JPH07110330A - Optical sensor - Google Patents

Abstract

PURPOSE:To enable the measurement of a substance to be performed in the state where one end of an optical fiber is immersed into a solution by a method wherein a porous shielding thin film is fixed on the tip face at the one end thereof through an antigen-antibody reaction layer and is immersed into the solution containing the substance to be measured while the other end thereof is put outside the solution. CONSTITUTION:Porous black thin films 3 and 4 are laminated on the tip face 2 of an optical fiber bundle 1 and fastened with an O ring 5. The tip face 2 is immersed into an antigen or antibody solution previously to absorb the antigen or antibody physically to make an antigen/antibody reaction layer thereon. The tip part of the fiber bundle 1 is inserted into a solution containing a substance 7 to be measured such as antigen or antibody with the thin films being immersed therein. As a result, the substance 7 is immobilized specifically on the tip face 2 passing through the thin films 3 and 4. So, a measuring person can observe emission or fluorescence from the substance 7 on the tip face 2 thereof from the other end face of the fiber bundle 1 while the end face 2 is immersed into the solution. The thin films 3 and 4 are black so as not to allow the transmission of light from the side of the solution 8 and the admission of the reflected light thereof thereby preventing effect of extraneous light.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is an optical device for capturing an unknown substance to be measured contained in a solution on a solid surface in contact with the solution and specifically fixing and quantifying the luminescence or fluorescence phenomenon of the substance. Regarding the sensor.

[0002]

2. Description of the Related Art Conventionally, as such an optical sensor,
There is one disclosed in JP-A-58-221167.
This optical sensor is a fluorescent immunoassay element (optical sensor) for optically measuring various antigens or antibodies by a fluorescent immunoassay method, and as disclosed in the publication, a transparent support (1 ) Is provided with a thin film (3) having light-shielding properties and porosity on one end surface via the detection layer (2), and further, an antigen-antibody reaction layer (4) is laminated on the back surface of the thin film (3). It has a four-layer structure. Then, for example, if the labeled antigen is added to the measurement target such as blood, then the measurement target containing the labeled antigen is subjected to a treatment such as wetting from the antigen-antibody reaction layer (4) side to measure the fluorescence. Since it is possible to measure the amount of unreacted labeled antigen from the transparent support (1) side and the amount of labeled antigen reacted from the antigen-antibody reaction layer (4) side, respectively, in the fluorescent immunoassay method. It is said to be an effective optical sensor.

[0003]

It can be said that the conventional optical sensor having the above structure is excellent in that it can easily measure an antigen or an antibody.

However, in recent years, in such a measurement field, it has been desired to rapidly perform a large amount of analysis processing, and an optical sensor having a structure as in a conventional example is used in combination with such a computer system. It was difficult to do. That is, in the optical sensor as in the conventional example, the operator cannot measure when the optical sensor is still immersed in the solution, and the optical sensor is once taken out of the solution. You can only measure from the beginning.
This causes the same complicated problem even when it is assembled in a computer system or the like, and in particular, real-time measurement while the optical sensor is immersed in the solution is extremely difficult.

The present invention has been made in view of the above problems, and it is possible to measure an unknown substance to be measured even in a state of being immersed in a solution, and to measure using a computer system or the like. And it aims at providing the optical sensor suitable for implementing an analysis process.

[0006]

In order to achieve such an object, the present invention is an arbitrary method in which one end side is immersed in a solution containing a substance to be measured and the other end side is outside the solution during measurement. A light guide member having a light-transmitting property such as an optical fiber having a length of, an antigen-antibody reaction layer having an antibody or an antigen immobilized on the end face of the one end of the light-transmitting light guide member, and the antigen A thin film having a porous property and a shielding property, which is fixed to the light guide member having a light transmitting property through an antibody reaction layer, is provided.

Further, an optical connector is provided at the other end of the light guide member having the light transmitting property, and is connected to an optical connector of an optical mechanism for detecting light provided in a computer system or the like.

[0008]

In the present invention having such a configuration, when one end of the light-transmitting light guide member on the side where the thin film is provided is immersed in the solution, the substance to be measured passes through the porous thin film. And reaches the tip end surface of the light guide member having light transmissivity and adheres thereto. Then, since the emission or fluorescence from the adhered substance to be measured can be observed from the other end surface of the light guide member having light transparency (the end surface located outside the solution), one end of the light guide member having light transparency is observed. It becomes possible to measure in real time while being immersed in the solution. In addition, the porous thin film allows the passage of the substance to be measured due to its innumerable pores due to its structural characteristics, but these innumerable pores have an extremely complicated and irregular shape and therefore do not transmit light. It is possible to optically measure only the measurement substance that is not allowed and adheres to the other end surface of the light guide member having light transmittance. Further, in the case of measuring the fluorescence of the substance to be measured, it becomes possible to inject light from the other end surface (the end surface located outside the solution) of the light guide member having light transmittance. Further, by connecting to a computer system or the like through an optical connector provided on the other end surface of the light guide member having light transmittance, a wide range of applications are possible.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment according to the present invention will be described below with reference to the drawings. First, the structure will be described with reference to FIGS. 1 and 2.
A porous thin black film (black membrane filter) is provided on the tip surface 2 of a light guide member (hereinafter referred to as an optical fiber bundle in the description of the embodiments) 1 in which a large number of extremely thin optical fibers are closely packed and bundled. ) 3 and 4 are fixed in a laminated state. By attaching the O-ring 5 to the peripheral wall of the tip of the optical fiber bundle 1, the thin films 3 and 4 are attached to the optical fiber bundle 1.
Is fixed to the tip surface 2 of the.

