Patent Document

BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     The present invention relates to a molecular interaction detector and a molecule recovery device using the detector. More particularly, the present invention relates to a molecular interaction detector suitable for use in medical diagnosis, food inspection, etc., and to a molecule recovery device using the detector.  
         [0003]     2. Description of the Related Art  
         [0004]     Patent Document 1 (Japanese Patent No. 2815120) discloses one example of known detectors for detecting the molecular interaction. In the disclosed molecular interaction detector, the molecular interaction is measured by using a sensor that utilizes a resonance phenomenon of surface plasmon, i.e., a compression wave of free electrons propagating along an interface between a metal thin film and a dielectric. To fix a ligand to a sensor surface, alkane thiol is adsorbed onto a gold thin film by self-assembly. On that occasion, the alkane thiol having an active group to be covalently bound to a bioadaptable matrix is given as an organic linker molecule which contains a functional group capable of being bound to a metal and has the chain length with the number of atoms of not less than 10. The alkane thiol is coated with the bioadaptable porous matrix.  
         [0005]     Patent Document 2 (JP,A 2000-55920) discloses another example of known detectors for detecting the molecular interaction. In the disclosed molecular interaction detector, a substrate is divided into a plurality of regions, and on the substrate, polystyrene nanoparticles modified by different biomolecules are adsorbed in the form of a monolayer in each of the divided regions. An analyte (detection target) in a sample is colored by a fluorochrome, and protein specifically bound to the analyte is adsorbed onto the polystyrene nanoparticles. Excitation light is irradiated to the fluorochrome and an excited fluorescence signal is detected.  
         [0006]     Patent Document 3 (JP,A 2002-365210) discloses still another example of known detectors for detecting the molecular interaction. In the disclosed molecular interaction detector, to simply measure the biomolecular binding in a liquid, light is irradiated in a particular direction to a substrate onto which noble metal nanoparticles are adsorbed, and the absorption wavelength of the reflected light is measured. On that occasion, the surfaces of the noble metal nanoparticles are modified by thiol molecules each having a functional group so that any antibody having an amino group can be bound to the thiol molecule.  
       SUMMARY OF THE INVENTION  
       [0007]     In the detector disclosed in Patent Document 1, because the gold thin film is used as the metal thin film, thiol and sulfides are adsorbed onto gold by self-assembly. However, the inventors have found the following disadvantage. Since the alkane thiol, etc. having the chain length with the number of atoms of not less than 10 is employed, as the organic linker molecule, in the noble-metal nanoparticle sensor, there is a fear that when a buffer solution is added, the absorbance maximum wavelength (peak wavelength) is shifted and the reaction to be measured cannot be precisely measured.  
         [0008]     In the detector disclosed in Patent Document 2, because of using a noble-metal nanoparticle sensor for the measurement, there is a fear that protein, etc. capable of being physically adsorbed onto the noble metal surface are adsorbed onto a sensor chip, and materials having no relation with the reaction to be measured are non-specifically adsorbed onto the sensor chip. This impedes precise measurement. Further, in the biomolecule detecting method disclosed in Patent Document 3, because an insulator spacer layer is used when the metal nanoparticles are adsorbed onto the metal substrate, a complicated process is required to manufacture the noble-metal nanoparticle sensor.  
         [0009]     In view of the above-mentioned problems in the related art, an object of the present invention is to increase measurement accuracy in a molecular interaction detector. Another object of the present invention is to reduce deposition of materials having no relation with the measurement onto a sensor in a molecular interaction detector.  
         [0010]     To achieve the above objects, the present invention provides a molecular interaction detector for detecting molecular interactions by using a noble-metal free-electron thin film, wherein the noble-metal free-electron thin film is modified by an organic linker molecule, and the organic linker molecule has a linear or branched chemical structure having a functional group capable of being fixed to a surface of the noble-metal free-electron thin film and including a linear chain made of 1 to 5 atoms. The organic linker molecule also includes a functional group capable of being bound to a particular analyte contained in a sample solution to be measured.  
