JP2005181296A - Localized surface plasmon sensor, sensing apparatus, and sensing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sensor microminiaturized so as to have a measuring range of the order of μm, easily manufactured, and sensitively detecting an interaction such as a reaction between an antigen and an antibody in situ without a label such as a fluorescent dye. <P>SOLUTION: The localized surface plasmon sensor is formed with a metal particulate layer 4 having a dimension for exciting a localized surface plasmon resonance on an end face of an optical fiber 2. A molecular layer of molecules 6 complementary to detected molecules 5 is formed on a surface of the metal particulate layer 4. The detected molecules 5 adsorbed or bonded to the complementary molecules 6 are detected by using a change of a light inputted to the optical fiber 2 due to the surface plasmon resonance localized in the metal particulate layer 4. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a sensor. More specifically, the present invention relates to a localized surface plasmon sensor, a sensing device, and a sensing method using them, which detect a detection target molecule present in a minute region.

  In order to apply the knowledge gained from the rapid development of genetic engineering and life sciences to medicine, pharmacy, engineering, etc., it is necessary to develop technology for sensing protein and DNA interactions and antigen-antibody reactions with high sensitivity and high throughput. Development is required. In existing sensing devices, a method using a fluorescent label, a method using surface plasmon resonance measurement using a total reflection attenuation method, a sensing method using a crystal resonator (the molecular weight adsorbed on the crystal resonator A method of reading as a change) is used. Although these sensing methods are excellent methods, it is difficult to miniaturize to a micron size or smaller size while maintaining a high sensitivity that can detect hybridization of DNA because the probe structure is greatly limited.

  FIG. 13 is a diagram for explaining the principle of sensing using surface plasmon resonance. Reference numeral 101 denotes a surface plasmon sensor. A metal fine particle layer 104 (made of, for example, gold fine particles) as a transducer is formed on the end surface 103 of the substrate 102. Next, a molecular layer (for example, a monomolecular layer) of molecules 106 a and b (ligand) complementary (strong affinity) to the detection target molecules 105 a and b (analyte) is formed on the surface of the metal fine particle layer 104. To construct. Here, the transducer converts the adsorption and binding of complementary molecules, the change in refractive index, and the like into optical signals such as reflected light intensity, scattered light intensity, and resonance wavelength. When the substrate 102 on which this sensor is mounted is immersed in the sample solution, if there are analytes 105a and 105b, they are selectively adsorbed or bound to the ligands 106a and 106b by their interaction (affinity). Since the analytes 105a and 105b and the ligands 106a and 106b are complementary to each other, they have a relationship like a key and a keyhole, and the interaction between complementary substances (for example, 105a and 105b) is very strong. The interaction between non-complementary substances (eg 105a and 106b) is very weak. If the amount of the adsorbed or bound analyte 105 can be sensed in real time, it is possible to immediately analyze the sample solution and discuss the interaction between molecules. For example, when an oligonucleotide is used as the ligand 106, an oligo DNA having a complementary base sequence is hybridized and can be detected. By discussing the kinetics of hybridization, it is possible to discuss base mismatches. Further, in an experiment using an antibody as a ligand, the target antibody can be detected with high sensitivity. Based on the same principle, it is possible to detect lipid-protein and sugar-protein interactions, and the versatility of this technique is high.

  What is required of a means for tracking such interactions between biomolecules is (a) high sensitivity capable of detecting adsorption or binding of monomolecular layers of several nanometers, and (b) real-time detection in solution. (C) It is possible to detect with a simple device. Local plasmon resonance is capable of confining or propagating light energy in metal particles that are sufficiently small (several nm to 100 nm) compared to the wavelength of light. The resonance condition is sensitive to the state near the surface, and has a feature that the resonance condition changes greatly due to adsorption / bonding or desorption of a substance. Therefore, biosensing using localized plasmon resonance can detect the interaction of biomolecules without using fluorescent labels, and uses nanometer-sized metal microparticles. The miniaturization is expected.

  FIG. 14 shows a configuration example of a conventional surface plasmon sensor 111 (see Non-Patent Document 1). The prism 112 is closely attached to one surface of the first glass substrate 114 by the matching oil 113. A gold thin film 115 (about 50 nm thick) is deposited on the surface of the first glass substrate 114 opposite to the prism 112. A second glass substrate 116 is disposed on the side of the first glass substrate 114 on which the gold thin film 115 is deposited via a spacer 117, and is formed in a space between the first and second glass substrates 114, 116 and the spacer 117. The sample 119 containing the detection target molecule (analyte) is injected from the sample container 118. Incident light 120 is incident on the prism 112 from the normal to the surface of the first glass substrate 114 at an incident angle θ (a condition where total reflection occurs). Due to surface plasmon resonance, when the incident angle θ is in the vicinity of the resonance angle, the energy of the incident light is absorbed and the reflectance of the reflected light 121 decreases. The reflected light 121 is detected by a photodetector.

  The dispersion relation of surface plasmon resonance is sensitive to the surface state, and enables highly sensitive detection of substances adsorbed on the surface. For example, when an ultra-thin film having a refractive index of 1.5 and a thickness of 1 nm is adsorbed on the surface, the resonance angle is shifted by about 0.2 degrees to the high angle side, and a difference ΔR occurs in the reflectance. Using this surface plasmon sensor 111, adsorption and desorption can be observed with a sensitivity equal to or lower than that of a monomolecular layer even if the ultra-thin sample does not have a label such as a fluorescent dye. However, the gold thin film 115 needs to be modified on a wide surface of the first glass substrate 114, and is not miniaturized so as to be inserted into a minute region such as a living body.

FIG. 15 shows a configuration example of a conventional localized surface plasmon sensor 131 (see Non-Patent Document 2). Gold fine particles 133 are attached to the inner wall of the cell 132, a sample 135 containing a molecule to be detected (analyte) is injected into the cell 132, and light (incident light I ₀ ) enters the cell 132 (FIG. 15A )), Or the surface of the gold fine particle 133 on the substrate is covered with the PMMA film 134 and incident light (incident light I ₀ ) is incident (FIG. 15B), and the transmitted light I is detected, thereby causing surface plasmon resonance. Light absorption can be measured. Since the gold fine particle 133 is used, the localized surface plasmon is excited.

  Using this localized surface plasmon sensor 131, the peak of the absorption spectrum of the gold fine particles 133 depends on the refractive index of the sample 135 immersed or the film thickness of the polymer dielectric (PMMA film or the like) 134 covering the surface. It is shown that the peak shift becomes exponential with respect to the film thickness. This result is not an example of direct detection of a biological molecule, but suggests that biosensing in localized plasmon resonance can be realized with high sensitivity and has high quantitativeness. However, the gold fine particles 133 need to be modified on a relatively wide surface such as the inner wall of the cell 132, and are not miniaturized so that they can be inserted into a minute region such as a living body.

  FIG. 16 shows a configuration example of another conventional localized surface plasmon sensor 141 and a configuration example of a sensing device 140 using the sensor (see Non-Patent Document 3). Light emitted from the tungsten-halogen light source 142 is incident on a sensor 141 having an optical fiber core 146 in which a single-layer gold particle 145 is modified, via a collimator lens 143 and an optical fiber coupler 144. Gold particles 145 are modified on the side and tip of the optical fiber core 146. An optical fiber core 146 modified with a single layer of gold particles 145 is immersed in a cell 148 into which a sample 147 containing a molecule to be detected (analyte) is injected, and the light transmitted through the optical fiber core 146 is passed through the fiber. Detected by the optical spectrometer 149, the detection signal is transmitted to the computer 150 for processing. A change in the intensity of transmitted light is measured using a sample 147 having a different refractive index with a different sucrose concentration. This change is due to the binding of the molecule to be detected (analyte) and is attributed to localized surface plasmon resonance.

  This sensor uses an optical fiber and is smaller than the previous two examples. However, the gold fine particle 145 also modifies the side surface of the optical fiber 146, and mainly uses localized plasmon resonance excited by evanescent light in this portion. For this reason, it is not possible to measure a micro three-dimensional region below a micron order. Further, since the transmitted light is measured, it is not possible to perform processing such as inserting the tip of the optical fiber into the place where measurement is desired.

