JP2005121611A - Optical probe and manufacturing method for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve excitation efficiency, light-collecting efficiency, surface fixed capability of a protein and analysis accuracy of measurement in an optical probe for measuring, based on an optical technique. <P>SOLUTION: The optical probe 1 comprises an optically transparent synthetic resin, and is provided with a cylindrical optical waveguide 2 and a light-entering/exiting part 3 disposed in an upper part, having a convex lens profile and entering and exiting a light. A flange 4 is provided between the optical waveguide 2 and the light entering/exiting/ part 3 and is used for mounting the optical probe 1 to a measuring apparatus. The optical probe 1 has a controlled surface roughness at a particular region on a surface of the optical waveguide 2, and the surface roughness is made small at the light-entering/exiting sides, and is made large at the tip. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an optical probe used in an analytical measurement apparatus based on an optical technique and a method for manufacturing the same.

  Absorption of light by a sample or the resulting fluorescence is used as an analytical technique in various fields, and most of them are placed in a light-transmitting container (cell) and observed from outside the container. Is. However, as another method, an optical waveguide made of a light-transmitting material is used as an optical probe, and light is introduced into the optical probe or collected from the optical probe to optically observe the surface of the optical probe. ing. Such an optical probe is brought into contact with a sample or a reaction solution, and simultaneously or subsequently connected to an appropriate optical system, and absorption or fluorescence by the sample is monitored.

  Of these, the method using fluorescence is generally more sensitive, and in particular, the method using the light in the optical probe as total internal reflection light and the evanescent wave generated on the optical probe surface at that time as the excitation light, Can be selectively observed, and is used, for example, for highly sensitive immunoassay based on antigen-antibody reaction. A plate-like or columnar elongated optical probe can increase the number of total internal reflections per unit volume, and can perform efficient excitation.

  A form using a plate-like so-called slab type optical probe is frequently used for research, and there are many examples. However, the one that also serves as a cell wall as in Patent Document 1 is highly convenient and useful. As a form using a cylindrical optical probe, there is a type in which a part of an optical fiber directly connected to a light source or a detector is used as a sensor. However, as in Patent Documents 2 to 4, a part used as a sensor is physically independent. There is what I did.

  The above examples are optical probes that can be attached to and detached from the apparatus, and these probes are often manufactured by processing a synthetic resin by a molding method such as injection molding due to disposableness and cost. In this case, the injection molding die or the piece incorporated in the die is generally manufactured by cutting the pilot hole with a processing machine in accordance with a desired shape and then polishing the inner surface.

JP-A 63-273042 JP-A-5-5742 US Pat. No. 4,582,809 US Pat. No. 6,136,611

  However, it is difficult to arbitrarily set the surface roughness in the mold, that is, the surface roughness of the optical probe, in the manufacturing method using the mold described above. For this reason, the surface roughness is uniform, or an unintended partial surface roughness variation occurs.

  The roughness of the surface tends to cause the total internal reflection light to leak to the outside due to scattering, and becomes an obstacle when the evanescent wave is selectively used. However, there are cases where the pumping efficiency is better when normal light that is not evanescent waves is used.

  In addition, when the optical probe also serves as a condensing system, an effect of suppressing the reflection of fluorescence on the surface of the optical probe can be expected. Furthermore, moderate surface roughness can be helpful when immobilizing proteins or the like on the optical probe surface.

  An object of the present invention is to provide an optical probe capable of solving the above-described problems, improving the surface immobilization ability against an antigen, and performing effective measurement, and a method for manufacturing the same.

  In order to achieve the above object, an optical probe according to the present invention is made of an optically transparent material, has a light incident / exit portion and an optical waveguide portion, and a specific portion of the surface of the optical waveguide portion has a predetermined surface roughness. It is characterized by that.