         [0011]     In the above molecular interaction detector, preferably, the detector comprises a light source for emitting light, and a unit for detecting the light emitted from the light source and reflected by the noble-metal free-electron thin film. Further, the noble-metal free-electron thin film has concaves and convexes formed in a film surface and having a size of not larger than a wavelength of the light source. Preferably, a detergent is added to the sample solution to be measured.  
         [0012]     In the above molecular interaction detector, preferably, the detector comprises a substrate on which the noble-metal free-electron thin film is formed, and a large number of nanoparticles adsorbed in the form of a single layer onto the substrate and having an essentially one diameter in the range of 5 nm to 100 μm. Further, each of the nanoparticles is made of an insulating high polymer selected from among at least polystyrene, styrene/butadiene, polyvinyltoluene, styrene/divinylbenzene, and vinyltoluene/tert-butylstyrene, or it is made of an insulating non-metal material selected from among at least silicon, silicon oxide, gallium arsenide, and glass. Moreover, a noble-metal free-electron thin film is formed on surfaces of the nanoparticles on the side opposed to the side where the nanoparticles are adsorbed onto the substrate.  
         [0013]     In the above molecular interaction detector, preferably, the functional group of the organic linker molecule capable of being bound to the particular analyte contained in the sample solution to be measured is one selected from among hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy, and vinyl groups.  
         [0014]     To achieve the above object, the present invention also provides a molecular recovery device comprising the above molecular interaction detector and one of an ultrasonic wave generating unit and a laser beam generating unit. In the molecular recovery device, preferably, the device further comprises a mass spectrometer for measuring a substance separated from a noble-metal free-electron thin film when one of an ultrasonic wave generated from the ultrasonic wave generating unit and a pulsated laser beam generated from the laser beam generating unit is irradiated to the molecular interaction detector.  
         [0015]     According to the present invention, since the molecular interaction is detected by using a sensor having a coated noble-metal free-electron thin film which is modified by alkane thiol having a chain length with the number of atoms of not more than 5, a signal not taking part in the reaction to be measured can be suppressed. As a result, measurement accuracy is increased. In addition, with the use of a detergent, a substance not taking part in the reaction to be measured can be suppressed from being deposited on a sensor chip. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]      FIG. 1  is a schematic view showing one example of a molecular interaction detector according to the present invention;  
         [0017]      FIG. 2  is a front view of a noble-metal nanoparticle sensor used in the molecular interaction detector shown in  FIG. 1 ;  
         [0018]      FIGS. 3A and 3B  are each a graph showing an example of detection by the noble-metal nanoparticle sensor;  
         [0019]      FIG. 4  is a graph showing an example of spectroscopic measurement by the noble-metal nanoparticle sensor;  
         [0020]      FIG. 5  is a graph showing an example of spectroscopic measurement by the noble-metal nanoparticle sensor;  
         [0021]      FIG. 6  is an explanatory view for explaining one method of measuring adsorption of protein;  
         [0022]      FIG. 7  is a graph showing an example of spectroscopic measurement by the noble-metal nanoparticle sensor;  
         [0023]      FIG. 8  is a graph showing an example of spectroscopic measurement by the noble-metal nanoparticle sensor;  
         [0024]      FIG. 9  is an explanatory view for explaining another method of measuring adsorption of protein;  
         [0025]      FIG. 10  is a graph showing an example of spectroscopic measurement by the noble-metal nanoparticle sensor;  
         [0026]      FIG. 11  is a graph showing an example of spectroscopic measurement by the noble-metal nanoparticle sensor;  
         [0027]      FIG. 12  is a graph showing an example of spectroscopic measurement by the noble-metal nanoparticle sensor; and  
         [0028]      FIG. 13  is an explanatory view for explaining a method of stripping, from a substrate, gold nanoparticles including adsorbed protein. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0029]     Several examples of a molecular interaction detector according to the present invention will be described below as preferred embodiments with reference to the drawings.  
         [0030]      FIG. 1  is a schematic view showing one example of a molecular interaction detector  100  according to the present invention. In this example, as described later in detail, a noble-metal nanoparticle sensor  104  is employed which contains a detergent in the mobile phase and is modified by alkane thiol having the chain length with the number of atoms of not more than 5. An analyte (target to be measured) is a biomolecule.  
