JP5286515B2 - Sensor chip and sensor chip manufacturing method - Google Patents

Description

  The present invention relates to a sensor chip and a sensor chip manufacturing method.

  A sensor that measures a refractive index change around a metal fine particle using plasmon resonance, which is one of resonance phenomena between metal fine particles and light, has been developed (for example, see Patent Document 1).

  Here, as in Japanese Patent Application Laid-Open No. H10-228707, the principle of detecting the refractive index change around the metal fine particles using plasmon resonance will be described. When metal fine particles such as gold nanoparticles and silver nanoparticles are irradiated with light, plasmon resonance, which is one of resonance phenomena, occurs at a predetermined resonance wavelength. As a result, since scattering and absorption by the metal fine particles irradiated with light increase, a resonance peak appears in the spectrum when light interacting with the metal fine particles is detected. Since the resonance wavelength causing the resonance peak depends on the refractive index around the metal fine particle, the change in the refractive index around the metal fine particle can be detected by detecting the shift of the resonance peak.

  In the localized plasmon resonance sensor (sensor chip) described in Patent Document 1, a plurality of gold nanoparticles as metal fine particles are fixed on the surface of a glass substrate, and light is irradiated from the back side of the glass substrate. The resonance peak is acquired by detecting the transmitted light. Then, the refractive index change around the metal fine particles is detected from the change in the resonance peak.

Such a sensor chip is used as a sensor chip for detecting an analyte such as DNA or protein as follows. That is, when probe molecules that specifically bind to the analyte are immobilized on the metal microparticles and the sample solution is fed, if the analyte is contained in the sample solution, it is affected by the analyte bound to the probe molecule. The refractive index in the vicinity of the metal fine particles changes and the resonance wavelength shifts. Therefore, if a change in the resonance peak before and after the sample solution is sent is detected, the analyte can be detected, and can be used as a sensor chip for detecting an analyte such as DNA or protein.
Japanese Patent Application Laid-Open No. 2000-356587

  However, in the method using the resonance phenomenon between the metal fine particles and the light, the resonance phenomenon between the metal fine particles and the light is detected when adjacent metal fine particles meet among the plurality of metal fine particles fixed on the glass substrate. Therefore, the spectrum of light interacting with the metal fine particles to be detected is distorted, and as a result, the detection sensitivity may be lowered.

  Therefore, an object of the present invention is to provide a sensor chip capable of detecting a change in refractive index around a metal fine particle with high sensitivity and a method for manufacturing the sensor chip.

  A sensor chip according to the present invention is a sensor chip for detecting a refractive index change around a metal fine particle by utilizing a resonance phenomenon between the metal fine particle and light, and is formed on the substrate, the substrate, and the metal chip. A first film for fixing fine particles, a plurality of metal fine particles fixed on the sensor region of the first film, and an adjacent metal among the plurality of metal fine particles fixed on the first film And a blocking agent disposed between the fine particles.

  In the sensor chip having the above configuration, since the blocking agent is fixed between adjacent metal fine particles on the sensor region, association between adjacent metal fine particles is suppressed. As a result, when the refractive index change around the metal fine particle is detected by irradiating the metal fine particle with light and using the above resonance phenomenon, the distortion of the spectrum of the light interacting with the metal fine particle is suppressed, and the refraction around the metal fine particle is suppressed. The rate change can be detected with high sensitivity. For this reason, a specific analyte can be detected with high sensitivity by fixing, for example, probe molecules that specifically bind to the analyte to the metal fine particles.

  In addition, as said board | substrate, what is transparent with respect to the light of the wavelength band containing the wavelength which produces the resonance phenomenon in a metal microparticle can be considered, and a glass substrate is illustrated.

  In the sensor chip according to the present invention, the first film is preferably made of a silane compound, and the blocking agent is preferably a compound having a carboxyl group.

  A silane compound has an amino group and forms an amide bond with a carboxyl group. Therefore, since the blocking agent having a carboxyl group can be reliably fixed to the first film, association between the metal fine particles is suppressed. As a result, as described above, the refractive index change around the metal fine particles can be detected with high sensitivity.

  In this case, the blocking agent is preferably formed from any one of glycolic acid, sodium acetate, 16-hydroxyhexadecanoic acid and polyethylene glycol. Since these have a carboxyl group at the terminal, the blocking agent is reliably fixed to the first film.

  Furthermore, in the sensor chip according to the present invention, it is preferable that the sensor chip further includes a second film that is formed on the surface of the metal fine particles and fixes the subject. Thus, the subject can be fixed in the vicinity of the metal fine particles using the second film. Therefore, the refractive index change around the metal fine particles due to fixation of the subject can be detected with high sensitivity, and as a result, the subject can be detected with high sensitivity.

  The second film may be formed of mercaptoundecanoic acid.

  Furthermore, the sensor chip according to the present invention preferably has a plurality of sensor regions on the first film, and the metal fine particles and the blocking agent are fixed on each sensor region. In this case, the sensor chip has a plurality of sensor regions to which the metal fine particles and the blocking agent are fixed. Therefore, different analytes can be detected in each sensor region by fixing, for example, probe molecules that specifically bind to various analytes to the metal fine particles on each sensor region, and as a result, sensor chips A plurality of objects can be detected at one time.

  Furthermore, in the sensor chip according to the present invention, it is preferable that the metal fine particles have a particle size of 50 nm to 100 nm.

  The sensor chip manufacturing method according to the present invention is a method for manufacturing a sensor chip for detecting a refractive index change around a metal fine particle by utilizing a resonance phenomenon between the metal fine particle and light, and (1) a substrate. Particle fixing for fixing metal fine particles to a sensor region at a predetermined particle density by bringing a sensor region of a first film for fixing metal fine particles formed thereon into contact with a metal fine particle solution containing metal fine particles. And (2) a blocking agent fixing step of fixing the blocking agent to the sensor region by bringing the sensor region in which the metal fine particles are fixed into contact with the blocking agent introduction solution.