  FIG. 17 shows a configuration example of another conventional surface plasmon sensor 151 (see Patent Document 1). On the end surface 153 of the optical fiber 152, a conical convex portion 156 (or a concave portion) that is sharpened only at the core 155 portion with the end surface of the clad 154 as a reference surface is provided. A metal thin film 157 is fixed to the sharpened conical surface of the convex portion 156 (or the concave portion) to obtain a surface plasmon resonance (SPR) measurement surface 158. When light is incident on the conical convex portion 156 (or concave portion) from the core 155 of the optical fiber 152, the surface plasmon wave 160 is generated when the angle formed by the incident light 159 and the conical surface is the resonance angle, and the reflected light is reflected. By detecting 161, surface plasmon resonance can be detected. In FIG. 17, D indicates the optical fiber diameter, and d indicates the core diameter.

  Since the metal thin film 157 is fixed to the end face 153 of the optical fiber 152 and the end face portion of the core 155 is used as the detection portion, measurement at a minute portion or measurement for a minute amount of sample is possible. In addition, the position of the surface plasmon resonance measurement surface can be freely set, and interactions between substances in the living body can be measured in real time. However, it is necessary to form a sharp conical convex portion 156 (or concave portion) on the end face 153, and the manufacturing process including the adjustment of the angle becomes complicated accordingly. In addition, since a flat metal film is used, even if localized surface plasmon occurs, it is considered that the portion is limited to the convex portion or the apex portion of the concave portion, and the localized surface plasmon is always highly sensitive. It cannot be said that is used.

JP 2001-165852 (paragraphs 0017-0030, FIG. 1-8) “Surface plasmon resonance measurement of ultra-thin organic films” Kotaro Kajikawa Surface Science Vol. 21, no. 10, pp630-634, 2000 “Local Plasmon sensor with gold colloid monolayers deposited up glass substrates” Takayuki Okamoto and Ichirou Yamaguchi, OpticalL et al. 25, no. 6, pp 372-374, March 15,2000 “Colloidal Gold-Modified Optical Fiber for Chemical and Biochemical Sensing” Shu-Fang Cheng and Lai-Kwan Chau, Anal. Chem. 2003, 75, 16-21

  What is required of a high-sensitivity sensor, particularly means for tracking the interaction between biomolecules, is (a) high sensitivity capable of detecting adsorption or binding of a monomolecular layer of about several nm, and (b) real-time detection in a solution. (C) The detection is possible with a simple device. The use of localized plasmon resonance is a technique that satisfies these conditions. However, any of the existing methods (except for the sensor of FIG. 17) is difficult to reduce to the micrometer order.

  Therefore, using the localized surface plasmon without using the conventional surface plasmon resonance, and forming the detection portion on the end face of the optical fiber to reduce the size of the sensing probe (or sensor head), and the optical fiber coupler We decided to pursue miniaturization of the optical system using In addition, it was decided to pursue a sensor that is easy to manufacture and inexpensive, without forming a sharpened convex part (or concave part) on the end face or adjusting the angle.

  In the present invention, (1) the measurement area can be reduced to the micrometer order (target of 200 μm or less), (2) it is easy and inexpensive to manufacture, and in particular, the angle of the surface on which the metal is fixed in the sensor head. (3) It is possible to detect antigen-antibody reaction and interactions such as DNA and protein with high sensitivity on the spot without labeling with fluorescent dye, etc. (4) An optical fiber is attached to the sensor head. That it can be used to introduce into a microscopic area, that it can be carried and can expand the measurable range, and that a large optical system is not required. (5) The sensor head is inexpensive enough to be disposable. Sensors and sensors that satisfy the following conditions: (6) Do not use reagents and materials that are harmful to living organisms, and be safe even if the light source is viewed directly with the naked eye It is an object to provide an operating device.

  In addition, the present invention realizes a localized surface plasmon sensor capable of detecting with high sensitivity even when detection of a small amount of sample such as a protein solution is required based on the localized surface plasmon in gold fine particles. With the goal. Another object of the present invention is to make it possible to measure a wide range of light spectrum at a time in order to increase the efficiency of measurement and to ensure higher reliability of data.

  In order to solve the above-mentioned problem, the localized surface plasmon sensor according to claim 1 has a dimension in which localized surface plasmon resonance is excited on the end face 3 of the optical fiber 2 as shown in FIG. And a molecular layer of molecules 6 complementary to the detection target molecule 5 on the surface of the metal fine particle layer 4, and the optical fiber 2 by surface plasmon resonance localized in the metal fine particle layer 4. The detection target molecule 5 adsorbed or bound to the complementary molecule 6 is detected using the change in the light input to.

  Here, complementary means that the affinity is strong. Adsorption means physical adsorption, and bond means chemical bond. Localized surface plasmon resonance is a wave of localized electrons excited in a fine structure of metal (for example, 1 nm to 1 μm, generally about several tens of nm to 100 nm). Happens on the surface. The metal fine particles are excited if the particle diameter is about the wavelength or less.

  With this configuration, the measurement region (detection portion) can be reduced to the micrometer order, and it is necessary to form conical protrusions and recesses for fixing metal fine particles in the sensor head, and the inclination angle of the cone It is easy to manufacture and inexpensive, and can be used to detect antigen-antibody reactions and interactions such as DNA and proteins with high sensitivity on the spot without labeling with fluorescent dyes. It is done.

  Further, the localized surface plasmon sensor according to claim 2 has a function of the molecule 6 complementary to the detection target molecule 5 to a dimension that excites the surface plasmon resonance localized on the end face 3 of the optical fiber 2. A molecule to be detected which is adsorbed or bonded to a complementary molecule 6 using a change in light input to the optical fiber 2 by surface plasmon resonance localized in the metal fine particle layer 4. 5 is detected.

  When configured in this manner, as in the case of claim 1, the measurement region (detection portion) can be miniaturized on the order of μm, and the conical convex portion or concave portion for fixing the metal fine particles in the sensor head is provided. It is not necessary to form or adjust the angle of inclination of the cone, and it is easy and inexpensive to manufacture. It is highly sensitive to interactions such as antigen-antibody reactions and DNA and proteins without labeling with fluorescent dyes. The effect that it can be detected in the field is obtained.

  According to a third aspect of the present invention, in the localized surface plasmon sensor according to the first or second aspect, the metal fine particle layer 4 contains a noble metal as a main component. Such a configuration is suitable for obtaining localized surface plasmons. In particular, when gold and silver are used, highly sensitive measurement is possible.

  According to a fourth aspect of the present invention, in the localized surface plasmon sensor according to any one of the first to third aspects, the thickness of the metal fine particle layer 4 is 1 nm to 1 μm. Such a configuration is suitable for obtaining localized surface plasmons. Of these, a thickness of about 1 nm to 100 nm is more preferable.

  According to a fifth aspect of the present invention, in the localized surface plasmon sensor according to any one of the first to fourth aspects, the diameter of the detection portion on the end face 3 of the optical fiber is 5 mm or less. Of these, 200 μm or less is preferable because a fine region can be detected, and 100 μm or less is preferable because a fine region can be further detected. Further, since an optical fiber having a core diameter of 5 μm exists, there is a possibility of miniaturization up to a diameter of 1 μm of the detection portion, and 1 μm or more is preferable.

  Further, the localized surface plasmon sensing device according to claim 6 is a localized surface plasmon sensor 1 according to any one of claims 1 to 5 (for example, as shown in FIG. 2). An optical fiber constituting the surface plasmon sensor 1 is defined as a first optical fiber 2), a light source 8 that emits light that excites the surface plasmon resonance, and a photodetector 9 that detects light. An optical fiber coupler 10 that can divide and output light, a second optical fiber 11 that guides the light emitted from the light source 8 to the optical fiber coupler 10, and a first optical fiber 2 and an optical fiber coupler 10 are connected. The light emitted from the light source 8 is guided from the optical fiber coupler 10 to the end face 3 of the first optical fiber 2 of the localized surface plasmon sensor 1 (the end face is defined as the first end face), and from the first end face 3 Reflected or scattered It comprises a third optical fiber 12 for guiding light to the optical fiber coupler 10, and a fourth optical fiber 15 for guiding the light reflected from the first end surface 3 from the optical fiber coupler 10 to a photodetector 9.

  Here, the light scattered from the first end face 3 enters the metal fine particle layer 4 through the first end face 3, is scattered by the excited localized surface plasmon resonance, and passes through the first end face 3. Light that passes through and returns to the first optical fiber. Further, the light reflected from the first end face 3 is irradiated to the metal fine particle layer 4 through the first end face 3 and reflected, and then passes through the first end face 3 and passes through the first optical fiber 2. It also includes the light returned to.