         [0031]     The molecular interaction detector  100  includes the noble-metal nanoparticle sensor  104  as a sensor for detecting molecular interactions. A sensor chip  104   a  of the noble-metal nanoparticle sensor  104  has a recess A formed at its center. The bottom of the recess A is formed to be a flat surface. Opposite lateral surfaces of the recess A are sloped. Light emitted from a light source  103  is irradiated to the recess A through an optical multi-fiber probe  101 . The optical multi-fiber probe  101  is branched at an intermediate position between one end facing the light source  103  and the other end from which the light is irradiated toward the sensor chip  104   a . A branched fiber tip  101   a  is connected to a spectrophotometer  102 . The spectrophotometer  102  is connected to a data processing unit  107  through an A/D converter  106 .  
         [0032]     The recess A of the noble-metal nanoparticle sensor  104  is shown in enlarged scale in the lower side of  FIG. 1 . In the noble-metal nanoparticle sensor  104 , a noble-metal thin film  109  is coated over the surface of a flat substrate  108  by vapor deposition. A monolayer of substantially spherical nanoparticles  110  is formed in planar arrangement and is adsorbed onto the noble-metal thin film  109 . Another noble-metal thin film  111  is formed on the upper surfaces of the nanoparticles  110  by vapor deposition.  
         [0033]     In the noble-metal nanoparticle sensor  104 , 1 μM to 10 mM of sodium thioglycolate  112  dissolved in water is permeated into the noble-metal thin film  111  such that the thioglycolate  112  is adsorbed onto the noble-metal thin film  111 . Also, 1 μM to 10 mM of 10-carboxy-1-decanethiol  113  dissolved in 10% (v/v) of ethanol is permeated into the noble-metal thin film  111  such that the 10-carboxy-1-decanethiol  113  is adsorbed onto the noble-metal thin film  111 . The thioglycolate  112  and the 10-carboxy-1-decanethiol  113  contain carboxyl groups as functional groups.  
         [0034]     In the molecular interaction detector  100  constructed as described above, the light emitted from the light source  103  passes through the optical multi-fiber probe  101  and is irradiated to a noble-metal nanoparticle sensor surface  105  of the sensor chip  104   a . A part of the reflected light from the noble-metal nanoparticle sensor surface  105  is returned through the optical multi-fiber probe  101  and enters the spectrophotometer  102 . The spectrophotometer  102  measures an absorption wavelength characteristic based on the reflected light from the noble-metal nanoparticle sensor surface  105 . The measured result is sent to the data processing unit  107  through the A/D converter  106  and is stored therein.  
         [0035]     Details of the noble-metal nanoparticle sensor  104  will be described below with reference to  FIG. 2 . After coating the noble-metal thin film  109  over the surface of the substrate  108 , a monolayer of the nanoparticles  110  made of a high polymer, SiO 2 , TiO 2  or the like is adsorbed onto the noble-metal thin film  109 . Then, a noble metal, e.g., gold, silver, copper or platinum, is vapor-deposited or sputtered to form the noble-metal thin film  111  on the nanoparticles  110 . Because the surfaces of the substrate  108  and the nanoparticles  110  are coated with the noble metals, the substrate  108  and the nanoparticles  110  noticeably develop colors.  
         [0036]     An example of measuring the biomolecular interaction by using the noble-metal nanoparticle sensor  104  will be described below in connection with the case of detecting an antigen—antibody reaction.  FIGS. 3A and 3B  are graphs for explaining the principle in measurement of the biomolecular interaction by using the spectrophotometer  102 .  FIG. 3A  is a graph showing absorption wavelength characteristics, and  FIG. 3B  is a graph showing change of peaks of the absorption wavelength characteristics over time, which are detected as shown in  FIG. 3A .  