Further, the optical sensor having such a structure is formed in the next step. First, as shown in FIG. 2, the optical fiber bundle 1
The tip surface 2 is immersed in the antigen or antibody solution 6 to physically adsorb the antigen or antibody to the tip surface 2. Alternatively, the tip surface 2 is previously silanized and then immersed in the antigen or antibody solution 6 to chemically bond the antigen or antibody to the tip surface 2. As a result, an extremely thin antigen-antibody reaction layer is formed on the tip surface 2. Next, the O-ring 5 is attached with the thin films 3 and 4 in close contact with the tip surface 2.

Next, an example of the usage of such an optical sensor will be described with reference to FIGS. 3 and 4. First, as shown in FIG. 3, the tip of the optical fiber bundle 1 is inserted so that the thin films 3 and 4 are immersed in a solution (for example, blood or serum) 8 containing a measurement substance 7 such as an antigen or an antibody. . Solution 8
When immersed in, the measurement substance 7 is specifically fixed to the tip surface 2 through the pores of the thin films 3 and 4 due to the porosity of the thin films 3 and 4.

Then, when the measurer looks at the other end of the optical fiber bundle 1, the other end face of the measured substance 7 attached to the tip face 2 is measured.
The emission or fluorescence from can be observed. Furthermore,
It is possible to observe the tip portion of such an optical sensor even in a state where it is immersed in the solution 8 as shown in the drawing. Further, the porous thin films 3 and 4 allow the passage of the substance to be measured 7 through the innumerable holes due to their structural characteristics, but these innumerable holes are extremely complicated and irregularly shaped, Furthermore, since the thin films 3 and 4 of this embodiment are black, they do not allow the transmission of light or the incidence of reflected light from the solution 8 side. Therefore, it is possible to clearly observe the light emission or fluorescence from only the measurement substance 7 attached to the tip surface 2 without being affected by the external light.

Next, the case of mounting on a computer system or the like having a signal processing function and performing mechanical measurement will be described with reference to FIG. 4. In this case, in this case, an optical input device provided on the computer system or the like is used. The optical mechanism and the other end of the optical fiber bundle 1 are connected by an optical connector 9.

In this optical mechanism, the portion 10a connected by the optical connector 9 is composed of a single optical fiber bundle facing the other end surface of the optical fiber bundle 1, and the optical guide path is branched from the middle into two parts. A light guiding optical cable (light guide) 10 having the optical fiber bundles 10b and 10c, and an optical filter 11 provided on an end surface of the optical fiber bundle 10b.
a, 11b, 11c and an optical lens 11d, an optical system 12 having optical filters 12a, 12b and an optical lens 12c provided on the end face of the optical fiber bundle 10c, and an optical system 12 provided behind the optical system 12. A photo detector (PMT) 13 is provided.

When light from an excitation light source (not shown) such as a halogen lamp is made incident on the optical system 11, light having a specific wavelength selected by the optical filters 11a to 11c is transmitted through the optical fiber bundle 10b to the optical fiber. Sent to bundle 1,
On the other hand, the light emission or fluorescence from the measurement substance 7 is sent to the optical system 12 via the optical fiber bundle 1 and the optical fiber bundle 10c, and the light of the specific wavelength selected by the optical system 12 is imaged by the photodetector 13. To be done. Then, the detection signal output from the photodetection device 13 is transferred to a signal processing device such as a computer system for signal analysis and the analysis result is displayed on a monitor (not shown). .
The solution 7 is put in a container 15 placed on a stirring device 14 and stirred.

According to such a configuration, real-time measurement is realized, and since the analysis processing is performed by the computer system, various types of analysis can be performed at high speed and rationally.

Next, a specific example in which the apparatus shown in FIG. 4 is applied will be described.

