JP2009192274A - Light waveguide device, fluorometric analyzer and detection method of chemical substance using them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide both of the light waveguide device and a fluorometric analyzer for quantitatively detecting the quantity of fluorescence from fluorescent protein with high reproducibility, especially the micro-change in the quantity of fluorescence caused by an external factor with high reproducibility, and a detection method of a chemical substance using them. <P>SOLUTION: The exciting light of the fluorescent protein is introduced into the light waveguide 8 from the core incident side end surface 2a of the light waveguide device 1 equipped with the light waveguide 8 having a core 2 comprising sol-gel slilica to which the fluorescent protein is internally fixed to detect the fluorescence of the fluorescent protein from the core emitting side end surface 2b of the light waveguide device 1. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an optical waveguide device, a fluorescence analyzer, and a chemical substance detection method using the same.

  A fluorescent protein is a protein that emits fluorescence when irradiated with light having an excitation wavelength, and there exists a natural living body that expresses the fluorescent protein. As a representative fluorescent protein, GFP (Green Fluorescent Protein, molecular mass 27 kDa) which is a protein emitting green fluorescence isolated from Aequorea jellyfish is known.

  In recent years, a method for further increasing the amount of fluorescence by expressing a fluorescent protein on the surface of yeast has been developed as compared with the fluorescence from the fluorescent protein expressed in the conventional yeast.

  Conventionally, as a method for quantitatively evaluating the amount of fluorescence from a fluorescent protein, a visual method using a microscope is generally used, but a method capable of quantitatively confirming a change in the amount of fluorescence due to an external factor with high reproducibility. Is not established yet.

  The use of optical waveguides and optical fibers has been studied as a method that can be expected to provide quantitative evaluation with high reproducibility of the amount of fluorescence from fluorescent proteins. Conventionally, in the field of optical waveguides and optical fibers, a technique for observing fluorescence by forming an amplifier by fixing an inorganic rare earth such as erbium inside, and a technique using a stress luminescent material are known (see Patent Document 1). ).

  However, although the fluorescence intensity can be expected to be improved if the fluorescent protein can be immobilized in the optical waveguide, it is technically difficult to immobilize the biologically derived fluorescent protein in the optical waveguide, which has not been realized yet. Currently. That is, when using an optical waveguide, it is necessary to control the optimal refractive index, to reduce the light loss, and to maintain the living state of the living body expressing the fluorescent protein. However, the optical waveguide satisfies all such requirements. Has not yet been proposed.

  As a technique using an optical waveguide for fluorescence detection, a method of detecting a change in the amount of fluorescence due to a biological reaction by immobilizing a biocatalyst such as an enzyme on the end of an optical fiber cladding, or on the core surface or cladding surface of an optical waveguide is examined. Has been.

In addition, a method in which a living body expressing a fluorescent protein mixed with a buffer solution is used as the core of the optical waveguide has been studied.
JP 2004-71511 A

  However, the former method employs a method of using evanescent light (leakage light) for fluorescence detection. However, in this case, the fluorescence intensity is weak and the concentration of fluorescence is inefficient. was there. Therefore, quantitative evaluation with high reproducibility is difficult, and detection by a naked eye observation or a silicon detector that can be driven at a low voltage at low cost is difficult.

  In the latter method, a method using a special optical waveguide called ARROW (Anti-resonant reflecting optical waveguide) has been adopted because of the refractive index relationship with the buffer mixed with a living body expressing a fluorescent protein. However, there is a problem that the coupling loss with the single mode optical fiber used in the long-distance optical communication, the waveguide loss of the optical waveguide itself is large, and the fluorescence is weak. Furthermore, all-solid-state packaging using optical fibers is difficult, and it is necessary to use a large-sized photomultiplier tube that drives the detection system at a high voltage. For this reason, this optical waveguide employs a method of observing fluorescence from the side of the optical waveguide. However, this optical waveguide has less advantage compared to conventional fluorescence microscopes, and the fluorescent protein mixed in the buffer solution is fluorescent. When the naked eye was observed with a microscope, the observable period was limited to about 1-2 days.