  With this configuration, it is possible to attach and use an ultra-compact sensing probe 1 (having a sensor head at the tip) of the order of μm using the optical fiber 2 and further downsize the optical system. Moreover, the sensing probe 1 (that is, the sensor head at the tip thereof) can be introduced into a minute region, and measurement at a minute part or measurement of a minute amount of sample is possible. Furthermore, since the sensing probe 1 is portable, the measurable range is widened. It is also possible to measure interactions between substances in the living body in real time.

  Further, in the localized surface plasmon sensing device 7 according to claim 6, for example, the localized surface plasmon sensor 1, the third optical fiber 12, and the like as shown in FIG. Are connected by a splicer 18 so as to be detachable. If comprised in this way, the sensing probe 1 (with a sensor head) can be replaced | exchanged using the splicer 18, and it is hardly necessary to align at the time of replacement | exchange of the sensing probe 1, and replacement | exchange is easy.

  A localized surface plasmon sensing device according to claim 8 is a localized surface plasmon sensor 1 according to any one of claims 1 to 5 (which constitutes the localized surface plasmon sensor 1). A first optical fiber 2), a light source 8 that emits light that excites surface plasmon resonance, a photodetector 9 that detects light, and an optical fiber that can divide and output input light. The coupler 10, the second optical fiber 11 that guides the light emitted from the light source 8 to the optical fiber coupler 10, and the fourth that guides the light from the localized surface plasmon sensor 1 to the photodetector 9 from the optical fiber coupler 10. The first optical fiber 2 is connected to the optical fiber coupler 10, and the light emitted from the light source 8 is transmitted from the optical fiber coupler 10 to the first optical fiber of the localized surface plasmon sensor 1. 2 to the end face 3 (the end face is the first end face), the light reflected or scattered from the first end face 3 is guided to the optical fiber coupler 10, and the fourth optical fiber 15 is the first end face 3. The light reflected or scattered from the light is guided from the optical fiber coupler 10 to the photodetector 9. With this configuration, sensing with the microminiature sensing probe 1 of the order of μm using the optical fiber 2 becomes possible, and the sensor probe 1 can be configured without using the splicer 18.

  Further, the invention according to claim 9 is detected by the photodetector 9 in the localized surface plasmon sensing device according to any one of claims 6 to 8, for example, as shown in FIG. From the intensity, phase, or amount of resonance wavelength of the localized surface plasmon resonance, the film thickness, adsorption amount or binding amount of the detection target molecule 5 or the localized surface plasmon sensor 1 A computing device 20 is provided for calculating the refractive index of a substance containing surrounding complementary molecules 6. If comprised in this way, based on the detected optical signal, quantitative detection will be attained. In addition, since the localized surface plasmon resonance wavelength varies depending on the refractive index of the surrounding gas, liquid, etc., the localized surface plasmon sensor 1 also functions as a refractive index sensor.

  Further, in the localized surface plasmon sensing device according to claim 9, for example, as shown in FIG. 2, the invention described in claim 10 is a lock-in detection that locks in a signal output from the photodetector 9. An amplifier 19 is provided, and the arithmetic unit 20 acquires a signal output from the photodetector 9 via the lock-in amplifier 19. If comprised in this way, a sensitivity can be improved using the lock-in amplifier 19, and it is suitable for trace amount detection.

  The invention according to claim 11 is the localized surface plasmon sensing device according to any one of claims 6 to 10, wherein the light source 8 has a wavelength in the wavelength range of 550 nm to 740 nm. Emits light. As the light source, a light emitting diode (LED), a laser, or the like may be used alone, or a superluminescent diode, a halogen lamp, or the like may be used in combination with a filter or a spectroscope. If comprised in this way, the sensitivity of a sensor can be made high.

  In addition, the localized surface plasmon sensing device 7A according to claim 12 includes a localized surface plasmon sensor 1 according to any one of claims 1 to 5, as shown in FIG. An optical fiber constituting the localized surface plasmon sensor 1 is defined as a first optical fiber 2), a light source 21 that emits light including a predetermined wavelength range that excites surface plasmon resonance, and a predetermined wavelength range. A spectrometer 22 that detects an optical spectrum of light, an optical fiber coupler 10 that can divide and output input light, and a second optical fiber 11 that guides light including a predetermined wavelength range from the light source 21 to the optical fiber coupler 10. And the first optical fiber 2 and the optical fiber coupler 10 are connected, and light including a predetermined wavelength range is transmitted from the optical fiber coupler 10 to the first optical fiber of the localized surface plasmon sensor 1. A third optical fiber 12 that guides the light including a predetermined wavelength range reflected or scattered from the first end surface 3 to the optical fiber coupler 10; And a fourth optical fiber 15 that guides light including a predetermined wavelength range reflected from the first end face 3 from the optical fiber coupler 10 to the spectrometer 22.

  If comprised in this way, the sensing by the microminiaturized sensing probe 1 of the micrometer order using the optical fiber 2 will be attained, and the optical spectrum data over a wide range of wavelengths can be measured at a time. In addition, it is possible to measure the interaction between substances in the living body with high sensitivity in real time.

  Further, the invention according to claim 13 is the localized surface plasmon sensing device according to claim 12, for example, as shown in FIG. 9, wherein the localized surface plasmon sensor 1 and the third optical fiber 12 are connected. The splicer 18 is connected so as to be detachable. If comprised in this way, a sensor head can be replaced | exchanged using the splicer 18, and it is hardly necessary to align at the time of replacement | exchange of a sensor head, and replacement | exchange is easy.

  A localized surface plasmon sensing device according to claim 14 is a localized surface plasmon sensor 1 according to any one of claims 1 to 5 (which constitutes the localized surface plasmon sensor 1). A first optical fiber 2), a light source 21 that emits light including a predetermined wavelength range that excites surface plasmon resonance, and a spectrometer that detects an optical spectrum of light including the predetermined wavelength range. 22, an optical fiber coupler 10 that can divide and output input light, a second optical fiber 11 that guides light including a predetermined wavelength range from the light source 21 to the optical fiber coupler 10, and a localized surface plasmon sensor 1. And a fourth optical fiber 15 for guiding the light from the optical fiber coupler 10 to the spectrometer 22, and the first optical fiber 2 is connected to the optical fiber coupler 10 and has a predetermined wavelength range. The contained light is guided from the optical fiber coupler 10 to the end face 3 of the first optical fiber 2 of the localized surface plasmon sensor 1 (the end face is defined as the first end face) and reflected or scattered from the first end face 3. Light including a predetermined wavelength range is guided to the optical fiber coupler 10, and the fourth optical fiber 15 transmits light including the predetermined wavelength range reflected or scattered from the first end surface 3 from the optical fiber coupler 10 to the spectrometer 22. Lead. With this configuration, sensing with the microminiature sensing probe 1 of the order of μm using the optical fiber 2 becomes possible, and the sensor probe 1 can be configured without using the splicer 18. Moreover, optical spectrum data over a wide range of wavelengths can be measured at a time. In addition, it is possible to measure the interaction between substances in the living body with high sensitivity in real time.

  In the localized surface plasmon sensing device according to any one of claims 12 to 14, the predetermined wavelength range is a wavelength of 550 nm to 740 nm. If comprised in this way, the sensitivity of a sensor can be made high.

  Further, in the sensing method using the localized surface plasmon sensor according to claim 16, the metal fine particle layer 4 having a size capable of exciting the localized surface plasmon resonance is formed on the end face 3 of the optical fiber 2, A step of preparing a localized surface plasmon sensor 1 in which a molecular layer of a molecule 6 complementary to a detection target molecule 5 is formed on the surface of the metal fine particle layer 4 and light for exciting surface plasmon resonance on the end face of the optical fiber 2 And the localized surface plasmon resonance based on the light that excites the surface plasmon resonance in the metal fine particles 4 of the localized surface plasmon sensor 1, and the reflected light from the end face 3 of the optical fiber 2 or The step of detecting the scattered light and the end face 3 of the optical fiber 2 of the localized surface plasmon sensor 1 are immersed in the sample 17 including the detection target molecule 5, and the detection target molecule 5 is absorbed by the complementary molecule 6. Alternatively, the localized surface plasmon resonance based on the light that excites the surface plasmon resonance is excited in the metal fine particle 4 of the localized surface plasmon sensor 1 immersed in the sample 17, and the step of coupling is performed. The step of detecting reflected light or scattered light from the end face 3, reflected light or scattered light when the end face 3 of the optical fiber 2 is not immersed in the sample 17, and the end face 3 of the optical fiber 2 in the sample 17 A step of detecting the detection target molecule 5 by comparing the reflected light or the scattered light when immersed.

