted light;

e) an emission filter for selectively transmitting only fluorescence emitted by the fluorescent dye; And

f) Optical fibers that pass light emitted by light emitting diodes or fluorescent dyes.

In the optical sensor probe according to the present invention, a quartz plate may be used as the sensor film, and the fluorescent dye may be immobilized on the surface of the quartz plate using the sol-gel method. In the case of the ruthenium complex used for oxygen detection or fluoresceamine used for pH detection, tetraethylorthosilicate and 3-glycidoxypropyl trimethoxysilane are first mixed and stored on nitrogen. The tetramethylammonium hydroxide solution is mixed with high purity ethanol and ice cooled, followed by mixing and stirring the solution to prepare a sol-gel solution. The ruthenium complex solution dissolved in high purity ethanol was mixed with the obtained sol-gel solution in a 1: 1 ratio, stirred, coated on the surface of the sensor film, and then air dried at room temperature and dried at 80 ° C. for immobilization. To immobilize the HPTS used for carbon dioxide detection, 3-aminopropyl-triethoxysilane, 3-glycidoxypropyl trimethoxysilane, and tetraethylorthosilicate and hydrophobic polymer hydride to diffuse only carbon dioxide in the air Roxy-terminated poly (dimethylsiloxane) is used. First mix tetraethylorthosilicate with 3-glycidoxypropyl trimethoxysilane, hydroxy-terminated poly (dimethylsiloxane) and 3-aminopropyl-triethoxysilane, 3-glycidoxypropyl trimethoxysilane Ice-cooled and stirred to prepare a sol-gel solution. Tetramethylammonium hydroxide is mixed with high purity ethanol, ice-cooled and stirred, then mixed with the sol-gel solution and stirred to prepare a sol-gel solution. HPTS dissolved in high purity ethanol is mixed with a sol-gel solution, stirred at room temperature, coated on the surface of the sensor membrane, and then dried and immobilized as described above. On the other hand, immobilization can also be performed using silicon, coating a solution of silicon and fluorescent dye dissolved in toluene on the surface of the sensor film and air dried at room temperature.

In the optical sensor probe according to the present invention, the optical fiber may be composed of a first optical fiber for passing the light emitted from the light emitting diode and a second optical fiber for passing the light emitted from the fluorescent dye (see FIG. 1). In addition, the optical fiber may be composed of one strand of optical fiber passing through the light emitted from the light emitting diode and the light emitted from the fluorescent dye, in which case the light emitting diode and the photodiode are positioned at a right angle, at a 45 ° in the middle A dichroic filter can be placed that reflects the excitation light and transmits incident light at 180 ° (see FIG. 2). In addition, the optical sensor probe according to the present invention preferably further includes an amplifier for amplifying the signal coming into the photodiode (see FIGS. 1 and 2). 3 to 5 show the main body of the optical sensor probe according to the present invention.

Sensor probe according to the present invention is preferably made of a stainless steel outer container, to be applicable in the general bioreactor. In addition, the sensor film and the optical fiber is detachable from the optical sensor probe, by separating the optical fiber portion to combine the probe only with the bioreactor to enable high temperature and high pressure sterilization, and by using the sensor film exchanged according to the analysis target It is more desirable to be able to detect and monitor various analytes such as dissolved oxygen, dissolved carbon dioxide, and pH.

Therefore, the optical sensor probe according to the present invention can be mounted on a bioreactor to monitor online the dissolved oxygen, dissolved carbon dioxide, and pH in the reactor in situ, and can be usefully used for various environmental monitoring and process monitoring. In addition, it is possible to monitor several analytes at the same time using a single sensor probe, and because the analysis using light can be very useful especially in the field of analysis sensitive to electromagnetic fields.

Hereinafter, the present invention will be described in detail by way of examples, which are intended to aid the understanding of the present invention but do not limit the scope of the present invention in any way.

Preparation Example 1 Immobilization of Ruthenium Complex and Fluoresceinamine by Sol-Gel Method

In order to immobilize the ruthenium complex or fluoresceamine on the surface of the quartz plate, 0.6 ml of tetraethylorthosilicate (98%) and 0.6 ml of 3-glycidoxypropyl trimethoxysilane (98%) were mixed, followed by nitrogen. Stored in the stomach. 0.4 ml of tetraethylammonium hydroxide (25%) solution was mixed with 1.5 ml of high purity ethanol, the solution was ice-cooled, mixed with the solution and stirred at room temperature for 3 hours to obtain a sol-gel solution. The obtained sol-gel solution and the ruthenium complex solution dissolved in high purity ethanol at a concentration of 5 mg / l were mixed in a 1: 1 ratio, then stirred at room temperature for one day, and the solution was coated on the quartz plate surface. The coated quartz plate was dried in air for 5 days at room temperature and then fixed for 2 days at 80 ° C.