         [0037]     The spectrophotometer  102  detects the reflected light from the recess A in the noble-metal nanoparticle sensor  104  and measures the absorbed light intensity per wavelength. For example, when a solution of 1 μM to 10 mM of the sodium thioglycolate  112  is supplied to the recess A, an absorption wavelength characteristic  118  is obtained. A peak wavelength of the absorption wavelength characteristic  118  is x 1 . Next, when another solution, e.g., a buffer solution containing antigen protein, is added to the solution of the sodium thioglycolate  112 , an absorption wavelength characteristic  119  is obtained and its peak wavelength (x 2 ) is shifted toward the longer wavelength side. Subsequently, when still another solution, e.g., a buffer solution containing antibody protein that is specifically bound to the antigen protein to be detected, is added, an absorption wavelength characteristic  120  is obtained and its peak wavelength (x 3 ) is further shifted toward the longer wavelength side.  
         [0038]     In the measurement process described above, the peak wavelength (absorption maximum wavelength) is changed as shown in  FIG. 3B . More specifically, the peak wavelength is first changed from the peak wavelength x 1  obtained when only the solution of the sodium thioglycolate is added, to the peak wavelength x 2  obtained when the other solution, e.g., the buffer solution containing antigen protein, is added, with the lapse of a predetermined time. The amount of the fixed antigen protein can be measured from the extent by which the peak wavelength has shifted. Then, when the buffer solution containing antibody protein specifically bound to the antigen protein to be detected is added, the peak wavelength is further shifted to x 3  with the lapse of a predetermined time. The amount of the specifically bound antigen protein can be obtained by measuring the extent by which the peak wavelength has shifted from x 2  to x 3 .  
         [0039]     Practical examples of the measurement using the spectrophotometer  102  will be described below with reference to  FIGS. 4 and 5 . The measurement is performed under the conditions shown in  FIG. 1 . More specifically, in the noble-metal nanoparticle sensor  104 , the gold thin film  109  is coated over the flat substrate  108  by vapor deposition. Further, the gold thin film  111  is coated over the nanoparticles  110  by vapor deposition. The thioglycolate  112  and the 10-carboxy-1-decanethiol  113  are separately adsorbed onto different regions of the gold thin film  111  on the nanoparticles  110 .  
         [0040]      FIG. 4  shows an experimental result for change of the shift amount (Δx) of the peak wavelength corresponding to  FIG. 3B . When the sodium thioglycolate (thiol)  112  was adsorbed onto the nanoparticles  110 , the peak of absorption spectrum measured by the spectrophotometer  102  was shifted by ΔP 1  as shown in  FIG. 4 . Then, a solution of PBS (Phosphate Buffered Saline) (i.e., 10 mM of Phosphate and 150 mM of NaCl) at pH 7.4 was added to the recess A. As a result of measuring the reflected light from the nanoparticles  110  through the optical multi-fiber probe  101 , the peak wavelength was further shifted by ΔP 2 .  
         [0041]      FIG. 5  shows an experimental result when the 10-carboxy-1-decanethiol  113  was adsorbed onto the nanoparticles  110 . The peak wavelength was shifted by ΔP 3 . Then, as in the case of using the thioglycolate, the solution of PBS (i.e., 10 mM of Phosphate and 150 mM of NaCl) at pH 7.4 was added to the recess A. As a result of measuring the reflected light from the recess A by the spectrophotometer  102 , the peak wavelength was further shifted by ΔP 4 .  
         [0042]     According to this example of the present invention, since the sodium thioglycolate is used as an organic thiol molecule, it is possible to suppress a non-specific signal due to the buffer solution (BPS), which is measured in the case of using the 10-carboxy-1-decanethiol. Also, various kinds of proteins can be fixed to the noble-metal nanoparticle sensor by selecting functional groups, such as hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy, and vinyl groups, contained in the organic linker molecule depending on the kind of protein to be captured.  
         [0043]     Another example of the molecular interaction detector  100  according to the present invention will be described below with reference to  FIGS. 6-8 .  FIG. 6  shows the adsorbed state of alkane thiol, and  FIGS. 7 and 8  are each a graph showing peak shift characteristics of the reflected light measured by the spectrophotometer  102 . This example differs from the above-described example only in alkane thiol adsorbed onto a gold thin film  202 . An aqueous solution of 1 μM to 10 mM of sodium thioglycolate  201  is supplied to the nanoparticles  110  on which the gold thin film  202  is formed by vapor deposition. A molecular monolayer of the thioglycolate  201  is thereby formed on the gold thin film  202 .  