  When configured in this way, the microminiature sensing probe 1 (having a sensor head at the tip) of the micrometer order can be used to enhance the antigen-antibody reaction and the interaction between DNA and protein without labeling with a fluorescent dye or the like. Sensitivity can be detected on the spot. The reflected light and scattered light are understood in the same manner as in the sixth aspect.

  The invention according to claim 17 is the sensing method using the localized surface plasmon according to claim 16, wherein the wavelength of the light that excites the surface plasmon resonance is in the wavelength range of 550 nm to 740 nm. Is the wavelength. If comprised in this way, the sensitivity of a sensor can be made high.

In addition, the sensing method using the localized surface plasmon sensor according to claim 18 forms the metal fine particle layer 4 having a dimension capable of exciting the localized surface plasmon resonance on the end face 3 of the optical fiber 2, A step of preparing a localized surface plasmon sensor 1 in which a molecular layer of a molecule 6 complementary to a molecule to be detected 5 is formed on the surface of the metal fine particle layer 4, and the end face 3 of the optical fiber 2 includes a predetermined wavelength range. The step of introducing light, the localized surface plasmon resonance based on the light including a predetermined wavelength range is excited in the metal fine particles 4 of the localized surface plasmon sensor 1, and the reflected light from the end face 3 of the optical fiber 2 Alternatively, the step of detecting the spectrum of the scattered light and the end surface 3 of the optical fiber 22 of the localized surface plasmon sensor 1 are immersed in the sample 17 including the detection target molecule 5, and the detection target molecule is complemented by the complementary molecule 5. Suck 5 Alternatively, the step of coupling and exciting the localized surface plasmon resonance based on the light including a predetermined wavelength range in the metal fine particles 4 of the localized surface plasmon sensor 1 immersed in the sample 17, The step of detecting the spectrum of reflected light or scattered light from the end face 3, the spectrum of reflected light or scattered light when the end face 3 of the optical fiber 2 is not immersed in the sample 17, and the end face 3 of the optical fiber 2 are A step of detecting the detection target molecule 5 by comparing the spectrum of reflected light or scattered light when immersed in the sample 17.
If comprised in this way, the sensing by the microminiaturized sensing probe 1 of the micrometer order using the optical fiber 2 will be attained, and the optical spectrum data over a wide range of wavelengths can be measured at a time.

  According to a nineteenth aspect of the present invention, in the sensing method using the localized surface plasmon sensor according to the eighteenth aspect, the predetermined wavelength range is a wavelength of 550 nm to 740 nm. If comprised in this way, the sensitivity of a sensor can be made high.

  As described above, the localized surface plasmon sensor and sensing device according to the present invention (including the invention of each claim) have the following effects. (1) A sensor head having a diameter of 200 μm or less can be produced, for example, with a detection portion (corresponding to the core portion of the end face of the optical fiber) that can be miniaturized on the order of μm. (2) There is no need to form conical convex portions or concave portions for fixing metal fine particles in the sensor head, and there is no need to adjust the inclination angle of the cone, and the manufacturing is easy and inexpensive. (3) Without labeling with a fluorescent dye or the like, antigen-antibody reaction and interaction of DNA, protein, etc. can be detected in situ with high sensitivity. (4) By using an optical fiber for the sensor head, the sensor head can be introduced into a minute region. Moreover, it is portable, so that the measurable range can be expanded. In addition, the optical system can be made small. (5) The sensor head can be replaced using a splicer, and there is almost no need for alignment when replacing the sensor head, and replacement is easy. (6) Do not use reagents and materials that are harmful to the body. In addition, when a light-emitting diode is used as the light source, safety to the living body is high. Among these, the first sentence of (1) to (4) is the effect according to the present invention, and the second sentence and the following of (4) are the effect of the preferred embodiment of the present invention.

  Further, according to the present invention, even when detection of a small amount of sample such as a protein solution is desired based on the localized surface plasmon in the gold fine particles, a small amount of sample can be detected, and A highly sensitive localized surface plasmon sensor equivalent to a conventional surface plasmon sensor (which requires a large amount of sample) can be provided. Moreover, according to the preferable aspect of this invention, a wide optical spectrum can be measured at once.

  Biosensing using localized plasmon resonance uses nanometer-sized metal microparticles, so the micrometer of the sensing portion or nanometer-sized miniaturization is expected. Furthermore, since biosensing using localized plasmon resonance can achieve high sensitivity with a simple optical system, the detection part is sub-micron-sized, and the super-high-density biochip is two-dimensionally arranged. The construction of an array is expected. If a massively parallel measurement system using this chip can be realized, a large amount of information analysis can be expected on a small biochip compared to an existing DNA chip or the like. These will be new tools in fields that require a large amount of DNA information such as bioinformatics, and are expected to make a significant contribution to fields such as custom-made medicines.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 shows the configuration of a localized surface plasmon sensor according to the first embodiment. Reference numeral 1 denotes a localized surface plasmon sensor (sensing probe). A metal fine particle layer 4 as a transducer is formed on the end face 3 (first end face) of the optical fiber 2 (first optical fiber), and is complementary to the detection target molecule 5 (analyte) on the metal fine particle layer 4. A molecular layer of molecules 6 (ligands) is formed. Typically, gold fine particles are used as the metal fine particles, and the ligand 6 is a monomolecular layer. For example, as a model of an antigen-antibody reaction, an example in which avidin 5 as an analyte is detected using a sensing probe 1 whose surface is modified with a biotin monolayer as a ligand 6 can be mentioned. Biotin and avidin are complementary antigen-antibodies, and the interaction is strong.

  FIG. 2 shows the configuration of the localized surface plasmon sensing device 7 in the first embodiment. In creating a localized surface plasmon sensor, the detection portion of the sensing probe 1 was miniaturized to a size of 200 μm or less, and the object was to enable detection with a simple optical system. Here, a simple optical system as shown in FIG. 2 was prepared, and detection was performed using an optical fiber sensing probe using localized surface plasmons.

  Reference numeral 7 denotes a localized surface plasmon sensing device. As the light source 8, a green light emitting diode (LED) having a wavelength of 520 nm close to the actual resonance wavelength was used. The reason is that the stability can be easily improved as compared with the laser, the safety to the living body is high, and it is suitable for practical use at a low cost. The LED was modulated at 1.087 kHz using a suitable oscillator. The detection part of the sensing probe 1 (corresponding to the core part of the end face of the optical fiber) was 50 μm in diameter.

  The localized surface plasmon sensing device 7 includes a light source 8 that emits light that excites surface plasmon resonance, a photomultiplier tube 9 that functions as a photodetector that detects light, and light that divides input light and outputs the light. The fiber coupler 10, the second optical fiber 11 that guides the light emitted from the light source 8 to the optical fiber coupler 10, the first optical fiber 2 and the optical fiber coupler 10 are connected, and the light emitted from the light source 8 is A third light guides from the optical fiber coupler 10 to the end face 3 (first end face) of the first optical fiber 2 of the localized surface plasmon sensor 1 and guides the light reflected from the first end face 3 to the optical fiber coupler 10. And the second end face 13 treated to reduce the reflected light, and the other is reflected from the first end face 3 and the fifth optical fiber 14 connected to the optical fiber coupler 10. Light And a fourth optical fiber 15 leading from Bakapura 10 to the photodetector 9. Note that the fifth optical fiber 14 may be omitted.

  Here, the first end face 3 side of the first optical fiber 2 of the sensing probe 1 can be immersed in the sample 17 containing the detection target molecule 5 and the like housed in the cell 16, and the first optical fiber 2 is the other side. The third optical fiber 12 and the splicer 18 are detachably connected on the end face side. The photomultiplier tube 9 as a photodetector is connected via a lock-in amplifier 19 to an arithmetic unit 20 that analyzes an optical signal detected by the photomultiplier tube 9.

  That is, the light emitted from the light emitting diode (LED) 8 enters the second optical fiber 11 and is introduced into the optical fiber coupler 10 (F-CPL-M22855, manufactured by Newport). After splitting the incident light using the optical fiber coupler 10, one light is introduced into the localized surface plasmon sensor 1 (sensing probe) connected via the third optical fiber 12 and the mechanical splicer 18, It reaches the first end face 3 of the first optical fiber 2 modified with the ligand 6. The other light reaches the end face 13 (second end face) of the fifth optical fiber 14. Since the reflected light from the second end face 13 has a significant effect on the measurement, the matching oil was used to scatter the light at the second end face 13 and minimize the reflected light therefrom.