  The present invention has been made in view of the circumstances as described above, and can detect the amount of fluorescence from a fluorescent protein quantitatively with high reproducibility, in particular, a minute change in the amount of fluorescence due to external factors. It is an object of the present invention to provide an optical waveguide device, a fluorescence analyzer, and a method for detecting a chemical substance using the optical waveguide device that can quantitatively detect the above.

  The present invention is characterized by the following in order to solve the above problems.

  1stly, the optical waveguide device of this invention is equipped with the optical waveguide which has a core which consists of sol-gel silica which fixed fluorescent protein inside.

  Second, in the first optical waveguide device, the fluorescent protein is GFP (Green Fluorescent Protein).

  Thirdly, the fluorescence analyzer of the present invention includes the first or second optical waveguide device, an excitation light source for introducing excitation light of the fluorescent protein into the optical waveguide from the core incident side end surface of the optical waveguide device, And a photodetector for detecting fluorescence of the fluorescent protein from the end surface on the core exit side of the optical waveguide device.

  Fourth, in the third fluorescence analyzer, the optical waveguide of the optical waveguide device is a single mode optical waveguide, and at least one of the core incident side end surface and the core output side end surface is connected to the single mode optical fiber. It is characterized by.

  Fifth, the method for detecting a chemical substance of the present invention is a method for detecting a chemical substance using the third or fourth fluorescence analyzer, wherein the optical waveguide of the optical waveguide device is brought into contact with the chemical substance to be detected. In a possible state, the excitation light from the excitation light source is introduced into the optical waveguide, the fluorescence of the fluorescent protein from the optical waveguide is detected by the photodetector, and the pH change in the optical waveguide due to the chemical substance that has penetrated into the optical waveguide A chemical substance is detected by a change in the amount of fluorescence from a fluorescent protein based on the above.

  Sixth, in the fifth chemical substance detection method, the chemical substance is a phosphorus compound.

  According to the optical waveguide device and the fluorescence analyzer of the present invention, since the fluorescent protein is fixed inside using sol-gel silica as the material of the optical waveguide, the fluorescence in the optical waveguide from the end surface on the core incident side in the linear optical waveguide. By exciting the protein, the fluorescence from the fluorescent protein can be detected in the traveling direction of the optical waveguide from the end surface on the core exit side, and highly efficient fluorescence detection becomes possible.

  Thereby, the amount of fluorescence from the fluorescent protein can be quantitatively detected with high reproducibility, and in particular, a minute change in the amount of fluorescence due to an external factor can be quantitatively detected with high reproducibility.

  In addition, by doping the sol-gel silica as an optical waveguide with the fluorescent protein, the fluorescent protein can be maintained in the optical waveguide with a long lifetime, and microorganisms such as yeast expressing the fluorescent protein can survive in the optical waveguide for a long period of time. It can also be made.

  In addition, because strong fluorescence can be detected, an inexpensive and small semiconductor laser and silicon detector were used without using a large and expensive high-power laser or a detection system using a photomultiplier tube driven at a high voltage. Optical integration is possible.

  In addition, by making the optical waveguide of the optical waveguide device a single mode optical waveguide, it is possible to dramatically improve the light irradiation intensity of the excitation light to the fluorescent protein and the single mode used in long-distance communication. Since it can be coupled with an optical fiber, a long-distance fiber sensor network can be constructed.

  According to the method for detecting a chemical substance of the present invention, by bringing the optical waveguide of the optical waveguide device into contact with the chemical substance to be detected, the solid, liquid, or gaseous chemical substance is contained in the sol-gel silica optical waveguide. By observing the change in the amount of fluorescence from the fluorescent protein based on the pH change in the optical waveguide, it is possible to detect chemical substances such as phosphorus compounds.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  1A and 1B are diagrams schematically showing an optical waveguide device according to an embodiment of the present invention, in which FIG. 1A is a perspective view and FIG. 1B is a sectional view in the waveguide axis direction. As shown in the figure, an optical waveguide device 1 according to this embodiment includes a clad 3 provided on a substrate 5 and a core 2 surrounded by the clad 3 and extending in the waveguide axis direction. It is produced using a lithography technique or the like.