Preparation Example 2 Immobilization of HPTS by Sol-Gel Method

In order to immobilize the HPTS on the surface of the quartz plate, 3-aminopropyl-triethoxysilane, 3-glycidoxypropyl trimethoxysilane and tetraethylorthosilicate and hydroxy- which is a hydrophobic polymer to diffuse only carbon dioxide in the air Terminal poly (dimethylsiloxanes) were used to form the sol-gel. First 0.6 ml tetraethylorthosilicate and 0.4 ml 3-glycidoxypropyl trimethoxysilane, 0.4 ml hydroxy-terminated poly (dimethylsiloxane) and 0.6 ml 3-aminopropyl-triethoxysilane, 3-glycidoxypropyl trimethoxysilane was mixed and stirred under ice-cooling to prepare a sol-gel solution. 0.4 ml of tetraethylammonium hydroxide (25%) was mixed with 1.5 ml of high purity ethanol and stirred with ice. The sol-gel solution was prepared by mixing with the sol-gel solution and stirring for 3 hours. 5 mg / L HPTS dissolved in high purity ethanol was mixed with the sol-gel solution, stirred at room temperature for one day, coated on the surface of the quartz plate, and dried and immobilized in the same manner as in Preparation Example 1 above.

Preparation Example 3 Immobilization of Ruthenium Complex, HPTS and Fluoresceinamine Using Silicon

The quartz plate surface was coated with a solution of silicon and fluorescent dye dissolved in toluene and immobilized by air drying at room temperature.

Example 1: Fabrication of an optical sensor probe consisting of two strands of optical fiber

A blue light emitting diode (blue LED) emitting light of 470 nm was used as a light source, and an optical band pass filter was placed in front of the LED to irradiate only excitation light that the fluorescent dye could respond to. In addition, an optical band pass filter was installed to selectively receive only light having a desired wavelength (fluorescence generated by a fluorescent dye) among the light incident on the detector (silicon photodiode). According to Preparation Examples 1 to 3, the fluorescent dye was immobilized on a quartz plate having a thickness of 2 mm and a diameter of 12 mm. In addition, the LED is connected to the electronic control unit to emit a constant amount of light from the LED, and a lock-in amplifier is connected to the photodiode to amplify the signal coming into the detector. Probes were fabricated.

Example 2: Fabrication of an optical sensor probe consisting of one strand of optical fiber

The light source and the detector are installed in the same manner as in Example 1, but in order to use a single optical fiber, the LED and the photodiode are positioned at right angles, and a dike that reflects the excitation light at 45 ° in the middle and transmits the incident light at 180 °. The roic filter was positioned to produce an optical sensor probe as shown in FIG. 2.

The optical sensor probe as described above may be mounted on a bioreactor to monitor dissolved oxygen, dissolved carbon dioxide, and pH in the reactor online, and may be useful for various environmental monitoring and process monitoring. In addition, it is possible to monitor several analytes at the same time using a single sensor probe, and because the analysis using light can be very useful especially in the field of analysis sensitive to electromagnetic fields.

Claims (9)

a) a sensor film in which a fluorescent dye is immobilized on the surface; b) light emitting diodes emitting light of a constant wavelength; c) a photodiode for detecting emitted light; d) an optical fiber connecting the sensor film and the light emitting diode and the photodiode, or the sensor film and the photodiode, which pass light emitted by the light emitting diode or the fluorescent dye; e) an excitation filter positioned in front of the light emitting diode, which selectively transmits only excitation light which can be sensitive by a fluorescent dye among light emitted from the light emitting diode; And f) an emission filter located in front of the photodiode, which selectively transmits only fluorescence emitted by the fluorescent dye: It includes, The fluorescent dye is fixed on the surface of the sensor film using a sol-gel (sol-gel) method or silicon, the optical sensor probe. The optical fiber of claim 1, wherein the optical fiber passes through the light emitted from the light emitting diode and connects the sensor film and the light emitting diode, and the second optical fiber passes through the light emitted from the fluorescent dye and connects the sensor film and the photodiode. Optical sensor probe consisting of. The optical fiber according to claim 1, wherein the optical fiber is composed of one strand of optical fiber passing through the light emitted from the light emitting diode and the light emitted from the fluorescent dye and connecting the sensor film and the photodiode, wherein the light emitting diode and the photodiode are positioned at right angles. And a dichroic filter reflecting the excitation light at 45 ° in the middle thereof and transmitting the incident light at 180 ° in the middle thereof. The optical sensor probe according to claim 1, wherein the fluorescent dye is selected from the group consisting of ruthenium complex, HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) and fluoresceamine. delete The optical sensor probe of claim 1 further comprising an outer container made of stainless steel. The optical sensor probe of claim 1 further comprising an amplifier for amplifying the signal coming into the photodiode. The optical sensor probe of claim 1, wherein the sensor film and the optical fiber are detachable from the optical sensor probe. A method for detecting dissolved oxygen, dissolved carbon dioxide, or pH in an in-situ manner in a bioreactor using an optical sensor probe according to any one of claims 1 to 4 and 6 to 8.

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