  The signal light scattered and reflected on the first end face 3 side of the sensing probe 1 returns from the second optical fiber 11 connected through the mechanical splicer 18 to the optical fiber coupler 10. In the optical fiber coupler 10, the signal light is combined with the light (trace light) returned from the second end face 13 of the fifth optical fiber 14, and the difference is detected by the photomultiplier tube 9 as a photodetector. The detected signal is amplified by the photomultiplier tube 9 and detected by using a lock-in amplifier 19 (SR80, Stanford Research System) in order to increase the S / N ratio. It is converted into a digital signal using a conversion board (not shown) and processed by a computer. In the arithmetic unit 20, the film thickness, the amount of adsorption or the amount of binding of the detection target molecule 6 from the intensity, phase, or amount of resonance wavelength of the localized surface plasmon resonance detected by the photomultiplier tube 9, Alternatively, the refractive index of a substance containing complementary molecules 5 around the localized surface plasmon sensor is calculated.

  Local plasmon resonance is a wave of localized electrons excited in a minute structure of a metal (for example, a region of about 1 nm to 1 μm, generally several tens of nm to 100 nm), and occurs on a metal fine particle or a rough metal surface. . The metal fine particles are excited when the particle diameter is approximately 1/4 or less of the wavelength. Further, even a metal thin film is excited if the surface roughness is about the wavelength. For example, the reason why gold colloid particles exhibit a wine red color in water is that localized plasmons are excited in gold fine particles (colloid). In many cases, local plasmon resonance is analyzed to approximate a sphere and a spheroid. Consider the optical response of a metal microsphere that is sufficiently small compared to the wavelength of light. Fine spherical metal particles with a radius a having a complex dielectric constant ε1 (λ) are placed in a medium having a dielectric constant ε2 (λ). Note that ε1 (λ) is strongly dependent on wavelength λ. An actual sensor uses a large number of fine metal particles, but when the distance between the fine particles is large, the interaction can be ignored, so the optical response of one fine particle can be considered.

と書くことができる。 The polarizability α of the metal fine particles is

Can be written.

  At a wavelength λ where the absolute value of the denominator of (Expression 1) is minimum, the localized plasmon becomes a resonance state, and the magnitude of the polarizability becomes maximum. If the interaction is negligible, the scattered light intensity can be obtained from the light scattering cross section of the gold fine particles and the number of particles.

  FIG. 3 is a diagram for explaining the resonance wavelength dependence of the scattered light intensity. The solid line in the figure plots the scattered light intensity of metal particles having a diameter of 15 nm in water against the wavelength λ. A peak due to resonance is seen at the wavelength λ = 420 nm. Although the resonance wavelength is slightly different from the actually measured value (λ = 490 to 520 nm), it is understood that the model used (Drude-Lorentz model) cannot completely reproduce the dielectric constant of gold.

  In the sensor according to the present embodiment, the ligand molecule covers the metal microsphere as a dielectric layer. Further, the adsorption of the analyte on the ligand by the interaction corresponds to an increase in the thickness of the dielectric layer equivalently. Even in the case where such a dielectric layer covers a metal microsphere (shell type structure), the polarizability α (λ) as a whole can be analytically determined. The broken line in FIG. 3 shows the intensity of scattered light when a 5 nm thick dielectric layer having a refractive index of 1.5 covers gold fine particles. It can be seen that the scattered light spectrum changes greatly due to the adsorption of the dielectric.

  FIG. 4 is a diagram for explaining the dependence of scattered light intensity on the dielectric (detection target) film thickness. The scattered light intensity when the wavelength of the light source is fixed at the resonance wavelength λ = 420 nm is obtained with respect to the film thickness d, and is plotted as a ratio of change with respect to the scattered light intensity when there is no dielectric layer. Scattered light intensity changes by 5% due to adsorption of a 1 nm dielectric. By observing changes in the intensity of scattered light while fixing the wavelength in this way, it is possible to monitor the adsorption of a substance on the surface with high sensitivity, so that localized plasmon resonance can be applied to a sensor.

  FIG. 5 shows an example of a detection result obtained by detecting avidin using the localized surface plasmon sensing device 7 in the present embodiment. As an example of biosensing detection, the interaction of streptavidin, a kind of protein, on the surface modified with a biotin molecule, which is a model molecular system for antigen-antibody reaction, was traced. That is, the results of tracking the interaction between avidin 5 and biotin 6 having a strong interaction are shown. A sensing probe 1 modified with a biotin 6 monomolecular film as a ligand on the surface of a gold fine particle 4 deposited by modifying a silane coupling agent on the optical fiber end face 3 is used as a borate buffer solution (1.5 μM of avidin 5 as an analyte). ) And the adsorption process was measured. The vertical axis represents signal (scattered light) intensity (arb. Unit), and the horizontal axis represents time (sec).

  First, after confirming that the signal is stabilized by adding a solvent to the cell 16 for background measurement, the sample solution 17 is injected into the cell 16 (indicated by an arrow A in FIG. 5), and the signal is observed. The scattered light intensity immediately increased and reached a constant value within a few minutes, indicating that the adsorption reaction of avidin 5 to biotin 6 was completed (indicated by arrow B in FIG. 5). Although the size of avidin 5 is about 5 nm, it is known to actually act as a dielectric layer of about 4 nm, and the amount of change in signal obtained is larger than that of octadecanethiol molecules described later. From the measurement result of this adsorption process, it can be seen that the interaction between molecules is captured with sufficient sensitivity and S / N ratio.

  The combination of the analyte and the ligand is not limited to avidin and biotin, but DNA-DNA, DNA-RNA (ribonucleic acid), DNA-protein, DNA-sugar, DNA-organic compound, protein-protein, lipid-protein, It is also possible to detect interactions such as sugar-protein and protein-organic compounds.

  In the second embodiment, the metal fine particle also serves as a complementary molecule 6 (ligand). In this case, in FIG. 1 showing the configuration of the localized surface plasmon sensor, the metal fine particle layer 4 has a molecule 6 (ligand) complementary to the molecule to be detected 5 (analyte) and having a high affinity. It also serves as (or includes) a molecular layer. For example, when gold is used as the metal fine particle and octadecanethiol (ODT) is used as the molecule to be detected (analyte), the ODT is adsorbed on the gold surface at a high density and is self-assembled. A molecular film is formed. Therefore, in this case, the gold fine particle surface itself deposited on the optical fiber end face 3 can be regarded as the ligand 6. Therefore, in order to evaluate the performance as a biosensor, the adsorption reaction to the sensing probe 1 was detected using ODT as a well-defined sample.

FIG. 6 shows an example of detection results obtained by detecting octadecanethiol using the localized surface plasmon sensing device 7 (see FIG. 2) in the present embodiment. The vertical axis represents signal (scattered light) intensity (arb. Unit), and the horizontal axis represents time (sec). First, after confirming that the signal is stabilized by adding a solvent to the cell 16 for background measurement, the sample solution 17 is injected into the cell 16 (indicated by an arrow A in FIG. 6) and the signal is observed. The scattered light intensity immediately increased and reached a constant value in about 1 minute, indicating that the adsorption was completed (indicated by arrow B in FIG. 6). By comparison with a calibration curve measured in advance with reference to the result of the adsorption density of gold fine particles by a scanning electron microscope (SEM), the amount of change in signal and noise obtained, and the ODT molecule 5 on the gold fine particles 5 From this amount, the sensitivity of this sensing device (system) could be estimated as 10 pg / mm ² . That is, the sensitivity was almost the same as that of a biosensor using a commercially available surface plasmon resonance using the total reflection attenuation method.

  In addition to ODT, there are analytes that are complementary (strong affinity) to the metal surface. For example, for gold, organic sulfur compounds with thiol and disulfide groups, primary amines with amino groups, secondary amines, tertiary amine compounds interact, and for silver, in addition to these compounds , Compounds with carboxyl groups interact.

  Next, a third embodiment will be described. The localized surface plasmon sensing device 7A of the present embodiment uses a red light emitting diode (LED) as the light source 8, but the other configuration is the same as that of the first embodiment (see FIG. 2). .

  In the simulation using isolated metal spheres, the resonance wavelength of localized plasmon resonance is close to 420 nm, but this uses the simplified Drude-Lorentz model as a dielectric model of gold as described above. This is because the actual dielectric constant of gold cannot be accurately reproduced. When calculation is performed with the measured value of the dielectric constant of gold, the resonance wavelength of localized plasmon resonance appears in the vicinity of 520 nm. When a dielectric is formed on the gold surface, the resonance wavelength shifts to the longer wavelength side, and the scattered light intensity on the longer wavelength side also increases. This is supported by the experimental data described in the following embodiment (see FIG. 10). Therefore, for example, a red LED (λ = 623 nm) is suitable as the light source, and the sensitivity of the sensor can be made higher than that of the first embodiment, and detection is facilitated.