  The core 2 constituting the optical waveguide 8 is obtained by directly mixing (doping) a fluorescent protein into a gel-like or porous sol-gel silica that has been subjected to a low temperature treatment, and fixing it.

  The sol-gel silica is preferably treated by low-temperature treatment. For example, by performing heat treatment at 80 ° C. using γ-methacryloxypropyltrimethoxysilane and a refractive index adjusting agent zirconium (IV) -n-propoxide, Decomposition and degeneration begin, and alcohol evaporates at the same time as vitrification. The same vitrification also starts with UV irradiation.

The porosity of sol-gel glass has been reported in the past (R. Armon et al. “Sol-gel as reaction matrix for bacterial biological activity”, Journal of Sol-Gel Science and Technology, 19, 289-292, 2000.) In general, the porous radius of sol-gel silica in low-temperature treatment becomes large (Sakuo Sakuo, “Application of the sol-gel method”, Agne Jofu Co., Ltd. p. 93). In the present invention, for example, a sol-gel having a porous radius of about 10 to 100 nm is used. The use of silica is considered. The change of waveguide loss due to reaction with water vapor in the air has also been reported (ON Mishechkin et al. “Passivation of organic-inorganic hybrid sol-gel”, Journal of Sol-Gel Science and Technology, 34, 47). -51, 2005.)
A fluorescent protein is a protein that emits fluorescence when irradiated with light having an excitation wavelength, and there exists a natural living body that expresses the fluorescent protein. Representative examples of the fluorescent protein include GFP (Green Fluorescent Protein, molecular mass 27 kDa), which is a protein emitting green fluorescence isolated from Aequorea jellyfish. Since GFP is a protein unlike general inorganic and organic fluorescent substances, it can be introduced into living cells and organisms in the form of genes.

  The clad 3 can be made of, for example, sol-gel silica in the same manner as the core 3 in consideration of the refractive index and the like based on the chemical composition.

  In the optical waveguide device 1 of the present embodiment, the excitation light is incident from the incident side end surface 2a of the core 2 constituting the optical waveguide 8, and the fluorescent protein in the core 2 is excited, so that the waveguide travels from the emission side end surface 2b. The fluorescence from the fluorescent protein can be taken out in the direction.

  The sol-gel silica doped with the fluorescent protein can be gelled or porous when subjected to low-temperature treatment, so that all solids, liquids, and gases can permeate the sol-gel silica. Therefore, when a microorganism such as an enzyme that expresses fluorescent protein is doped in sol-gel silica, the microorganism can survive inside sol-gel silica by exchanging oxygen from the outside, and the life of fluorescent protein in optical waveguide device 1 is improved. be able to.

  It is also possible to infiltrate the chemical substance into the optical waveguide 8 by bringing a solid, liquid, or gaseous chemical substance into contact with the optical waveguide device 1 from the outside. For example, when a phosphorus compound such as pesticide or sarin is brought into contact with the optical waveguide device 1 by contacting the optical waveguide device 1, the fluorescence from the fluorescent protein is caused by the reaction of organophosphorus hydrolase adhering to microorganisms such as yeast. The amount changes, and the phosphorus compound can be detected from this change in fluorescence.

  FIG. 2 is a diagram schematically showing a fluorescence analyzer according to an embodiment of the present invention. The fluorescence analyzer 10 of the present embodiment includes the optical waveguide device 1 of FIG. 1, and further uses an Ar ion laser 11 (wavelength 488 nm) as an excitation light source for the fluorescent protein doped in the core 2 of the optical waveguide device 1. I have.

  A single mode optical fiber 13 is optically connected to the incident side end face 2 a of the core 2 of the optical waveguide device 1, and pump light from the Ar ion laser 11 is transmitted from the laser fiber coupler 12 to the single mode optical fiber 13. And is incident on the core 2 of the optical waveguide device 1.