         [0044]     Then, a solution of 0.2 M of WSC/0.05 M of NHS is added. Here, WSC means N-ethyl-N′-(3 dimethylaminopropyl)-carbodiimide hydrochloride, and NHS means N-hydroxy succimide. This WSC/NHS solution is an aqueous solution obtained by dissolving them in an extra-pure water (MiLiQ). The nanoparticles  110  coated with the gold thin film  202  is immersed in the WSC/NHS solution for 7 minutes to activate a carboxyl group  203 . Streptoavidin  204  is added to the activated carboxyl group  203  such that an amino group of the streptoavidin  204  is coupled to the carboxyl group  203 . The streptoavidin  204  is dissolved in 10 mM of acetic acid buffer at pH 4.5 and is used in concentration of 100 μg/mL.  
         [0045]     A solution of 50 mM of Tris-HCl (pH 7.5) and 0.15 M of NaCl is used as the mobile phase. Biotinylated second protein  205  specifically bound to first protein  206  is added as a sample. The biotinylated second protein  205  is captured by the streptoavidin  204 . Further, a detergent Tween 20 (registered trade name) is added to the mobile phase in final concentration of 0.1%.  
         [0046]     Each of data  207  and  209  indicated by dark lines in  FIGS. 7 and 8  represents the peak wavelength resulting when the first protein  206  is added after capturing the biotinylated second protein  205 . Each of data  208  and  210  indicated by light lines in  FIGS. 7 and 8  represents the peak wavelength resulting when the first protein  206  is added without capturing the biotinylated second protein  205 .  
         [0047]     When the first protein  206  is added to the solution to which is added the biotinylated second protein  205 , the first protein  206  is captured by the biotinylated second protein  205  in a way like hybridization. For comparison, an experiment of adding 1 μM of the first protein  206  alone without adding the biotinylated second protein  205  was also conducted (see the data  208 ). The result of measuring an absorption spectrum by the spectrophotometer  102  with the first protein  206  adsorbed onto the second protein  205  is shown by the data  207  in  FIG. 7 . Note that  FIG. 7  plots a peak shift, i.e., a shift of the absorption maximum wavelength.  
         [0048]     As a comparative experiment, the streptoavidin  204  was adsorbed onto the nanoparticles  110  coated with gold by vapor deposition without using not only the organic linker molecule, i.e., the linker molecule such as the thioglycolate, but also the detergent. Then, the first protein  206  was adsorbed onto the biotinylated second protein  205 . The result of measuring an absorption spectrum by the spectrophotometer  102  in that case is shown by the data  209  in  FIG. 8 . The result in the case of adsorbing the first protein  206  without capturing the second protein  205  is also shown in  FIG. 8  (see the data  210 ). As seen from  FIGS. 7 and 8 , according to this example, non-specific adsorption caused in the case of not capturing the biotinylated second protein  205  can be suppressed by adding the detergent to the mobile phase and by using the thioglycolate as the organic linker molecule.  
         [0049]     Still another example of the molecular interaction detector  100  according to the present invention will be described below with reference to  FIGS. 9-12 . This example differs from the above-described examples in alkane thiol adsorbed. With reference to  FIG. 9 , the following description is made of a method of measuring the amount of adsorbed protein when first protein and biotinylated second protein specifically bound to the first protein are adsorbed onto the nanoparticles  110  coated with the gold thin film  202  by vapor deposition. A suspended aqueous solution of 1 μM to 10 mM of sodium thioglycolate or aminoethane thiol is supplied to the recess A. A monolayer of organic thiol molecules  301  of the thioglycolate or the aminoethane thiol is thereby formed on the surface of the vapor-deposited gold thin film  202  coated over the nanoparticles  110 .  