Next, creation of a sensing probe will be described. This can be applied in common to the first and third embodiments. The fixing of the gold fine particle layer 4 to the end face 3 of the fiber 2 can also be applied to the second embodiment. Gold fine particles can be synthesized, for example, by reduction of NaAuCl ₄ . Keeping an aqueous solution of 0.254 mM (100 mL) NaAuCl ₄ at 95 ° C. in a water bath, adding 2.5 mL of an aqueous citric acid solution (33.3 mM) and stirring, the color of the solution changes to ruby red within 1 minute. changed. When the solution was further stirred for 10 minutes and then cooled to room temperature, for example, gold fine particles having an average diameter of about 20 nm were obtained. The average diameter was measured with a transmission electron microscope. The end face 3 of the optical fiber 2 cracked cleanly is held at room temperature for 10 minutes in a solution obtained by adding 5 vol% acetic acid to an ethanol solution of N- (2-aminoethyl) 3-aminopropyl-trimetaoxysilane, and 120 A silane coupling agent was attached in an oven at 0 ° C., and then immersed in the gold fine particle aqueous solution, and the gold fine particle layer 4 was fixed to the end face 3 of the optical fiber 2. With such a procedure, about 20% or more of the end face of the fiber was covered with gold fine particles with a reproducibility of 80% or more (this is sufficient for experimental use).

  In order to measure the affinity between avidin and biotin, the surface of the gold fine particle layer 4 of the sensing probe 1 needs to be modified with biotin. The sensing probe 1 is immersed in an ethanol solution of 11-amino-1-andecanethiol hydrochloride having a concentration of 1 mM for 10 minutes, rinsed with ethanol, immersed in Sulfosuccinimidel-D-biotin for 10 minutes, and rinsed with a buffer solution. It was done. As a result, the surface of the gold fine particle layer 4 of the sensing probe 1 could be modified with the thiol layer 6 labeled with biotin at the end as a ligand. On the other hand, avidin 5 as an analyte was dissolved in a tetraborate buffer solution, and a 20 μg / mL solution was prepared in a cuvette.

In FIG. 7, in order to obtain the sensitivity of the sensor, the return light intensity (specifically, the intensity of the return light including reflected light and scattered light) was measured by changing the refractive index of the surrounding medium (a mixture of glycerol and water). Results are shown. The sensing probe 1 used was covered with a gold fine particle layer 4. The refractive index of the surrounding medium can be changed by adding 0 to 2.5 wt% of glycerol to water. FIG. 7A shows the response of the sensor when the glycerol concentration is increased stepwise (from A to F) by 0.5 wt%. The vertical axis represents the light intensity, and the horizontal axis represents time. FIG. 7B is a plot of the return light intensity (vertical axis) against the refractive index (horizontal axis) of the surrounding medium. Each plot in FIG. 7B corresponds to a refractive index change of 5.5 × 10 ⁻⁴ at each step shown in FIG. The standard deviation of the measurement value obtained by repeating the measurement once per second at each refractive index 60 times is 6 × 10 ⁻⁴ . The return light intensity signal is substantially proportional to the refractive index, and its slope is 29.9 RIU- ¹ . Dividing 6 × 10 ⁻⁴ by 29.9 gives a refractive index resolution of 2 × 10 ⁻⁵ , which corresponds to a general dielectric layer adsorption of 20 pg / mm ² . Thus, it can be seen that the sensitivity is equivalent to that of a sensor using a conventional propagation surface plasmon using a total reflection attenuation method or a grating. However, it can be concluded that the present sensor has sufficient performance that can be applied to a biosensor. In actual measurement with a sensor using a conventional propagation type plasmon, a sample must be put in a solution cell, a flow path, etc. must be formed and the sample must flow therethrough, and a considerable amount of sample must be prepared. However, in this surface plasmon sensor, it is sufficient that the solution is exposed to a detection surface having a diameter of about several μm to 1 mm, and even a sample of several μL or less can be detected.

  FIG. 8 shows the measurement results of the affinity of avidin for biotin. The vertical axis represents return light intensity (specifically, the intensity of return light including reflected light and scattered light), and the horizontal axis represents time. The sensing probe 1 in which the end face 3 of the optical fiber 2 was modified with a gold fine particle layer 4 covered with a thiol monolayer 6 labeled with biotin at the end was used. Before injecting the avidin solution, the sensing probe 1 was immersed in a tetraborate buffer solution for several minutes to confirm the stability of the return light intensity. Next, 20.4 μL of an avidin solution having a concentration of 1 mg / mL was added to 1 mL of a tetraborate buffer solution to obtain an avidin solution having a concentration of 20 μg / mL. From FIG. 8, the return light intensity signal rapidly increased by the injection of avidin 5, and a clear reaction indicating the affinity between biotin and avidin was obtained. The return light intensity signal becomes almost constant at a time of about 2000 s, and the signal increase rate after binding of biotin and avidin is 15.6% as compared to before binding.

  FIG. 9 shows the configuration of a localized surface plasmon sensing device 7A according to the fourth embodiment. The light source in the first embodiment is replaced with a halogen lamp 21, and a spectrometer 22 (USB-2000: manufactured by Ocean Optics Inc.) is used instead of the photomultiplier tube 9 as a photodetector. Removed. Further, although not essential, the fifth optical fiber 14 is removed by replacing the optical fiber coupler 10 from a 2 × 2 coupler to a 2 × 1 coupler. Other configurations are the same as those of the first embodiment. When this configuration is used, the spectrum of reflected light or scattered light over a predetermined wavelength range can be detected.

  FIG. 10 shows an example in which the spectrum of return light (specifically, return light including reflected light and scattered light) is measured at a wavelength of 400 to 700 nm using the localized surface plasmon sensing device 7A in the present embodiment. The vertical axis represents the return light intensity, and the horizontal axis represents the wavelength. The solid line is the spectrum of the return light when the sensing probe 1 in which the gold fine particles 4 are deposited on the optical fiber end face 3 is immersed in water. The baseline (horizontal axis) is the spectrum of the return light when the sensing probe 1 in which the gold fine particles 4 are not deposited on the optical fiber end face 3 is immersed in water. The spectrum of FIG. 10 is normalized by the baseline. is there. Although strong localized plasmon resonance is observed at a wavelength of about 550 nm, it is shifted to the red side (long wavelength side) by 30 nm as compared with the case of measurement in the air due to the refractive index of the surrounding water. A broken line is a spectrum when the gold fine particle layer 4 on the end face 3 of the optical fiber is modified with thiol 6 whose end is labeled with biotin, and the biotin monomolecular layer 6 is a very thin dielectric layer (1.6 nm thickness). In addition, the return light intensity is slightly changed. A dotted line is a result of detecting avidin 5, and is a spectrum when gold fine particles 4 are completely covered with avidin 5 which is an analyte. A large change is seen in the return light intensity over the wavelength range of 550 to 680 nm. The profile of the measurement results agrees well with the theoretical simulation based on pseudo-state approximation. From this result, it is understood that the wavelength range of 550 to 680 nm is optimal for biosensing, and a red LED (λ = 623 nm) is suitable as the light source.

  FIG. 11 is a diagram in which the amount of change in return light intensity is plotted against the wavelength of light based on FIG. The vertical axis indicates the amount of change, and the horizontal axis indicates the wavelength. In the figure, ○ indicates a case where the sensing probe 1 in which the gold fine particles 4 are not deposited on the optical fiber end surface 3 is immersed in water, and □ indicates a thiol in which the gold fine particles 4 on the optical fiber end surface 3 are labeled with biotin. ◇ indicates data when the gold fine particle layer 4 and the thiol 6 labeled with biotin at the end are completely covered with avidin 5 as an analyte. There is a slight change in the data modified with thiol 6 over the wavelength range of 550 to 680 nm, and in the data in which biotin and avidin are bound, the change amount is about 1.2 times or more, and further, the wavelength range is 590. At ˜640 nm, the change is about 1.3 times or more, and a larger change is observed. The maximum change amount is obtained at a wavelength of about 610 nm.