  In the sensor chip manufactured by this method, the metal fine particles are fixed on the sensor region in order to fix the blocking agent on the sensor region where the metal fine particles are fixed at a predetermined particle density using the metal fine particle solution. The blocking agent is fixed to the non-existing part. Thereby, a blocking agent is arrange | positioned between adjacent metal microparticles. As a result, when the refractive index change around the metal fine particle is detected by irradiating the metal fine particle with light and using the above resonance phenomenon, the distortion of the spectrum of the light interacting with the metal fine particle is suppressed, and the refraction around the metal fine particle The rate change can be detected with high sensitivity. For this reason, a specific analyte can be detected with high sensitivity by fixing, for example, probe molecules that specifically bind to the analyte to the metal fine particles.

  Furthermore, in the sensor chip manufacturing method according to the present invention, the first film is preferably formed of a silane compound, and the blocking agent is preferably a compound having a carboxyl group.

  A silane compound has an amino group and forms an amide bond with a carboxyl group. Therefore, the blocking agent having a carboxyl group can be reliably fixed to the first film, and the association between the metal fine particles is suppressed. As a result, as described above, the refractive index change around the metal fine particles can be detected with high sensitivity.

  In this case, the blocking agent is preferably composed of any one of glycolic acid, sodium acetate, 16-hydroxyhexadecanoic acid and polyethylene glycol. Since these have a carboxyl group at the terminal, the blocking agent is reliably fixed to the first film.

  In the sensor chip manufacturing method according to the present invention, the second film for immobilizing the subject is brought into contact with the sensor region to which the metal fine particles are fixed and the second film forming solution, thereby It is preferable to further include a film forming step for forming on the surface. As a result, the subject can be fixed in the vicinity of the metal microparticles using the second film. Therefore, the refractive index change around the metal fine particles due to fixation of the subject can be detected with high sensitivity, and as a result, the subject can be detected with high sensitivity.

  The second film may be made of mercaptoundecanoic acid.

  Furthermore, in the sensor chip manufacturing method according to the present invention, when the particle size of the metal fine particles fixed to the substrate is less than 50 nm, the metal fine particles fixed to the substrate are brought into contact with the particle growth solution to reduce the particle size of the metal fine particles. It is preferable to further include a particle size increasing step for increasing to 50 nm to 100 nm.

  By increasing the particle size of less than 50 nm to the above range by the particle size increasing step, the resonance phenomenon between the metal fine particles and light is more efficiently generated, so that the refractive index change around the metal fine particles can be detected with higher sensitivity. . In addition, it is preferable to implement this particle size increase process before a blocking agent introduction | transduction process from a viewpoint of increasing the particle size of a metal microparticle reliably.

  Furthermore, in the sensor chip manufacturing method according to the present invention, the first film has a plurality of sensor regions, and in the particle fixing step, the metal fine particles are fixed at a predetermined particle density on each sensor region, In the agent fixing step, it is preferable to fix the blocking agent on each sensor region.

  In this case, the metal fine particles and the blocking agent are fixed on each sensor region. Since the sensor chip has a plurality of sensor areas, different objects can be detected in each sensor area, so that a plurality of objects can be detected at a time by the sensor chip. For example, different analytes can be detected in each sensor region by immobilizing probe molecules that specifically bind to various analytes, for example, on metal fine particles on each sensor region. In addition, a plurality of subjects can be detected.

The predetermined particle density is preferably 30 to 60 particles / μm ² .

  According to the sensor chip and the sensor chip manufacturing method of the present invention, it is possible to detect the refractive index change around the metal fine particles with high sensitivity.

  Hereinafter, embodiments of a sensor chip and a sensor chip manufacturing method according to the present invention will be described with reference to the drawings. In the following description, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the dimensional ratios in the drawings do not necessarily match those described.

  FIG. 1 is a schematic diagram showing a cross-sectional configuration of an embodiment of a sensor chip according to the present invention. The sensor chip 10A is a sensor element for detecting an object 20 (see FIG. 5) described later by using resonance scattering by plasmon resonance, which is one of the resonance phenomena between gold nanoparticles 14 as metal fine particles and light. It is. The subject 20 is exemplified by DNA and protein.

The sensor chip 10A includes a glass substrate 11 on which an aminosilane film (first film) 12 made of silane treatment and made of aminopropylsilane (silane compound) is formed on a surface 11a. As for the magnitude | size of the glass substrate 11, when it sees from the surface 11a side, about 18 mm x 18 mm is illustrated. On the sensor region 13 of the aminosilane film 12, gold nanoparticles 14 are fixed at a particle density of, for example, 30 to 60 particles / μm ² . Here, the sensor region 13 is a region that is used for detecting the analyte 20 by fixing a group of gold nanoparticles composed of a plurality of gold nanoparticles 14 in the aminosilane film 12. In other words, the region where the gold nanoparticle group is fixed becomes the sensor region 13. In the sensor chip 10 </ b> A shown in FIG. 1, the entire surface 12 a of the aminosilane film 12 functions as the sensor region 13. The particle diameter (diameter) d1 of the gold nanoparticles 14 is preferably 50 nm to 100 nm from the viewpoint of increasing the intensity of scattered light when the gold nanoparticles 14 are irradiated with light. On the surface of the gold nanoparticle 14, a self-assembled film (second film) 15 made of 11-mercaptoundecanoic acid (MUA) is formed. Hereinafter, the self-assembled film 15 is also referred to as a MUA film 15.

  As shown in FIG. 1, in the sensor chip 10 </ b> A, polyethylene glycol (PEG: polyethyleneglycohol) as a blocking agent 16 is formed on at least a part of the portion of the aminosilane film 12 where the gold nanoparticles 14 are not adsorbed. It is fixed. As a result, the blocking agent 16 is disposed between the adjacent gold nanoparticles 14. The blocking agent 16 may be a compound having a carboxyl group (COOH group) at its terminal, and examples thereof include glycolic acid, sodium acetate, 16-hydroxyhexadecanoic acid, etc. in addition to PEG. In the following description, the blocking agent 16 is PEG unless otherwise specified.