  The fluorescent protein in the core 2 of the optical waveguide device 1 excited by the excitation light from the Ar ion laser 11 emits fluorescence and is emitted to the outside from the emission-side end face 2 b of the core 2. On the exit side of the optical waveguide device 1, a microscope objective lens 14 and a dichroic filter 15 are arranged in this order as an optical system. After the fluorescence is condensed and the wavelength is selected by these, the target is detected by the silicon detector 16. Fluorescence is detected. Then, by using the silicon detector 16 or visually observing the fluorescence intensity and its change, it can be used as a sensor.

  In this embodiment, since the core 2 constituting the optical waveguide 8 of the optical waveguide device 1 is doped with a fluorescent protein such as GFP, the fluorescence intensity can be greatly improved. With a normal laser microscope, the range that can be irradiated with strong light intensity is only near the focal point and the fluorescence outside the focal point is weak, and the principle of a simple fluorescence measuring device using an LED and a silicon detector causes the fluorescence intensity to be weak and condensing. However, in this embodiment, strong fluorescence can be observed. For example, GFP in the core 2 can be observed using weak excitation light of about 20 mW at a wavelength of 488 nm. Fluorescence from can be detected by the silicon detector 16.

  In addition, the optical waveguide 8 is a single mode optical waveguide, and a structure in which light is directly coupled to the optical waveguide 8 of the waveguide device 1 from the single mode optical fiber 13 is applied. Thus, by using a single mode optical waveguide, for example, GFP in a 4 μm × 4 μm core 2 doped with a fluorescent protein such as GFP can be excited with a substantially equal light intensity over a length of 5 mm. .

  Furthermore, since the optical waveguide 8 of the optical waveguide device 1 can be optically connected by bonding to the optical fiber at the incident end face 2a of the core 2 or at both the incident end face 2a and the outgoing end face 2b, optical axis alignment becomes unnecessary. As a whole, the life of the fluorescence analyzer 10 can be extended and the size thereof can be reduced, and the portability can be improved.

  In addition, since the fluorescent protein is immobilized on the core 2 of the optical waveguide device 1, the fluorescence from the fluorescent protein passes through the optical waveguide 8 and exits from the exit-side end face 2 b in the direction of travel of the waveguide. Fluorescence can be easily collected. For example, when the fluorescence is transmitted through an optical fiber, transmission with low light loss is easy.

  Furthermore, since the fluorescence intensity can be significantly improved by doping the core 2 constituting the optical waveguide 8 of the optical waveguide device 1 with a fluorescent protein such as GFP, photomultiplier that requires a high voltage of about kV. Instead of using a large detector such as a tube, a small detector such as a silicon detector 16 is used, an inexpensive semiconductor laser or the like is used as an excitation light source, and a single mode optical fiber 13 with low optical loss is provided. When used in combination, the fluorescence analyzer 10 can be configured as a biosensor in which a fiber network for indoors and outdoors such as a building is constructed. That is, by utilizing the characteristics of the optical waveguide device 1 of the present invention, it is possible to construct a long-distance fiber sensor network.

  Further, due to the strong fluorescence of the fluorescent protein and the long lifetime inside the sol-gel silica, for example, it is possible to detect the naked eye for 30 days or more and observe the change in the fluorescence amount with the silicon detector 16.

  Further, an external phosphorus compound or the like is brought into contact with the optical waveguide device 1 to permeate into the optical waveguide 8, and the pH in the core 2 doped with the fluorescent protein is changed to observe a change in the amount of fluorescence based thereon. This makes it possible to detect phosphorus compounds and the like. Therefore, application as an outdoor biosensor for phosphorus such as agricultural chemicals is also possible.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.
<Example 1>
A GFP-doped sol-gel optical waveguide device having the structure shown in FIG. 1 was produced by the method shown in FIG.

A sol-gel having a chemical composition of Si / Zr mixing ratio = 88/12 mol% using γ-methacryloxypropyltrimethoxysilane (MAPTMS) and zirconium (IV) -n-propoxide (ZrPO) as a refractive index modifier. Using the solution for clad preparation, spin coat the solution twice on the SiO ₂ / Si substrate 5 shown in FIG. 3A, perform baking at 150 ° C./1 h, and then hydrolyze and degenerate with 0.1 N HCl. Vitrification was advanced to form a lower cladding 3a having a thickness of 8 μm.