         [0050]     Then, 10-1000 mg/mL of streptoavidin  302  is added to the suspended aqueous solution such that the streptoavidin  302  is bound to the thioglycolate or the aminoethane thiol. Further, biotinylated second protein  303  specifically bound to first protein  304  is added. The biotinylated second protein  303  is captured by the streptoavidin  302 . A detergent Tween 20 (registered trade name) is added in final concentration of 0.1% to the mobile phase that is a solution of 50 mM of Tris-HCl (pH 7.5) and 0.15 M of NaCl.  
         [0051]     When the first protein  304  specifically bound to the biotinylated second protein  303  is supplied to the sensor, the first protein  304  is captured by the biotinylated second protein  303  in a way like hybridization. For comparison, an experiment of capturing, to the streptoavidin  302 , biotinylated variant second protein having no molecular configuration capable of being bound to the first protein  304  was also conducted. Also in that experiment, sodium thioglycolate or aminoethane thiol was used as the organic thiol molecule  301 .  
         [0052]      FIG. 10  shows the result of measuring adsorption of the first protein  304  by the spectrophotometer  102  when the thioglycolate is used as the organic thiol molecule. Note that  FIG. 10  is a graph showing a peak shift of the absorption spectrum. Further,  FIG. 11  shows the result of measuring a peak shift of the absorption spectrum caused with adsorption of the first protein  304  when the aminoethane thiol is used as the organic thiol molecule, and  FIG. 12  shows the result of measuring a peak shift of the absorption spectrum caused with adsorption of the first protein  304  when the organic thiol molecule is not used.  
         [0053]     In those graphs, each of data  305 ,  307  and  309  indicated by dark lines represents the peak wavelength resulting when the first protein  304  specifically bound to the biotinylated second protein  303  is added after capturing the biotinylated second protein  303 . Each of data  306 ,  308  and  310  indicated by light lines represents the peak wavelength resulting when the first protein  304  is added after capturing the biotinylated variant second protein having no configuration capable of being bound to the first protein  304 .  
         [0054]     As seen from  FIGS. 10-12 , according to this example, non-specific adsorption caused in the case of capturing the biotinylated variant second protein  303 , which has no molecular configuration capable of being bound to the first protein  304 , can be suppressed by adding the detergent to the mobile phase and using the thioglycolate or the aminoethane thiol as the organic thiol molecule.  
         [0055]     Other example of a molecule interaction detector according to the present invention will be described below with reference to  FIGS. 13A and 13B  which show the recess A in enlarged scale. In a noble-metal nanoparticle sensor  104 , as in the example shown in  FIG. 1 , a noble-metal thin film  402  is coated over a substrate  401 . A monolayer of nanoparticles  403  made of a high polymer, SiO 2 , TiO 2  or the like is formed on the noble-metal thin film  402  coated over the substrate  401 . Then, a noble metal, e.g., gold, silver, copper or platinum, is vapor-deposited or sputtered from above the nanoparticles  403 , to thereby form a noble-metal thin film  404  on the upper surfaces of the nanoparticles  403 . Because the surfaces of the substrate  401  is coated with the noble-metal thin film  402 , the substrate  401  and the nanoparticles  403  noticeably develop colors.  
         [0056]     Second protein  405  is physically adsorbed onto the nanoparticles  403  coated with the noble-metal thin film  404 . Then, a sample containing first protein  406  specifically bound to the second protein  405  is supplied to the recess A. As shown in  FIG. 13A , the first protein  406  is captured on the surfaces of the nanoparticles  403 . An ultrasonic wave with transmission frequency of 50 kHz and output of 10 W is irradiated toward the substrate  401  in water for 5 seconds. As shown in  FIG. 13B , the nanoparticles  403  are stripped from the substrate  401  with the irradiation of the ultrasonic wave. The nanoparticles  403  stripped from the substrate  401  with the first protein  406  adsorbed on them are recovered by using a filter (not shown). The recovered nanoparticles  403  are treated with a buffer solution of 10 mM of glycine—hydrochloric acid at pH 3 for elution of the first protein  406 . The eluted first protein  406  is analyzed by, e.g., a mass spectrometer (not shown). According to this example, it is possible to recover protein and to reliably confirm the recovery of the protein.

Technology Category: 3