  Next, a method for manufacturing the sensor chip 10A will be described with reference to FIGS. As shown in FIG. 2, the manufacturing process of the sensor chip 10A includes the silane treatment process S1, the particle fixing process S2, the blocking agent introduction process S3, the particle size increasing process S4, and the MUA film forming process (film forming process) S5. And have. In FIG. 2, the main processing of each process described below is illustrated with a specific example.

  Each step will be described with specific numerical values such as concentrations, solution names, and treatment times as examples. 3 and 4 are schematic views showing each step of the sensor chip manufacturing method. FIG. 4C shows a chemical formula of MUA described later. The sensor region 13 is the entire surface of the aminosilane film 12 as described above.

  In the silane treatment step S1, the cleaned glass substrate 11 is immersed in a 5% 3-aminopropyltrimethoxysilane (APTMS) toluene solution for 5 minutes. Thereby, the aminosilane film 12 is formed on the surface of the glass substrate 11 as shown in FIG. Next, the glass substrate 11 on which the aminosilane film 12 is formed is ultrasonically cleaned 6 times with ethanol and 3 times with pure water.

  In the particle fixing step S2, the gold nanoparticles 14 are fixed on the aminosilane film 12 at a predetermined particle density. The glass substrate 11 on which the aminosilane film 12 is formed is immersed in a gold nanoparticle solution having a concentration of 10 nM for about 2 hours. The particle size d2 of the gold nanoparticles 14 in the gold nanoparticle solution is less than about 50 nm. As a result, as shown in FIG. 3B, gold nanoparticles 14 having a particle size d2 of less than about 50 nm are fixed to a part of the adsorption sites of the aminosilane film 12.

  What is necessary is just to utilize what was synthesize | combined by the citric acid reduction method, for example, and the said gold nanoparticle solution is specifically prepared as follows. That is, an aqueous solution of about 1 mM tetrachloroauric (III) acid tetrahydrate is stirred while being heated on a hot plate or the like. After sufficiently boiling, about 10 mM trisodium citrate aqueous solution is added, and the mixture is sufficiently heated and stirred, cooled to room temperature, and then filtered to a gold nanoparticle solution filtered through a membrane filter having 0.20 μm pores.

The particle density of the gold nanoparticles 14 can be controlled by adjusting at least one of the concentration of the gold nanoparticle solution and the immersion time, and the particle density is 30 to 60 particles / μm ^{2 under} the above conditions. Here, the predetermined particle density is set to a particle density of 30 to 60 particles / μm ² , but the predetermined particle density is a particle density in which the gold nanoparticles 14 are not fixed to all adsorption sites of the aminosilane film 12. Good.

  In the particle size increasing step S4, the glass substrate 11 on which the gold nanoparticles 14 and the blocking agent 16 are fixed is mixed with a 0.4 mM hydroxylamine hydrochloride solution and a 0.24 mM tetrachloroauric (III) acid tetrahydrate solution. Immerse 5 times in the particle growth solution. Thereby, as shown in FIG.3 (c), the particle size of the gold nanoparticle 14 increases and it can adjust to the particle size d1 of 60 nm-70 nm. In FIG.3 (c), the gold nanoparticle before a particle size increases is shown with the broken line for description. In addition, the particle growth solution is renewed for each immersion. Further, the particle size can be increased by increasing the number of immersions. Therefore, although the number of immersions is 5 here, the number of immersions may be determined according to the desired particle size, and the relationship between the particle size and the number of immersions may be obtained in advance. The particle size is preferably 50 nm to 100 nm.

  In the blocking agent introduction step S4, a solution obtained by dissolving PEG (molecular weight 2000) 16 having 20 mg / ml EDAC and 20 mg / ml COOH group in 0.1 M MES buffered Saline (hereinafter referred to as “blocking agent introduction solution”). The glass substrate 11 on which the gold nanoparticles 14 are fixed is dipped in about 30 minutes. As a result, as shown in FIG. 4A, PEG 16 is adsorbed on at least a part of the adsorption sites where the gold nanoparticles 14 are not fixed on the aminosilane film 12. The EDAC is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, and MES is 2-morpholinoethane. It is a sulfonic acid monohydrate (2-morpholinoethanesulfonic acid, monohydrate). Moreover, 0.1 M MES buffered Saline is a solution in which 0.1 M MES and 0.9% (w / v) sodium chloride are dissolved to adjust the pH to 4.5-5.

  In the MUA film forming step S5, the glass substrate 11 on which the blocking agent 16 and the gold nanoparticles 14 are fixed is taken out after being immersed in ethanol for about 1 to 2 minutes, and is taken out, and 10 mM MUA produced by dissolving MUA in ethanol. Immerse in an ethanol solution (second film forming solution) for about 10 minutes. Next, in order to remove excess MUA, it is again immersed in ethanol for 20 minutes and then taken out. The glass substrate 11 is immersed in pure water for about 1 minute and then taken out and further washed with pure water. Thereby, as shown in FIG. 4B, the MUA film 15 is formed on the surface of the gold nanoparticle 14, and the sensor chip 10A is obtained. In FIG. 4 (b), as shown in FIG. 4 (c), MUA is simply indicated by a carboxyl group at the end. The same applies to FIGS. 10 and 11.

  In addition, in the said manufacturing method, since the particle size increase process S3 is implemented before block agent introduction | transduction process S4, the particle size of the gold nanoparticle 14 can be increased reliably. However, the particle size increasing step S3 may be performed after the blocking agent introducing step S4.

  Next, a method for detecting the subject 20 using the sensor chip 10A will be described with reference to FIG. FIG. 5 is a conceptual diagram of a process for detecting a subject.