  The method of manufacturing an optical probe according to the present invention includes an optical transparent member made of an optically transparent material, having a light incident / exiting portion and an optical waveguide portion, and having a predetermined surface roughness at a specific location on the surface of the optical waveguide portion. A master having the same shape as the probe is manufactured, and manufactured by injection molding using a mold incorporating a piece processed by electrolytic casting using the master.

  Furthermore, the manufacturing method of the optical probe according to the present invention is the manufacturing of an optical probe made of an optically transparent material and having a light incident / exit part and an optical waveguide part. The surface roughness of the front end side of the wave portion is increased.

  The optical probe of the present invention has a predetermined surface roughness at a specific location on the surface of the optical waveguide section, so that the excitation efficiency, fluorescence condensing efficiency, surface fixing ability of proteins, etc. can be improved according to the purpose. Thus, it is possible to improve the accuracy of analysis and measurement based on an optical method, particularly a fluorescence method.

  Further, according to the method for manufacturing an optical probe, an optical probe in which the surface roughness of the optical waveguide portion is freely changed can be obtained.

  The optical probe according to the present invention is characterized in that the surface roughness is small on the incident / exit side of the optical waveguide section and large on the distal end side.

  The optical probe according to the present invention is characterized in that the optical waveguide section has a cylindrical shape or a plate shape.

  The optical probe according to the present invention is characterized in that a mounting portion for the measuring device is provided, and the mounting portion is detachable from the measuring device.

  FIG. 1 is a side view of the optical probe 1 according to the first embodiment. The optical probe 1 is made of, for example, an optically transparent synthetic resin material, and mainly enters and exits a cylindrical optical waveguide portion 2 and an upper portion thereof. And a light incident / exit part 3 having a convex lens, and a flange 4 is formed between the optical waveguide part 2 and the light incident / exit part 3 to be used as an attachment part when mounted on a measuring apparatus to be described later. .

  This optical probe 1 has a controlled surface roughness at a specific location on the surface of the optical waveguide section 2, and this surface roughness can be smaller on the light incident / exit side and larger on the tip side. preferable.

  Although it does not specifically limit about the resin material used for the optical probe 1, Polystyrene, polymethylmethacrylate, a polycarbonate etc. are mentioned, You may use a mold release agent and an external lubricant in the range which does not have a trouble. Since the optical probe 1 must intentionally control the light scattering element, it is necessary to pay sufficient attention to the inflow of other light scattering elements such as dust in the resin and moisture that causes bubbles. is there.

  The optical probe 1 is often used in a fluorescence method using an evanescent wave in which excitation light is generated on the surface as internally reflected light. For such usage, it is common to ensure the maximum smoothness of the surface of the optical probe 1. Light scattering factors due to surface scratches and roughness are loss or stray light.

  However, the present inventors have found that the sensitivity may be improved within a range where some scattering does not hinder. It is found in sandwich method measurements, such as bacteria that bind the labeled antibody to this antigen after it has been captured by the antibody. The principle has not been elucidated in detail, but the explanatory diagram of FIG. 2 shows the assumed concept. Note that FIG. 2 is schematic, and relative sizes such as bacteria, antibodies, and surface roughness are not drawn strictly.

  Since the evanescent wave has an effective exudation only in the range D of several hundreds of nanometers from the surface, the excitation by this is as much as 1 μm. In the bacteria F captured by the capture antibody E, usually, as shown in (a) Only the labeled antibody G existing near the optical waveguide 2 can be excited. However, as shown in (b), when leakage light H and diffracted light I derived from scattering due to roughness on the surface of the optical waveguide 2 are mixed, these lights reach the far side of the bacteria F. It is assumed that many labeled antibodies G can be excited. At this time, unbound free labeled antibody G or fluorescent impurities may be excited, but these can be reduced by a washing operation.

  In this way, if the scattered light due to the surface roughness is positively used, it is possible to excite an object far from the surface of the optical probe 1. However, the scattered light is leakage light H or diffracted light I, which attenuates the propagation of the excitation light in the optical probe 1 and reduces the surface area that can be excited in the optical probe 1. Reduce.