  Next, a side cladding 3b having a film thickness of 4 μm was spin-coated on the lower cladding 3a, and etching was performed by UV irradiation and IPA through the mask 6 to pattern the side cladding 3b. Thereafter, by baking at 80 ° C./24 h, the core forming groove 4 was formed in the side cladding 3b as shown in FIG.

On the other hand, 2 mg of model GFP was mixed with 1 ml of a solution for preparing a sol-gel core after filtering (MAPTMS, ZrPO: Si / Zr mixing ratio = 85/15 mol%), and allowed to stand at room temperature for 5 minutes, followed by stirring. This solution was spin-coated in a range including the core-forming groove 4 and then UV-irradiated for 9 minutes at an irradiation intensity of 9 mW / cm ² using a mercury lamp 365 nm line as shown in FIG. Thereafter, the end face was cleaved to obtain a straight end face, and thus an optical waveguide device 1 having a core 2 having a cross section of 4 μm × 4 μm was produced.
<Example 2>
A fluorescence analyzer 10 having the configuration shown in FIG. 2 was configured using the optical waveguide device 1 manufactured in Example 1, and fluorescence observation was performed. An Ar ion laser 11 (wavelength 488 nm) is used as an excitation light source of GFP doped in the core 2 of the optical waveguide device 1, and the single mode optical fiber 13 bonded to the incident side end face 2 a of the core 2 of the optical waveguide device 1 is used. Excitation light is introduced into the optical waveguide 8 through the laser fiber coupler 12.

  On the exit side of the optical waveguide device 1, a microscope objective lens 14 (× 20 times), a dichroic filter 15 (High Transmission at 511 nm, Low Transmission at 488 nm), and a silicon detector 16 are sequentially arranged as an optical system.

  With respect to this fluorescence analyzer 10, fluorescence was observed by exciting GFP doped in the optical waveguide 8 of the optical waveguide device 1. Laser light with an excitation wavelength of 488 nm from the Ar ion laser 11 was incident on the optical waveguide 8 of the optical waveguide device 1 through the single mode optical fiber 13, and fluorescence with a wavelength of 511 nm was measured by the silicon detector 16.

  FIG. 4 shows a fluorescence detection result by the silicon detector 16 after the background noise correction. As shown in the figure, strong fluorescence due to GFP in the optical waveguide 8 was observed depending on the excitation light intensity. In addition, strong fluorescence was confirmed by visual observation in single mode and slab mode.

  5 and 6 are graphs showing the results of transmission loss measurement of a GFP-doped sol-gel silica optical waveguide. The optical loss of a GFP-doped sol-gel silica optical waveguide (FIG. 5: waveguide length 5 mm, FIG. 6: waveguide length 10 mm) with respect to laser beams having wavelengths of 488 nm and 515 nm was measured.

  A transmittance of about 30% was obtained at a waveguide length of 5 mm with respect to a waveguide width of 5 μm at an excitation wavelength of 515 nm corresponding to GFP fluorescence. The optical waveguide loss of the device for the excitation wavelength of 515 nm is estimated to be <1 dB / 5 mm because the waveguide length is about −4 to −5 dB when the waveguide length is 5 mm and about −5 dB when the waveguide length is 10 mm. The optical waveguide loss of the device for the excitation wavelength of 488 nm is estimated to be about 4 dB / 5 mm because it is about −6 dB when the waveguide length is 5 mm and about −10 dB when the waveguide length is 10 mm.

  FIG. 7 is a graph showing the background noise measurement result of the silicon detector. In the figure, the horizontal axis indicates the input power (mW) from the single mode optical fiber 13 into the optical waveguide device 1, and the vertical axis indicates the background noise (μW) when the silicon detector 16 is set at 510 nm.