  First, a probe molecule introduction solution containing a probe molecule (conjugate) 21 that specifically binds to the analyte 20 on the sensor chip 10A is flowed on the sensor chip 10A. Fix on the MUA membrane 15. At this time, the gold nanoparticle 14 is irradiated with light of a predetermined wavelength band from the back surface 11 b side of the glass substrate 11, and the scattered light of the gold nanoparticle 14 is detected.

  When the gold nanoparticles 14 are irradiated with light, plasmon resonance, which is one of resonance phenomena, occurs at a predetermined resonance wavelength. As a result, the intensity of scattered light by the gold nanoparticles 14 irradiated with light increases. Therefore, the spectrum of scattered light (hereinafter referred to as “scattering spectrum”) has a resonance peak. The resonance scattering wavelength, which is the wavelength that causes this resonance peak, depends on the refractive index in the vicinity of the gold nanoparticle 14.

  Next, the sample liquid to be inspected is made to flow on the sensor chip 10A. When the sample 20 is contained in the sample solution, the sample 20 specifically binds to the probe molecule 21 as shown in FIG. Since the refractive index in the vicinity of the gold nanoparticle 14 changes due to the binding of the subject 20, the resonance scattering wavelength shifts and the position of the resonance peak changes. Therefore, it is possible to detect whether or not the sample 20 is contained in the sample solution based on a change in the resonance scattering wavelength. The presence or absence of a change in the resonance peak may be detected by a change in the scattered light intensity of the resonance scattering wavelength at the stage where the probe molecule 21 is fixed, or the resonance peak shift may be obtained by acquiring a scattered spectrum of the scattered light. It may be detected.

  The above-described feeding of the probe agent introduction solution and the sample solution onto the sensor chip 10A can be performed using the flow cell 30 shown in FIGS. FIG. 6 is a configuration diagram schematically showing the configuration of the flow cell, and shows a state in which the sensor chip 10A is set. FIG. 7 is a cross-sectional view taken along line VII-VII in FIG.

  As shown in FIGS. 6 and 7, the flow cell 30 is configured by fixing a cover portion 31 to a cell main body portion 33 with screws or the like. An opening 32 for irradiating the sensor chip 10A with light is formed at the center of the cover portion 31, and the sensor chip 10A can be set at the edge of the opening 32 on the cell body 33 side. It is configured. Further, a recess 34 is formed in a region of the cell main body 33 that faces the opening 32. A liquid feed port 35 for introducing the sample liquid into the flow cell 30 and a discharge port 36 for discharging the sample liquid to the outside of the flow cell 30 are formed on the bottom surface of the recess 34. A liquid supply pipe 41 and a discharge pipe 42 as shown in FIG. 6 are connected to the discharge port 36.

  When the sensor chip 10A is set in the flow cell 30, it is set in the opening 32 so that the back surface 11b of the glass substrate 11 is on the cover part 31 side, the O-ring 37 is disposed on the sensor chip 10A, and then the cover is placed. The part 31 and the cell main body part 33 are screwed. Thereby, a liquid feeding space 38 for feeding the sample liquid is formed in a region surrounded by the sensor chip 10A, the O-ring 37 and the bottom surface of the recess 34. The volume of the liquid feeding space 38 is exemplified by about 180 μl.

  Here, an imaging apparatus 50 for detecting a subject using the flow cell 30 will be described with reference to FIG. In FIG. 8, the display of the liquid feeding pipe 41 and the discharge pipe 42 is omitted, and the display of the flow cell 30 is also simplified.

  In the imaging apparatus 50, the white light output from the white light source 51 is converted into parallel light by the collimator optical system 52, and then converted into light having a predetermined wavelength band through the interference filter 53. An example of the interference filter 53 is one that passes light in a narrow band within 50 nm within a range of 500 to 800 nm including a resonance scattering wavelength that causes resonance scattering of the gold nanoparticles 14. The parallel light that has passed through the interference filter 53 is irradiated to the sensor chip 10A obliquely with respect to the sensor chip 10A through the opening 32. The scattered light scattered by the gold nanoparticles 14 on the glass substrate 11 irradiated with the light is detected by the CCD camera 55 through the video camera lens 54, and the detected data is input to a personal computer (PC) 56 as an analysis device. Is done. In addition, the hatching part in FIG. 8 is for showing scattered light.

  Since this imaging device 50 uses a CCD camera 55 as a so-called imaging device, an image of the sensor chip 10A can be acquired. In the imaging apparatus 50, light having a narrow wavelength range of about 50 nm including the resonant scattering wavelength of the gold nanoparticle 14 is irradiated through the interference filter 53, so that the scattering detected by the light receiving cell of the CCD camera 55. A change in refractive index in the vicinity of the gold nanoparticle 14 can be detected by a change in light intensity.

  It will be described with reference to FIG. 9 that the imaging apparatus 50 can measure the refractive index change in the vicinity of the gold nanoparticle 14 by changing the scattered light intensity. FIG. 9 is a diagram illustrating an example of the measurement result of the scattered light intensity in the imaging apparatus. In this measurement, the sensor chip 10A manufactured by the sensor chip manufacturing method described with reference to FIG. 2 with specific concentrations and solution names is used. Then, the sensor chip 10A is set in the flow cell 30, and the intensity of the scattered light is measured while flowing water having a refractive index of 1.33 and ethanol having a refractive index of 1.36 alternately into the flow cell 30. The wavelength band of light irradiated to the sensor chip 10A is set to 600 to 650 nm. In FIG. 9, the horizontal axis represents time (seconds) and the vertical axis represents scattered light intensity (au). Regions I, III, and V in the figure show the intensity of scattered light when water is being sent, and regions II and IV are the intensity of scattered light when ethanol is sent. Is shown. As understood from FIG. 9, the scattered light intensity is clearly different between the medium around the gold nanoparticles 14 and water and ethanol, and the refractive index change in the vicinity of the gold nanoparticles 14 is surely detected by the imaging device 50. It can be detected.