A halogen lamp was used as the excitation light source, and light of about 150 W was made incident on the optical system 11. A sharp cut filter for selecting 440 nm light was applied to the optical filters 11a and 11b, and a 460 nm bandpass filter was applied to the optical filter 11c. On the other hand, the optical filter 1
A sharp cut filter for selecting light of 500 nm is applied to 2a, a bandpass filter of 520 nm is applied to the optical filter 12b, and a photomultiplier tube (PMT) having a bialkali photocathode is applied to the photodetector 13.

A plastic optical fiber is used for the optical fiber bundle 1. First, as described with reference to FIG. 2, an anti-insulin antibody {porcine insulin antiserum (made by Shiba Goat) is applied to the front surface 2 of the optical fiber bundle 1 by protein A Affinity chromatography "protein A affinity chromatograf
y ”(using Amersham resin) and insulin sepharose affinity chromatography“ insulinsep ”
which was purified by "harose affinity chromatography" (using Sigma resin)} was physically adsorbed.

That is, pH 7.0, 2 μg / ml of 0.1
The tip surface 2 of the optical fiber bundle 1 is immersed for 12 hours or more in a solution of the M phosphate buffer solution containing the anti-insulin antibody at 4 ° C. The anti-insulin antibody was physically adsorbed on the. As a result, an extremely thin antigen-antibody reaction layer is formed on the tip surface 2. In addition, when storing after that, the tip surface 2
In a solvent of 0.1M phosphate buffer (pH 7.)
Store at 4 ° C or below in a solution containing 2% sodium azide.

Alternatively, when a glass optical fiber is applied to the optical fiber bundle 1, the tip surface 2 is treated with silane to chemically bond the anti-insulin antibody to the tip surface 2. still,
For detailed treatment method, see Analytical Biochemistry 137 (198
4) See 106-114.CF Mandenium et al.

Next, the optical fiber bundle 1 thus treated
After thoroughly cleaning the front end surface 2 with only the phosphate buffer solution, thin films (black membrane filters) 3 and 4 having the same shape as the front end surface 2 were fixed to the front end surface 2 with an O-ring 5. The thin films 3 and 4 have pores with a diameter of 0.45 μm.
A 0 μm thick nitrocellulose membrane filter (manufactured by Toyo Roshi Co., Ltd.) was used.

Next, the tip end surface 2 of the optical fiber bundle 1 and the thin films 3 and 4 are dipped in a block ace solution (manufactured by Snow Brand Milk Products Co., Ltd.) which is twice diluted with 0.1 M phosphate buffer (pH 7.0),
Blocking treatment was performed at 4 ° C. for 12 hours or more. In addition, when storing after that, it is immersed in a block ace solution (manufactured by Snow Brand Milk Products Co., Ltd.), which is diluted 2-fold with 0.1 M phosphate buffer (pH 7.0), containing 0.02% sodium azide, and stored at 4 ° C.
Save under.

An optical sensor is formed by the above processing. It should be noted that such an optical sensor can be formed in advance and stored in advance, rather than being formed each time measurement is performed, and since it can be disposable every time the measurement target is changed, It is a simple optical sensor.

Next, the optical fiber bundle 1 of such an optical sensor
2, the other end is connected to an optical guiding optical cable 10 of an optical mechanism via an optical connector 9, and the tip portion of the optical fiber bundle 1 is dipped in a solution 8 containing a measurement substance 7.

That is, serum solution or standard solution 8 (both 0.5 cc appropriately diluted with the above-mentioned 2-fold diluted block ace solution and 0.06 μg / ml FI)
Pig insulin labeled with TC {FITC-labeled p
orcin insulin “Porcine insulin from Wako Pure Chemical Co., Ltd. combined with fluorescein isothiocyanate (FITC) from Wako Pure Chemical Co., Ltd. The detailed manufacturing method is“ An tibodies.
atory Manual. ”pp.354-355. by E. Harlow and D. Lane
(1988) Cold Spring Harbor Laboratory.} And mixed with 0.5 cc of a 2-fold diluted block ace solution in a container (test tube) 15 having a diameter of about 1 cm, and an optical fiber bundle 1
The tip portion (including the tip surface 2 and the thin films 3 and 4) was inserted so as to be immersed in this solution, and immediately after this insertion, the photodetector 13 picked up an image and reproduced the image on the monitor. During the measurement, at least the container 15 and the optical sensor were placed in a dark box and constantly stirred at room temperature of 25 ° C. with a stirring device (Magnetic Stirrer) 14.

FIG. 5 shows the measurement result. The vertical axis represents the relative speed of fluorescence intensity within the measurement time of 5 minutes. It should be noted that a standard curve may be drawn with the fluorescence intensity as the vertical axis after the fluorescence altitude reaches the plateau. However, it is not used here because the measurement time for one sample is as short as one hour and lacks swiftness.