  This is a problem particularly on the surface near the incident / exit side of the optical waveguide section 2. If light is leaked excessively in the vicinity of the incident / exit side, the portion beyond that does not contribute to the measurement due to insufficient light quantity. Therefore, it is desirable to provide the surface roughness in the form of (a) distributing an appropriate roughness component uniformly or (b) providing a surface rougher than the incident / exit side on the tip side.

  FIG. 3 shows an example of the path of the excitation light in the form (b). The internal total reflection is mainly on the incident / exit side, and the rate of leakage as normal light increases toward the distal end side.

  Such a form of surface roughness is effective for the optical probe 1 that serves both for excitation and light collection. For an optical system that only propagates and emits excitation light in the optical probe 1 and performs photometry from, for example, the lateral direction outside the optical probe 1, scattered light from a rough surface becomes stray light, so it is not always effective. is not. Moreover, in the optical probe 1 that also serves as a condensing light, the roughness of the surface of the optical probe 1 suppresses the reflection of the fluorescence, and increases the uptake of fluorescence into the optical probe 1.

  The surface roughness is based on the wavelength of light to be used, and for the purpose of obtaining appropriate scattering, it is desirable that the surface roughness be approximately the same as or larger than the wavelength of light. Roughness smaller than the wavelength hardly contributes to scattering, but is suitable for improving the retention in fixing proteins. On the other hand, if the roughness is too large, the leakage light is concentrated at that point, which may reduce the overall efficiency.

  As a method of giving the surface roughness to the optical waveguide portion 2 of the optical probe 1 in a desired position and amount, first, a method of secondarily giving the molded product is conceivable. For example, a method of immersing in an erodible solution, polishing, sandblasting, and the like can be given. However, these secondary methods cause significant damage to the material for the resin optical probe 1. In addition, performing a secondary method on each optical probe 1 is low in productivity, and it is more desirable to simultaneously control the surface roughness during molding.

  In industrial production of the optical probe 1, a master having the same shape as the optical probe 1 having a surface roughness at a specific location is manufactured, and a die that incorporates a piece subjected to electrolytic casting using the master is used. An example is molding.

  In order to give the surface roughness at a desired position and amount at the time of molding, it is generally applied to the inner surface of the piece incorporated in the mold, but the diameter is small and the depth is long, and the length of the optical waveguide section 2 is long. It is difficult to finely process the inner surface of the hole and measure the surface roughness. Therefore, a method is effective in which a master, that is, a master having the same shape as the optical probe 1 of a molded product is prepared, a predetermined shape and a predetermined surface roughness are given to the master, and then transferred to the piece by electrolytic casting. . In this case, since the surface processing of the master is performed from the outside, various devices and methods can be easily used.

  FIG. 4 shows two process examples from master production to piece production. The material of the master 11 can be a metal material such as iron or SUS, a material such as cemented carbide or ceramic. First, as shown in (a), a master 11 is made from a material by machining with a cutting tool 12 or the like, and is polished to a desired surface roughness as shown in (b). By changing the type and roughness of the abrasive at the location where it is polished, or by changing the amount of polishing pressure, it is possible to give any roughness only by polishing, or any roughness can be given by embossing or honing. .

  As another method, the master 11 is made a little smaller by machining. As shown in (c), the master 11 is attached with a chemical nickel plating or copper plating layer 13 and attached to an ultra-precision turning machine. , (D), there is a method of performing mirror cutting with a diamond tool 14 or the like. At that time, it is possible to freely change the surface roughness by changing the tool feed pitch, the processing amount of the tool, etc. at an arbitrary place.