  In addition, since the imaging apparatus 50 uses the flow cell 30, measurement can be performed while feeding a sample solution or the like, so that various sample solutions can be inspected continuously. Moreover, since the volume of the liquid feeding space 38 of the flow cell 30 can be set to 180 μl, for example, it is possible to inspect a very small amount of sample liquid.

  Next, a method for detecting a subject using the imaging apparatus 50 will be specifically described with reference to probe molecules, a subject, a solution concentration, and the like.

  First, in the imaging apparatus 50, after setting the glass substrate 11 in the flow cell 30, white light is output from the white light source 51, and scattered light from the gold nanoparticles 14 on the glass substrate 11 is detected by the CCD camera 55. Keep it like that. Next, a probe molecule introduction solution containing 1 mM (+)-Biotinyl-3,6-dioxaoctanediamine (hereinafter simply referred to as “biotin”) and 0.1 M WSC is fed into the flow cell 30 through the feeding tube 41. To do. Thereby, as shown in FIG. 10, biotin as the probe molecule 21 is fixed on the MUA film 15. Therefore, the refractive index in the vicinity of the gold nanoparticles 14 changes, and the resonant scattering wavelength shifts. As described above, the intensity of the scattered light detected by the CCD camera 55 changes due to the shift of the resonant scattering wavelength.

  Next, a Phosphate Buffered Saline (PBS) solution containing 1.6 nM Streptavidin (hereinafter simply referred to as “streptavidin”) as the subject 20 is fed into the flow cell 30 through the liquid feed tube 41 as a sample solution. Since streptavidin has a strong interaction with biotin, it is specifically bound to biotin. As a result, the refractive index in the vicinity of the gold nanoparticle 14 changes, and the intensity of the scattered light further changes for the same reason as when the probe molecule 21 is fixed. Therefore, in the imaging apparatus 50, the subject 20 can be detected by detecting the change in the refractive index around the gold nanoparticle 14 by the change in the intensity of the scattered light detected by the CCD camera 55.

  Next, effects of the sensor chip 10A and the manufacturing method of the sensor chip 10A will be described.

  In the sensor chip 10A, after fixing the gold nanoparticles 14 on the aminosilane film 12, the empty adsorption sites to which the gold nanoparticles 14 are not adsorbed are blocked by adsorbing PEG as the blocking agent 16, When the sample liquid is fed onto the sensor chip 10A, the subject 20 is prevented from being fixed on the aminosilane film 12. As a result, the analyte 20 arranged in the vicinity of the gold nanoparticle 14 can be efficiently detected by the MUA film 15 and the probe molecule.

  Since the glass substrate 11 is immersed in the blocking agent introduction solution after the gold nanoparticles 14 are fixed and before the MUA film 15 is formed, the gold nanoparticles 14 are adsorbed at the adsorption sites on the sensor region 13. The blocking agent 16 is fixed to the part that is not. Therefore, the blocking agent 16 is disposed between the adjacent gold nanoparticles 14. Thereby, even if the predetermined solution for forming the MUA film 15 and the glass substrate 11 are brought into contact with each other in order to form the MUA film 15, the adjacent gold nanoparticles 14 are suppressed from being associated with each other.

  The effect of suppressing the association between the gold nanoparticles 14 by providing the blocking agent 16 will be specifically described in comparison with the case of the sensor chip without the blocking agent 16.

  When the blocking agent 16 is not provided, when the MUA film 15 is formed, the gold nanoparticles 14 are associated with each other due to the flow of the solution and the like, so that the scattering spectrum of the scattered light is distorted. When distortion occurs in this way, the change in scattered light intensity or the scattered spectrum in the vicinity of the resonance peak cannot be obtained accurately, and as a result, the detection sensitivity when detecting the subject 20 is lowered.

  On the other hand, in the sensor chip 10 </ b> A of the present embodiment in which the blocking agent 16 is provided, since the association between the gold nanoparticles 14 is suppressed by the blocking agent 16, no distortion occurs in the spectrum of the scattered light. Therefore, since the change in the scattered light intensity or the scattered spectrum in the vicinity of the resonance peak can be acquired more accurately, the subject 20 can be detected with high sensitivity.

From the viewpoint of surely disposing the blocking agent 16 between the adjacent gold nanoparticles 14, the particle density of the gold nanoparticles 14 in the sensor region 13 is preferably 30 to 60 / μm ² as described above. is there. If the particle density is less than 30, sufficient scattered light necessary for detecting the analyte 20 cannot be obtained. On the other hand, if the particle density is more than 60, the gold nanoparticles 14 are fixed together. This is because this meeting is likely to occur. The association of the gold nanoparticles 14 can be suppressed while generating sufficient scattered light to detect the analyte 20 with the particle density in the above range. As a result, the subject 20 can be detected with higher sensitivity.

  Here, it will be described with reference to FIGS. 12 to 15 that the blocking agent 16 preferably has a carboxyl group, such as PEG, glycolic acid, sodium acetate, and 16-hydroxyhexadecanoic acid.

  FIG. 12 is a diagram showing an absorption spectrum of the glass substrate 11 when PEG is used as the blocking agent 16. FIG. 13 is a diagram showing an absorption spectrum of the glass substrate 11 when glycolic acid is used as the blocking agent 16. FIG. 14 is a diagram showing an absorption spectrum of the glass substrate 11 when sodium acetate is used as the blocking agent 16. FIG. 15 is a diagram showing an absorption spectrum of the glass substrate 11 when 16-hydroxyhexanedecanoic acid is used as the blocking agent 16. 12 to 15 show an absorption spectrum after immobilizing the gold nanoparticles 14, an absorption spectrum after introducing the blocking agent 16, and an absorption spectrum after forming the MUA film 15, respectively. Each absorption spectrum is measured using an absorptiometer after the particle size increasing step S3, the blocking agent introducing step S4, and the MUA film forming step S5 in the flowchart shown in FIG.