  From the results of FIGS. 4 and 7, it can be seen that the detection of the fluorescence power of GFP, which is one digit higher than the background noise level, has succeeded. From the above, it has been shown that quantitative fluorescence observation using the silicon detector 16, which has been difficult in the past, is also possible.

  FIG. 8 is a graph of the lifetime measurement results showing the fluorescence power after 10 days have passed after the production of the optical waveguide device 1 and stored in a refrigerator. FIG. 9 is a similar graph showing fluorescence power after 19 days and FIG. 10 after 30 days. The optical waveguide device 1 showed strong fluorescence with the naked eye even after 10 days, and fluorescence was observed with the naked eye after 19 days and after 30 days.

  FIG. 11 is a graph obtained by observing a change in fluorescence after a buffer solution (pH = 4.0) was dropped onto the optical waveguide device 1 and allowed to enter the optical waveguide 8 doped with GFP. As shown in the figure, the fluorescence intensity from the optical waveguide 8 doped with GFP showed dependency on the buffer solution.

It is the figure which showed schematically the optical waveguide device in one Embodiment of this invention, (a) is a perspective view, (b) is sectional drawing of a waveguide axial direction. It is the figure which showed roughly the fluorescence analyzer in one Embodiment of this invention. It is the figure which showed the preparation processes of a GFP dope sol gel optical waveguide device. It is a graph which shows the fluorescence detection result by the silicon detector after background noise correction | amendment. It is a graph which shows the result of the transmission loss measurement of a GFP dope sol gel silica optical waveguide (waveguide length 5mm). It is a graph which shows the result of the transmission loss measurement of a GFP dope sol gel silica optical waveguide (waveguide length 10mm). It is a graph which shows the background noise measurement result of a silicon detector. It is a graph of the lifetime measurement result which shows the fluorescence power after storage of a refrigerator after preparation of an optical waveguide device, and 10 days progress. It is a graph of the lifetime measurement result which shows the fluorescence power after 19 days progress, after producing an optical waveguide device and storing with a refrigerator. It is a graph of the lifetime measurement result which shows the fluorescence power after 30 days after storing in a refrigerator after preparation of an optical waveguide device. It is the graph which observed the fluorescence change after making buffer solution dripped at an optical waveguide device, and making it infiltrate into the optical waveguide doped with GFP.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical waveguide device 2 Core 2a Incident side end surface 2b Outgoing side end surface 3 Clad 3a Lower clad 3b Side surface clad 4 Core formation groove part 5 Substrate 6 Mask 8 Optical waveguide 10 Fluorescence analyzer 11 Ar ion laser 12 Laser fiber coupler 13 Single mode Optical fiber 14 Microscope objective lens 15 Dichroic filter 16 Silicon detector

Claims (6)

  An optical waveguide device comprising an optical waveguide having a core made of sol-gel silica having a fluorescent protein immobilized therein.   The optical waveguide device according to claim 1, wherein the fluorescent protein is GFP (Green Fluorescent Protein).   The optical waveguide device according to claim 1, an excitation light source for introducing excitation light of fluorescent protein into the optical waveguide from the end surface on the core incident side of the optical waveguide device, and an end surface on the core exit side of the optical waveguide device A fluorescence analyzer comprising a photodetector for detecting fluorescence of a fluorescent protein.   The optical waveguide of the optical waveguide device is a single mode optical waveguide, and at least one of the core incident side end surface and the core output side end surface is connected to the single mode optical fiber. Fluorescence analyzer.   5. A method of detecting a chemical substance using the fluorescence analyzer according to claim 3 or 4, wherein the optical waveguide of the optical waveguide device is brought into contact with a chemical substance to be detected, and excitation light from an excitation light source is guided. Fluorescence of fluorescent protein from the optical waveguide after being introduced into the waveguide is detected by a photodetector, and the chemical substance is changed by the amount of fluorescence from the fluorescent protein based on the pH change in the optical waveguide due to the chemical substance that has penetrated into the optical waveguide. A method for detecting a chemical substance, comprising detecting   The chemical substance detection method according to claim 5, wherein the chemical substance is a phosphorus compound.

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