  The surface roughness is adjusted while measuring and evaluating the master 11 produced by any one of the above methods using a non-contact roughness meter or a shape measuring instrument. Thereafter, as shown in (e), electrocasting is performed in an electroforming liquid using the master 11 as a cathode, thereby obtaining an electroformed piece 15 having a hole that can be used for molding. Examples of the electrolytic casting include nickel electrolytic casting and copper electrolytic casting. The inner surface of the piece 15 produced as shown in (f) is finished to a desired roughness, and after being subjected to outer shape processing and the like, it is assembled into a mold and molded into an optical probe by molding such as injection molding. Get one.

  In actual manufacture, the master 11 was turned with a cutting tool 12 from a SUS round material with an NC lathe to obtain a predetermined shape. Next, the surface of the master 11 was polished without changing the shape. That is, the paper was coarsened in order from the coarser one, and finally polished with a polishing cloth to which diamond paste was attached. At this time, the surface roughness on the incident side and the tip side of the optical waveguide 2 was adjusted while evaluating with a non-contact roughness meter during the polishing process. This adjustment method was performed by the roughness of the diamond paste and the pressing pressure of the polishing cloth.

  The master 11 was held so as not to be deformed and placed in an electrolytic casting tank, and nickel electrolytic casting was performed. When a nickel layer having a sufficient thickness as the piece 15 was formed, it was taken out from the electrolytic casting layer and the master 11 was pulled out. After both ends of the piece 15 were machined, the outer shape was machined with a cylindrical grinding machine on the basis of the hole, so that it could be loaded into an injection mold.

  Through the above steps, the piece 15a having a uniform surface roughness (surface roughness Pv = 35 nm) regardless of the location of the optical waveguide section 2, and the two types of pieces 15b, 15c having different surface roughness depending on the location. (Incoming / outgoing side Pv = 35 nm and tip side Pv = 200 nm are continuously changed).

  In the injection molding, a single-point gate cold runner mold and an injection molding machine (SG50M, manufactured by Sumitomo Heavy Industries, Ltd., screw diameter φ22 mm) were used. The material used was a polystyrene resin dried at 80 ° C. for 4 hours or more. Table 1 shows the surface roughness Pv of the optical probes 1a, 1a, 1a of the molded products molded with the two holding pressures a, b for these three pieces 15a, 15b, 15c.

Table 1
No. Top surface roughness (nm) Holding pressure Surface roughness of molded product (nm)
[Incoming / outgoing side-tip side] (MPa) [Incoming / outgoing side-tip side]
1a 35-200 a 15-500
1b 35 (uniform) a 27
1c 35 (uniform) b 45-220

  A black paint was applied to the end faces of the optical probes 1a, 1b, and 1c in Table 1 for the purpose of suppressing reflection, and anti-E. Coli O157: H7 polyclonal antibody was immobilized on the surface of the cylindrical portion. And the optical system shown in FIG. 5 was used for excitation and photometry.

  The washing solution used was 0.01M phosphate buffer (pH 7.4) containing 0.5% polyoxyethylene (20) sorbitan monolaurate. The fluorescence-labeled antibody was prepared by allowing Cy5 bisfunctional reactive dye (Amersham Biosciences) to act on the antibody, and dissolved in the washing solution at 2 μg / ml. A solution containing 10 to 1000000 CFU / ml of E. coli O157: H7 was prepared as a sample, and measurement was performed for each optical probe 1a, 1b, and 1c as follows.

  These optical probes 1 were measured by inserting the optical probe 1 into the insertion portion 22 in the measurement port 21 shown in FIG. The measurement port 21 is provided with a liquid supply port 23 and a liquid discharge port 24 with respect to the insertion portion 22.

  In the measurement optical system, light (635 nm) from the semiconductor laser light source 31 passes through the lens 32, the half mirror 33, and the lens 34 and enters from the light incident / exit section 3 above the optical probe 1. The feedback light excited in the optical waveguide unit 2 is extracted in the horizontal direction by the half mirror 33, dispersed through the filter 35 and the lens 36, and detected by the photodiode 37.