  The method for immobilizing the blocking agent 16 in FIG. 12 is as described above. The immobilization method of the blocking agent 16 in FIG. 13 to FIG. 15 is 3.8 mg / ml glycolic acid, 4.1 mg / ml sodium acetate, respectively, instead of 20 mg / ml PEG in the blocking agent introduction step S4. PEG is the same as PEG except that 2.7 mg / ml 16-hydroxyhexadecanoic acid is dissolved.

  As shown in FIGS. 12 to 15, it can be seen that by using the blocking agent 16, no distortion is seen in each absorption spectrum, and the association of the gold nanoparticles 14 is suppressed. This is because PEG, glycolic acid, sodium acetate, 16-hydroxyhexadecanoic acid and the like have a carboxyl group. That is, the aminosilane film 12 has an amino group as an adsorption site for the gold nanoparticle 14, but the gold nanoparticle 14 is immobilized by using the compound having a carboxyl group as the blocking agent 16 as described above. Later empty adsorption sites can be blocked by forming amide bonds. As a result, for example, even when the MUA film 15 is formed, association of adjacent gold nanoparticles 14 can be reliably suppressed. Therefore, the blocking agent 16 may be any compound having a carboxyl group, and in addition to PEG, for example, glycolic acid, sodium acetate, and 16-hydroxyhexadecanoic acid can be used as described above. In addition, when detecting protein using the sensor chip 10A, it is preferable to use a PEG derivative that suppresses nonspecific adsorption of the protein to the blocking agent 16.

  Moreover, it is preferable that the particle size of the gold nanoparticle 14 is 50 nm-100 nm. FIG. 16 is a diagram showing a scattering spectrum of the gold nanoparticle 14 calculated using the Mie scattering theory with the refractive index of the medium around the gold nanoparticle 14 being 1.35. In FIG. 16, the horizontal axis indicates the wavelength (nm), and the vertical axis indicates the scattering cross section. The gold nanoparticles 14 are calculated with the particle diameters of 30 nm, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm, and 200 nm. As shown in FIG. 16, it can be seen that a resonance peak occurs when the particle size of the gold nanoparticle 14 is 50 nm or more. It can also be seen that the scattering spectrum is distorted when the particle size exceeds 150 nm.

  FIG. 17 is a diagram showing the refractive index dependence of the scattered light intensity. In FIG. 17, the horizontal axis represents the refractive index, and the vertical axis represents the scattering cross section. The refractive index dependency is based on the peak position on the long wavelength side when the scattering spectrum shown in FIG. 16 is differentiated, more specifically, the gradient of the scattering spectrum on the long wavelength side with respect to the resonance peak of the scattering spectrum. Acquired based on the wavelength position having the maximum value. At each particle size, the scattering cross section (scattered light intensity) is substantially linear with respect to the refractive index, and the slope of this straight line corresponds to the sensitivity of the sensor chip 10A as a sensor. The sensitivity of the sensor chip 10A is almost constant from about 80 nm in the particle size of the gold nanoparticles 14 and hardly changes for those having a size larger than 100 nm.

  16 and 17, it can be seen that a larger scattering cross section can be obtained when the particle size is larger, but even if the particle size is larger than 100 nm, improvement in the sensitivity of the sensor cannot be expected much. Therefore, when the analyte 20 is detected using the resonance scattering phenomenon, the gold nanoparticles 14 preferably have a particle size of 50 nm to 100 nm. When the gold nanoparticles 14 are synthesized using, for example, the citric acid reduction method as described above, a particle size of about 30 nm is obtained by a single synthesis process, while a particle size of 50 nm or more is obtained. It is difficult. Therefore, as described with reference to FIG. 2, for example, in order to obtain gold nanoparticles 14 having a particle diameter of 50 nm or more, it is necessary to increase the particle diameter after fixing the gold nanoparticles 14 to the glass substrate 11. is there. The increase in particle size is carried out by immersing the particle growth solution a plurality of times. As shown in the range of the particle diameter, considering the sensor sensitivity, the number of immersions of the glass substrate 11 in the particle growth solution can be reduced by setting the particle diameter to 100 nm or less. The sensor chip 10A can be efficiently manufactured while increasing the sensitivity.

  In addition, since the detection of the subject 20 using the sensor chip 10A uses a change in the intensity of scattered light due to plasmon resonance (or a change in the resonance peak position of the scattering spectrum), for example, when using surface plasmon resonance It is possible to detect the subject 20 with higher sensitivity than the above. Furthermore, unlike the so-called fluorescence method, it is not necessary to label the subject 20 with the fluorescent probe, so that the inspection cost for performing the inspection for the presence or absence of the subject 20 in the sample liquid can be reduced. In addition, unlike the fluorescence method, the preparation step in the previous stage for detecting the subject 20 does not include a step of labeling the fluorescent probe, and thus the subject 20 can be detected with simpler preparation.

  FIG. 18 is a plan view of another embodiment of a sensor chip according to the present invention. FIG. 19 is a schematic diagram showing a cross-sectional configuration along the line XIX-XIX in FIG.

  The sensor chip 10B shown in FIGS. 18 and 19 is a so-called microarray. The configuration of the sensor chip 10B is the same as the configuration of the sensor chip 10A shown in FIG. 1 in that it has a plurality of sensor regions 13 (six in FIG. 19) on the aminosilane film 12 formed on the surface of the glass substrate 11. Is different. In FIG. 19, the configuration on each sensor region 13 is described in a simplified manner. However, the configuration on each sensor region 13 is the same as the configuration on the sensor region 13 of the sensor chip 10A shown in FIG. is there. That is, the gold nanoparticle 14 is fixed at a predetermined particle density, and the adsorption site where the gold nanoparticle 14 is not adsorbed is blocked with the blocking agent 16. A MUA film 15 (not shown in FIG. 19) is formed on the surface of the gold nanoparticle 14. In addition, when the size of the glass substrate 11 when viewed from the surface 11a side of the glass substrate 11 is, for example, 18 mm × 18 mm and the sensor region 13 is circular as shown in FIG. 18, the sensor region As the diameter d3 of 13, about 500 μm is exemplified.