  FIG. 12 is a graph plotting the amount of change in return light intensity against the wavelength of light when the gold fine particle surface itself deposited on the optical fiber end face 3 is the ligand 6 and octadecanthiol 5 is used as the analyte. It is. The difference in plot data is due to the temporal change from the injection of the analyte. ○ is the data at the time of injection, □ is the data after 0.8 sec, ◇ is after 10.4 sec, x is after 126 sec, and + is the data after 1032 sec. . There is a slight change in the data after 10.4 sec over the wavelength range of 550 to 740 nm, and the amount of change is large in the data after 126 sec. In the wavelength range of 590 to 690 nm, a large change of about 1.2 times or more is observed in the data after 126 sec. Further, the maximum change amount is shown at a wavelength of about 630 nm, and the change amount is about 1.5 times or more.

  Although the embodiment of the present invention has been described above, the embodiment is not limited to the above example, and it is obvious that various modifications can be made without departing from the spirit of the present invention.

  For example, in the above-described embodiment, the end face of the optical fiber is not particularly referred to. However, the end face of the optical fiber may be formed perpendicularly to the axis of the fiber, or may be formed inclined, and needs to be flat. Absent. Even if it is formed inclined, it may not be flat (may have irregularities), or it may have no sharp conical surface, if the end face of the optical fiber is modified with metal fine particles, the inclination of the plasmon resonance angle This is because there are many metal surfaces having a plane, and it is considered that resonance can occur. Further, even if the shape and size of the metal fine particles are various, it is sufficient that there are a large number of metal surfaces having an inclined surface with a plasmon resonance angle.

  In the above embodiment, the case where the metal fine particles are gold has been described. However, other metal fine particles may be used. Among metals, noble metals are preferable, and gold is particularly preferable because gold has excellent chemical stability and silver has a large propagation length of surface plasmons and a sharp resonance absorption profile.

  Further, the metal fine particle layer formed on the end face of the optical fiber may be a single layer or a multilayer. Further, the gap between the metal fine particles may be filled with a dielectric (at this time, the surface of the metal particle layer is not covered). This is because if there is a metal capable of modifying the ligand on the surface of the metal particle layer, the model of (Equation 1) is established even in the dielectric medium, and the localized surface plasmon is excited.

  The size of the detection portion on the end face of the optical fiber can also be used in a wide range. It is possible to realize 200 μm or less with an optical fiber and 1 mm or less with a plastic fiber.

In the above embodiment, the wavelength of the light source is preferably 550 nm to 740 nm. However, the resonance wavelength of the surface plasmon can be changed by the combination of the peripheral medium, the metal fine particles, the analyte and the ligand, and is not limited to this range. Absent. It should be noted that the resonance wavelength shifts to the long wavelength side with sensing, and the intensity of the return light varies greatly on the long wavelength side.
As for the combination of the light source and the detection system, in addition to the combination of detecting the light source as a monochromatic light with a photodetector such as a photomultiplier tube and detecting the light source as a spectral light with a spectrometer, the light source as a spectral light and photomultiplier A combination of detecting with a photodetector such as a tube and using a spectrometer with a monochromatic light as the light source is also possible.

  Further, the localized surface plasmon sensing device has been described with respect to the case where the reflected light from the fifth optical fiber is minimized, but the reflected light is the same as the reflected light in a state where the tip of the sensor is not immersed in the sample liquid. And only the difference signal between the reflected light and scattered light may be transmitted to the photodetector. In this case, for example, the same sensor may be connected to the fifth optical fiber via a splicer. Further, the return loss may be measured. Further, the fifth optical fiber may not be provided. The detection light may be reflected light or scattered light, or a mixture of these.

  As described above, the combination of the analyte and the ligand is not limited to avidin and biotin, ODT and gold, but DNA-DNA, DNA-RNA (ribonucleic acid), DNA-protein, DNA-sugar, DNA-organic. Compound, protein-protein, lipid-protein, sugar-protein, protein-organic compound interaction detection, gold, silver-thiol and organosulfur compounds with disulfide groups, primary amines with amino groups, secondary amines It is also possible to detect the interaction of a tertiary amine compound or the like. The general protein size is about several nm to 10 nm, but the amount of change in the signal obtained also depends on the equilibrium constant of the binding between the analyte and the ligand. Therefore, the sensitivity of the sensor depends not only on the system but also on the selected ligand. Therefore, a light source or a light detector corresponding to the combination of the analyte and the ligand should be selected.

  The localized surface plasmon sensor according to the present embodiment is not limited to an analyte detection sensor, but also a refractive index sensor (detection of a change in sugar concentration, detection of a change in methanol / water ratio of a fuel cell, etc. ) Is also useful. In addition, if a material whose refractive index or extinction coefficient changes in response to humidity in the gas or whose volume changes is applied, it can also be a humidity sensor. Similarly, if a substance whose refractive index or extinction coefficient changes or its volume changes is applied when exposed to a specific gas, it is also useful as a gas sensor.

It is a figure which shows the structure of the localized surface plasmon sensor in 1st Embodiment. It is a figure which shows the structure of the localized surface plasmon sensing apparatus in 1st Embodiment. It is a figure for demonstrating the resonant wavelength dependence of scattered light intensity | strength. It is a figure for demonstrating the dielectric material (detection target) film thickness dependence of scattered light intensity. It is a figure which shows the example of the detection result which detected the avidin using the localized surface plasmon sensing apparatus in 1st Embodiment. It is a figure which shows the example of the detection result which detected the octadecane thiol using the localized surface plasmon sensing apparatus in 2nd Embodiment. It is a figure which shows the result of having measured the return light intensity by changing the refractive index of a surrounding medium. It is a figure which shows the measurement result of the affinity of avidin with biotin. It is a figure which shows the structure of the localized surface plasmon sensing apparatus in 3rd Embodiment. It is a figure which shows the example which measured the spectrum of the return light in 4th Embodiment. It is a figure which shows the variation | change_quantity of return light intensity with respect to the wavelength of light when biotin is used as a ligand and avidin is used as analyte in the fourth embodiment. It is a figure which shows the variation | change_quantity of a return light intensity with respect to the wavelength of light in the case of using gold decane surface itself as a ligand and using octadecanethiol as an analyte in 4th Embodiment. It is a figure for demonstrating the principle of sensing using surface plasmon resonance. It is a figure which shows the structural example of the conventional surface plasmon sensor. It is a figure which shows the structural example of the conventional localized surface plasmon sensor. It is a figure which shows the structural example of another conventional localized surface plasmon sensor and sensing apparatus. It is a figure which shows the structural example of another conventional surface plasmon sensor.

Explanation of symbols

1 Localized surface plasmon sensor (sensing probe)
2 First optical fiber 3 End face (first end face) of the first optical fiber
4 Metal fine particle layer 5 Detection target molecule (analyte)
6 Complementary molecules (ligands)
7, 7A Localized surface plasmon sensing device 8 Light source (LED)
9 Photodetector (photomultiplier tube)
DESCRIPTION OF SYMBOLS 10 Optical fiber coupler 11 2nd optical fiber 12 3rd optical fiber 13 End surface (2nd end surface) of 5th optical fiber
14 fifth optical fiber 15 fourth optical fiber 16 cell 17 sample 18 splicer 19 lock-in amplifier 20 arithmetic unit 21 light source (halogen lamp)
22 Spectrometer
DESCRIPTION OF SYMBOLS 101 Surface plasmon sensor 102 Substrate 103 End face 104 of substrate Metal fine particle layer 105, 105a, 105b Detection target molecule (analyte)
106, 106a, 106b A molecule (ligand) complementary to the molecule to be detected
111 Surface plasmon sensor 112 Prism 113 Matching oil 114 First glass substrate 115 Gold thin film 116 Second glass substrate 117 Spacer 118 Sample container 119 Sample 120 Incident light 131 Localized surface plasmon sensor 132 Cell 133 Gold fine particle 134 PMMA film.
135 Sample 140 Localized surface plasmon sensing device 141 Localized surface plasmon sensor 142 Light source 143 Collimator lens 144 Optical fiber coupler 145 Gold particle 146 Optical fiber core 147 Sample 148 Cell 149 Fiber optic spectrometer 150 Computer 151 Surface plasmon sensor 152 Optical fiber 153 End face 154 of optical fiber Clad 155 Core 156 Convex part (or concave part)
157 Metal thin film 158 SPR measurement surface 159 Incident light 160 Surface plasmon wave 161 Reflected light

Claims (19)