As is clear from the measurement results, in the immunoassay by the immobilized antibody-competition method, insulin immobilized on the solid phase (and FI) was treated after a long treatment time.
TC insulin) and solution-phase insulin (and FITC)
The fluorescence of either FITC is measured after separation of insulin (referred to as B / F separation), but this method is extremely rapid because it monitors the initial reaction process and B / F separation is not required. Insulin can be quantified.

As described above, according to this embodiment, an unknown substance to be measured can be measured even when it is immersed in a solution, and measurement and analysis processing using a computer system or the like can be realized. You can

In this embodiment, a photomultiplier tube (PMT) is used as the photodetector 13, but a two-dimensional solid-state image pickup device (CCD) or other image pickup device capable of two-dimensional image pickup is applied. By doing so, the two-dimensional image by the light emission or fluorescence from the measurement substance 7 may be analyzed.

[0031]

As described above, according to the present invention, an optical fiber or the like having an arbitrary length used such that one end is immersed in a solution containing a substance to be measured and the other end is outside the solution during measurement. Through a light guide member having a light-transmitting property, an antigen-antibody reaction layer having an antibody or an antigen immobilized on the tip surface of the one end of the light-transmitting light guide member, and the antigen-antibody reaction layer A thin film having a porous property and a shielding property fixed to the light guide member having the light transmitting property is provided, and an optical connector is provided at the other end of the light guide member and provided in a computer system or the like. It was designed to be connected to the optical connector of the optical mechanism for detecting light. Thereby, when one end of the light-transmitting light guide member on the side where the thin film is provided is immersed in the solution, the substance to be measured passes through the porous thin film and reaches the tip surface of the light guide member having light transmittance. Reach and attach. Since the image of the adhered substance to be measured can be observed from the other end surface (the end surface located outside the solution) of the light guide member having the light transmitting property, one end of the light guide member having the light transmitting property can be observed in the solution. Real-time measurement is possible while immersed in. Also, the porous thin film allows the passage of the substance to be measured due to its innumerable pores due to its structural characteristics, but these innumerable pores have an extremely complicated and irregular shape, which prevents the transmission of light. It is possible to optically measure only the measurement substance that is not allowed and adheres to the other end surface of the light guide member having light transmittance. Further, in the case of measuring the fluorescence of the substance to be measured, it becomes possible to inject light from the other end surface (the end surface located outside the solution) of the light guide member having light transmittance. Further, by connecting to a computer system or the like through an optical connector provided on the other end surface of the light guide member having light transmittance, a wide range of applications are possible.

[Brief description of drawings]

FIG. 1 is a partial cross-sectional view showing the structure of an embodiment of an optical sensor according to the present invention.

FIG. 2 is a partial cross-sectional view further showing the structure of one embodiment.

FIG. 3 is a main-portion cross-sectional view for explaining the function of the embodiment.

FIG. 4 is an explanatory diagram of a system configuration when the optical sensor according to the embodiment is applied to a computer system or the like.

FIG. 5 is a graph showing a measurement result to which one example is applied.

[Explanation of symbols]

1 ... Optical fiber bundle, 2 ... Tip surface, 3, 4 ... Thin film, 5 ... O
Ring, 6 ... Antigen or antibody solution, 7 ... Measuring substance, 8 ... Solution, 9 ... Optical connector, 10 ... Light guide, 10a-1
0c ... Optical fiber bundle, 11a to 11c, 12a, 12b
... Optical filters, 11d, 12c ... Optical lenses, 13 ...
Light detection device, 14 ... Stirring device, 15 ... Container.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takeshi Hayakawa 1126-1126 Nomachi, Hamamatsu, Shizuoka Prefecture Hamamatsu Photonics Co., Ltd.

Claims (2)

[Claims] 1. An optical sensor that captures an unknown measurement substance contained in a solution on a solid-phase surface in contact with the solution and specifically fixes and quantifies the luminescence or fluorescence phenomenon of the measurement substance. A light guide member having a light-transmitting property of an arbitrary length used so that one end side is immersed in a solution containing a substance and the other end side is outside the solution; and the above-mentioned light guide member having the light-transmitting property. An antigen-antibody reaction layer having an antibody or an antigen immobilized on one end surface thereof, and a thin film having porosity and shielding property fixed to the light guide member having light permeability through the antigen-antibody reaction layer An optical sensor comprising: 2. An optical sensor that captures an unknown measurement substance contained in a solution on a solid surface in contact with the solution, and specifically identifies and quantifies the luminescence or fluorescence phenomenon of the measurement substance. A light guide member having a light-transmitting property of an arbitrary length used so that one end side is immersed in a solution containing a substance and the other end side is outside the solution; and the above-mentioned light guide member having the light-transmitting property. An antigen-antibody reaction layer having an antibody or an antigen immobilized on one end surface thereof, and a thin film having porosity and shielding property fixed to the light guide member having light permeability through the antigen-antibody reaction layer And an optical connector provided at the other end of the light guide member having the above-mentioned optical transparency.