  In the particle fixing step S2 shown in FIG. 2, instead of immersing the glass substrate 11 in a gold nanoparticle solution having a concentration of 10 nM, the sensor chip 10A is manufactured by, for example, pipetting the gold nanoparticle solution into each sensor region 13. The gold nanoparticles 14 are fixed to each sensor region 13 by dripping using. Similarly, in the particle size increasing step S3, the gold nanoparticle on each sensor region 13 is dropped by dropping the particle growth solution onto each sensor region 13 to which the blocking agent 16 is fixed using, for example, a pipette. Increase particle size of 14. Further, similarly in the blocking agent introduction step S4, the gold nanoparticles 14 are not fixed in each sensor region 13 by dropping the blocking agent introduction solution into each sensor region 13 using, for example, a pipette. The blocking agent 16 is fixed to the part. Further, similarly in the MUA film forming step S5, the MUA film 15 is formed on the surface of the gold nanoparticle 14 in each sensor region 13 by dropping the MUA ethanol solution on each sensor region 13. The glass substrate 11 cleaning method and the like in the particle fixing step S2, the particle size increasing step S3, the blocking agent introducing step S4, and the MUA film forming step S5 are the same as in the method of manufacturing the sensor chip 10A shown in FIG. The explanation is omitted because there are.

  When the subject 20 is detected using the sensor chip 10A, first, a probe molecule introduction solution is dropped onto the plurality of sensor regions 13 using a pipette or the like, and the MUA film 15 in each sensor region 13 is then dropped. The probe molecule 21 is fixed to the substrate. At this time, a different probe molecule introduction solution is dropped for each sensor region 13 to fix different probe molecules 21 for each sensor region 13.

  Next, the sensor chip 10B to which the probe molecules 21 are fixed is set in the flow cell 30, and the imaging device 50 shown in FIG. 8 detects the scattered light in the same manner as the sensor chip 10A and Get an image. Then, the sample liquid to be inspected is fed into the flow cell 30 using the liquid feeding pipe 41 and the discharge pipe 42. If the sample solution contains the analyte 20 that specifically binds to the probe molecule immobilized on each sensor region 13, the analyte 20 binds to the corresponding probe molecule 21. As a result, the scattering intensity at the sensor region 13 where the subject 20 is bonded to the probe molecule 21 changes. Since the probe molecules 21 fixed to each sensor region 13 and the corresponding analyte 20 are known, the analyte 20 contained in the sample liquid is reliably detected. In this case, since the plurality of sensor regions 13 are provided on the sensor chip 10B, the plurality of subjects 20 can be detected by one measurement. As a result, the sample liquid can be inspected efficiently.

  The preferred embodiments of the sensor chip and the sensor chip manufacturing method according to the present invention have been described above. However, the sensor chip and the sensor chip manufacturing method according to the present invention are not limited to the above embodiment. For example, although the sensor chip 10A has a MUA film, it may be a sensor chip 10C that does not have a MUA film as shown in FIG. In FIG. 20, only one sensor region 13 is shown, but the same applies to the case where a plurality of sensor regions 13 are provided. When the subject 20 is detected using the sensor chip 10A shown in FIG. 20, the MUA ethanol solution is added to the flow cell 30 after the sensor chip 10C is set in the flow cell 30 and before the probe molecule introduction solution is sent. After forming the MUA film 15 on the surface of the gold nanoparticle 14 as described above, the probe molecule introduction solution and the sample solution may be sequentially fed as in the case of the sensor chip 10A as described above. . However, when the sensor chip 10 </ b> C has a plurality of sensor regions 13 and detects different subjects 20 for each sensor region 13, the MUA film 15 is formed in each sensor region 13 using the flow cell 30. Thereafter, the sensor chip 10C is once taken out from the flow cell 30 and a different probe molecule introduction solution is dropped on each sensor region 13, and then set in the flow cell 30 again to send the sample liquid and detect the subject 20. .

  Furthermore, although the metal fine particles 14 are the gold nanoparticles 14, the metal fine particles 14 are not particularly limited as long as they cause a resonance phenomenon with light, and for example, fine particles made of silver can be used. The first film 12 is an aminosilane film 12 made of a silane compound, as long as it is capable of adsorbing metal fine particles, and the blocking agent 16 is also a compound having a carboxyl group. There is no particular limitation as long as it can suppress the association between 14 and can be fixed to the first film 12. For example, when the first film is formed from a compound having a thiol group at the terminal, a compound having a thiol group or a compound having a maleimide group can be used as the blocking agent 16. Further, the second film 15 is the MUA film 15, but is not particularly limited as long as it can be formed on the surface of the metal fine particle 14 and the subject 20 can be fixed via the probe molecule 21 or the like. Mercaptopropionic acid can also be used. Furthermore, although the substrate 11 is the glass substrate 11, it is not particularly limited as long as it can transmit light in a wavelength band including the resonance wavelength of the metal fine particles.

  In the description of the detection method of the subject 20 using the sensor chips 10A to 10C, scattered light is detected. For example, light is incident on the glass substrate 11 of the sensor chips 10A to 10C and transmitted. It is also possible to detect the subject 20 by detecting light. In addition, when detecting the subject 20 using the sensor chips 10A to 10C, for example, the shift of the resonance scattering wavelength is actually detected by acquiring the scattering spectrum of the scattered light by the scattering spectrum measuring device, The subject 20 may be detected.