  A metal fine particle layer having a size capable of exciting localized surface plasmon resonance is formed on the end face of the optical fiber, and a molecular layer of a molecule complementary to the molecule to be detected is formed on the surface of the metal fine particle layer. A localized surface plasmon sensor that detects a detection target molecule adsorbed or bound to the complementary molecule using a change in light input to the optical fiber due to surface plasmon resonance localized in a fine particle layer.   A metal fine particle layer having a function of a molecule complementary to the molecule to be detected is formed to a size that excites the surface plasmon resonance localized on the end face of the optical fiber, and the surface plasmon localized in the metal fine particle layer is formed. A localized surface plasmon sensor that detects a detection target molecule adsorbed or bound to the complementary molecule using a change in light input to the optical fiber due to resonance.   The localized surface plasmon sensor according to claim 1, wherein the metal fine particle layer contains a precious metal as a main component.   The localized surface plasmon sensor according to any one of claims 1 to 3, wherein the metal fine particle layer has a thickness of 1 nm to 1 µm.   The localized surface plasmon sensor according to any one of claims 1 to 4, wherein a diameter of a detection portion at an end face of the optical fiber is 5 mm or less. The localized surface plasmon sensor according to any one of claims 1 to 5 (an optical fiber constituting the localized surface plasmon sensor is a first optical fiber) and the surface plasmon resonance A light source that emits light to be excited, a photodetector that detects light, an optical fiber coupler that can divide and output input light, and a second optical fiber that guides light emitted from the light source to the optical fiber coupler And connecting the first optical fiber and the optical fiber coupler to transmit light emitted from the light source from the optical fiber coupler to the end surface of the first optical fiber of the localized surface plasmon sensor (the end surface). A first end face), a third optical fiber for guiding light reflected or scattered from the first end face to the optical fiber coupler, and light reflected or scattered from the first end face And a fourth optical fiber guiding the light detector from the optical fiber coupler;
Localized surface plasmon sensing device.   The localized surface plasmon sensing device according to claim 6, wherein the localized surface plasmon sensor and the third optical fiber are connected by a splicer so as to be detachable. The localized surface plasmon sensor according to any one of claims 1 to 5 (an optical fiber constituting the localized surface plasmon sensor is a first optical fiber) and the surface plasmon resonance A light source that emits light to be excited, a photodetector that detects light, an optical fiber coupler that can divide and output input light, and a second optical fiber that guides light emitted from the light source to the optical fiber coupler And a fourth optical fiber for guiding light from the localized surface plasmon sensor from the optical fiber coupler to the photodetector;
The first optical fiber is connected to the optical fiber coupler, and the light emitted from the light source is transmitted from the optical fiber coupler to the end face of the first optical fiber of the localized surface plasmon sensor (the end face is defined as the first end face). And the light reflected or scattered from the first end face is guided to the optical fiber coupler, and the fourth optical fiber takes the light reflected or scattered from the first end face as the light. Leading from a fiber coupler to the photodetector;
Localized surface plasmon sensing device.   From the intensity, phase, or amount of resonance wavelength of the localized surface plasmon resonance detected by the photodetector, the film thickness, adsorption amount or binding amount of the molecule to be detected, or The localized surface plasmon sensing device according to any one of claims 6 to 8, further comprising an arithmetic unit that calculates a refractive index of a substance including the complementary molecule around the localized surface plasmon sensor.   10. The apparatus according to claim 9, further comprising: a lock-in amplifier that detects lock-in of a signal output from the photodetector, wherein the arithmetic device acquires a signal output from the photodetector via the lock-in amplifier. Localized surface plasmon sensing device.   The localized surface plasmon sensing device according to any one of claims 6 to 10, wherein the light source emits light of any wavelength within a wavelength range of 550 nm to 740 nm. The localized surface plasmon sensor according to any one of claims 1 to 5 (an optical fiber constituting the localized surface plasmon sensor is a first optical fiber) and the surface plasmon resonance A light source that emits light including a predetermined wavelength range to be excited, a spectrometer that detects an optical spectrum of light including the predetermined wavelength range, an optical fiber coupler that can divide and output input light, and the predetermined A second optical fiber that guides light including a wavelength range from the light source to the optical fiber coupler, and the first optical fiber and the optical fiber coupler are connected, and the light including the predetermined wavelength range is transmitted to the light. The predetermined surface that is guided from a fiber coupler to an end face of the first optical fiber of the localized surface plasmon sensor (the end face is a first end face) and is reflected or scattered from the first end face. A third optical fiber for guiding light including a wavelength range to the optical fiber coupler; and a second optical fiber for guiding light including the predetermined wavelength range reflected or scattered from the first end face to the spectrometer from the optical fiber coupler. 4 optical fibers;
Localized surface plasmon sensing device.   The localized surface plasmon sensing device according to claim 12, wherein the localized surface plasmon sensor and the third optical fiber are connected by a splicer so as to be detachable. The localized surface plasmon sensor according to any one of claims 1 to 5 (an optical fiber constituting the localized surface plasmon sensor is a first optical fiber) and the surface plasmon resonance A light source that emits light including a predetermined wavelength range to be excited, a spectrometer that detects an optical spectrum of light including the predetermined wavelength range, an optical fiber coupler that can divide and output input light, and the predetermined A second optical fiber for guiding light including a wavelength range from the light source to the optical fiber coupler; and a fourth optical fiber for guiding light from a localized surface plasmon sensor from the optical fiber coupler to the spectrometer. ;
The first optical fiber is connected to the optical fiber coupler, and transmits light including the predetermined wavelength range from the optical fiber coupler to the end surface of the first optical fiber of the localized surface plasmon sensor. The first end face), the light including the predetermined wavelength range reflected or scattered from the first end face is guided to the optical fiber coupler, and the fourth optical fiber is guided from the first end face. Directing reflected or scattered light including the predetermined wavelength range from the fiber optic coupler to the spectrometer;
Localized surface plasmon sensing device.   The localized surface plasmon sensing device according to claim 12, wherein the predetermined wavelength range is a wavelength of 550 nm to 740 nm. Localization by forming a metal particle layer of a size capable of exciting localized surface plasmon resonance on the end face of the optical fiber, and forming a molecular layer complementary to the molecule to be detected on the surface of the metal particle layer Preparing a surface plasmon sensor;
Introducing light that excites the surface plasmon resonance into the end face of the optical fiber;
Exciting the localized surface plasmon resonance based on the light that excites the surface plasmon resonance in the metal fine particles of the localized surface plasmon sensor, and detecting reflected light or scattered light from the end face of the optical fiber When,
Immersing an end face of the optical fiber of the localized surface plasmon sensor in a sample containing the molecule to be detected to adsorb or bind the molecule to be detected to the complementary molecule;
Reflected light from the end face of the optical fiber by exciting the localized surface plasmon resonance based on the light that excites the surface plasmon resonance in the metal fine particles of the localized surface plasmon sensor immersed in the sample Or detecting scattered light;
The detection is performed by comparing reflected light or scattered light when the end face of the optical fiber is not immersed in the sample with reflected light or scattered light when the end face of the optical fiber is immersed in the sample. Detecting the molecule of interest;
Sensing method using localized surface plasmon sensor.   The sensing method using the localized surface plasmon sensor according to claim 16, wherein the wavelength of the light that excites the surface plasmon resonance is any wavelength within a wavelength range of 550 nm to 740 nm. Localization by forming a metal particle layer of a size capable of exciting localized surface plasmon resonance on the end face of the optical fiber, and forming a molecular layer complementary to the molecule to be detected on the surface of the metal particle layer Preparing a surface plasmon sensor;
Introducing light including a predetermined wavelength range into the end face of the optical fiber;
The localized surface plasmon sensor of the localized surface plasmon sensor excites localized surface plasmon resonance based on light including the predetermined wavelength range in the metal fine particles, and detects a spectrum of reflected light or scattered light from the end face of the optical fiber. And a process of
Immersing an end face of the optical fiber of the localized surface plasmon sensor in a sample containing the molecule to be detected to adsorb or bind the molecule to be detected to the complementary molecule;
The localized surface plasmon sensor immersed in the sample excites localized surface plasmon resonance based on light including the predetermined wavelength range in the metal fine particles, and the reflected light from the end face of the optical fiber Or detecting a spectrum of scattered light;
The spectrum of reflected or scattered light when the end face of the optical fiber is not immersed in the sample is compared with the spectrum of reflected or scattered light when the end face of the optical fiber is immersed in the sample. And detecting the molecule to be detected;
Sensing method using localized surface plasmon sensor. The predetermined wavelength range is a wavelength of 550 nm to 740 nm;
A sensing method using the localized surface plasmon sensor according to claim 18.

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