The measurement procedure is as follows.
(1) 1 ml of the sample solution is fed into the measurement port 21 at 0.1 ml / min.
(2) 2 ml of the washing solution is fed into the measurement port 21 at 10 ml / min.
(3) The labeled antibody solution is injected into the measurement port 21 and allowed to stand for 5 minutes.
(4) 2 ml of the washing solution is fed into the measurement port 21 at 10 ml / min.
(5) Excitation and photometry of the optical probe 1 are performed using an optical system in a state where the cleaning liquid is filled in the measurement port 21.
(6) The sample (10-fold concentration) is exchanged in the measurement port 21, and the above (1) to (5) are performed.

  FIG. 6 shows the results of the measurement, and the sensitivity is improved in the low concentration region in the optical probe 1a given the surface roughness that is likely to cause scattering.

  In the production of the optical probe 1, as a second embodiment in which the inner surface roughness of the piece 15 is not used, there is a method of controlling by molding conditions of injection molding. This method is applied to a long and narrow molded product such as a cylinder. It is effective.

  Generally, in order to completely fill the resin up to the hole tip of the mold and transfer the surface roughness of the mold, it is necessary to set the injection pressure amount to a certain level or to fill the resin at a high speed.

  However, by slightly lowering the holding pressure from the molding conditions for complete filling, the filling pressure at the beginning of the molten resin is lowered, and as a result, the surface of the molded product can be made rougher than the surface roughness of the mold.

  Thereby, roughness can be given to the surface near the tip side of the optical probe 1 set on the cavity side of the mold. The holding pressure depends on the size and shape of the optical probe 1 and is not uniquely determined. However, if the holding pressure is too low, the entire surface roughness increases due to insufficient filling.

  The optical probe 1 applicable in the present invention is not limited to the cylindrical optical probe 1 shown in FIG. 1, but is also a plate-like optical probe 1 ′ formed by molding, for example, an optically transparent resin as shown in FIG. It may be. In this optical probe 1 ′, the bottom surface is the optical waveguide portion 2 ′, and the light incident / exit portion 3 ′ is provided on the bottom side surface. L is a sample.

It is a side view of the optical probe in an Example. It is a principle explanatory drawing. It is explanatory drawing of the path | route of the excitation light in an optical probe. It is the schematic of the example of a process which produces a piece from a master. It is a block diagram of a measurement optical system. It is a graph of the measurement result in an Example. It is sectional drawing of a plate-shaped optical probe.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 1 'Optical probe 2, 2' Optical waveguide part 3, 3 'Light incident / exit part 4 Flange 11 Master 13 Plating layer 15 piece 21 Measurement port 31 Semiconductor laser light source 32 Half mirror 35 Filter 37 Photodiode

Claims (6)

  An optical probe comprising an optically transparent material, having a light incident / exiting portion and an optical waveguide portion, and having a specific surface roughness at a specific portion of the surface of the optical waveguide portion.   The optical probe according to claim 1, wherein the surface roughness is small on the incident / exit side of the optical waveguide and large on the tip side.   The optical probe according to claim 1, wherein the optical waveguide portion is formed in a columnar shape or a plate shape.   The optical probe according to any one of claims 1 to 3, wherein a mounting portion for the measuring device is provided, and the mounting portion is detachable from the measuring device.   A master having the same shape as an optical probe, which is made of an optically transparent material, has a light incident / exit portion and an optical waveguide portion, and has a predetermined surface roughness at a specific portion of the surface of the optical waveguide portion, A method of manufacturing an optical probe, wherein the optical probe is manufactured by injection molding using a mold incorporating a piece processed by electrolytic casting.   When manufacturing an optical probe made of an optically transparent material and having a light incident / exit section and an optical waveguide section, the surface roughness on the tip side of the optical waveguide section is increased by controlling the holding pressure in injection molding. An optical probe manufacturing method characterized by the above.

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