  Furthermore, in the sensor chips 10A to 10C, the metal particle 14 is preferably 50 nm to 100 nm in particle diameter, but the metal particle diameter may be less than 50 nm. However, as described above, when using resonance scattering, the range of the particle diameter is preferable from the viewpoint of more efficiently generating scattered light.

Further, the particle density (predetermined particle density) of the metal fine particles 14 on the sensor region 13 is preferably 30 to 60 particles / μm ² for the reason described above, but is not limited thereto. The predetermined particle density may be a particle density that does not block all the adsorption sites in the sensor region 13 of the first film 12 with the metal fine particles 14.

  Furthermore, the numerical values such as the composition of the solution and the processing time specifically exemplified in the method for manufacturing the sensor chip 10A described with reference to FIGS. 2 to 4 are examples, and the method for manufacturing the sensor chip of the present invention is limited thereto. Is not to be done.

  In the above description, the sensor chips 10A to 10C detect the analyte 20 such as DNA or protein. However, the sensor chips 10A to 10C can be used for measuring the refractive index of a solution in which a predetermined solvent is dissolved. It is. In this case, the second film 15 may not be provided.

It is the schematic which shows the structure of one Embodiment of the sensor chip based on this invention. It is a flowchart of the manufacturing method of a sensor chip. It is the schematic which shows the process of the manufacturing method of a sensor chip. It is the schematic which shows the process of the manufacturing method of a sensor chip. It is a conceptual diagram of the method of detecting the test object using a sensor chip. It is a block diagram which shows the structure of a flow cell. It is sectional drawing along the VII-VII line of FIG. It is a block diagram which shows the structure of an imaging device schematically. It is a figure which shows an example of the measurement of the refractive index change of the metal microparticle vicinity using an imaging device. It is a schematic diagram which shows the state by which the probe molecule was fixed to the sensor chip. FIG. 11 is a schematic diagram showing a state in which an analyte is adsorbed on the probe molecule of FIG. It is a figure which shows the light absorbency change when using PEG as a blocking agent. It is a figure which shows the light-absorbency change at the time of utilizing glycolic acid as a blocking agent. It is a figure which shows the light absorbency change when using sodium acetate as a blocking agent. It is a figure which shows the light absorbency change when utilizing 16-hydroxyhexadecanoic acid as a blocking agent. It is a figure which shows the particle size dependence of the scattering spectrum of a metal microparticle. It is a figure which shows the refractive index dependence of scattered light intensity | strength. It is a top view of other embodiments of a sensor chip concerning the present invention. It is the schematic which shows the structure along the XIX-XIX line | wire of FIG. It is the schematic which shows the structure of other embodiment of the sensor chip based on this invention.

Explanation of symbols

  10A, 10B, 10C ... sensor chip, 11 ... glass substrate, 11a ... surface of glass substrate, 12 ... aminosilane film (first film), 13 ... sensor region, 14 ... gold nanoparticles (metal fine particles), 15 ... MUA Film (second film), 16 ... blocking agent, 20 ... analyte, d1 ... particle diameter (particle diameter of metal fine particles).

Claims (12)

A sensor chip for detecting a refractive index change around the metal fine particles by utilizing a resonance phenomenon between the metal fine particles and light,
A substrate,
A first film formed on and in contact with the substrate, and for fixing the metal fine particles;
A plurality of the fine metal particles fixed on the sensor region of the first film;
A blocking agent fixed on the first film and disposed between adjacent metal fine particles among the plurality of metal fine particles;
With
The blocking agent is fixed to at least a part of the first film in a portion where the metal fine particles are not adsorbed in the sensor region of the first film.
A sensor chip characterized by that. The first film is formed of a silane compound;
The sensor chip according to claim 1, wherein the blocking agent is a compound having a carboxyl group.   3. The sensor chip according to claim 2, wherein the blocking agent comprises any one of glycolic acid, sodium acetate, 16-hydroxyhexadecanoic acid, and polyethylene glycol.   The sensor chip according to claim 1, further comprising a second film that is formed on a surface of the metal fine particle and fixes the subject.   The sensor chip according to claim 4, wherein the second film is made of mercaptoundecanoic acid. A plurality of sensor regions on the first film;
The sensor chip according to claim 1, wherein the metal fine particles and the blocking agent are fixed on each sensor region.   The sensor chip according to claim 1, wherein a particle diameter of the metal fine particles is 50 nm to 100 nm. A method of manufacturing a sensor chip for detecting a change in refractive index around the metal fine particles by utilizing a resonance phenomenon between the metal fine particles and light,
A sensor region of a first film, which is formed on the substrate so as to be in contact with the substrate and fixes the metal fine particles, and a metal fine particle solution containing the metal fine particles are brought into contact with the sensor region. A particle fixing step of fixing metal fine particles at a predetermined particle density;
By bringing the sensor region in which the metal fine particles are fixed into contact with the blocking agent introduction solution, a blocking agent is applied to at least a part of the first film where the metal fine particles are not adsorbed in the sensor region. A blocking agent fixing step for fixing;
A sensor chip manufacturing method comprising:   Forming a second film for immobilizing an object on the surface of the metal fine particles by bringing the sensor region in which the metal fine particles are immobilized into contact with the second film forming solution; The sensor chip manufacturing method according to claim 8, further comprising:   When the particle size of the metal fine particles fixed to the substrate is less than 50 nm, the particle size of the metal fine particles is increased to 50 nm to 100 nm by bringing the metal fine particles fixed to the substrate into contact with a particle growth solution. The sensor chip manufacturing method according to claim 8, further comprising a particle size increasing step. The first film has a plurality of the sensor regions,
In the particle fixing step, the metal fine particles are fixed at the predetermined particle density on each sensor region,
11. The sensor chip manufacturing method according to claim 8, wherein, in the blocking agent fixing step, the blocking agent is fixed on each sensor region. 12. The sensor chip manufacturing method according to claim 8, wherein the predetermined particle density is 30 to 60 particles / μm ² .

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