JP3774460B2 - Substance detection method - Google Patents

Description

  The present invention relates to a method for detecting a substance using a biosensor. More specifically, the present invention relates to a protein or the like labeled with a gold colloid by utilizing the sensitizing action of a surface plasmon resonance (SPR) sensor with a gold colloid. It is an invention that makes it possible to detect a living body related substance with high sensitivity.

  Conventionally, an antibody labeled with a fluorescent molecule or a radioisotope has been used to detect an interaction between biomolecules such as an antigen-antibody reaction. However, such labeling of antibodies requires complicated steps such as pretreatment and washing operations, and it is difficult to detect and quantify substances quickly and in real time. In particular, in the case of labeling with isotopes, there is a concern about the influence on the human body at the time of experimental operation or radiation exposure from waste liquid. In order to avoid these problems in the detection of antigen-antibody reaction and the like, there is a demand for means that can perform simple, safe, and real-time detection.

  As such means, the following techniques have been proposed so far.

Labeling method using a metal colloid This method is a method in which a metal colloid such as a gold colloid is bound to a specific binding portion to be labeled, and the metal colloid is detected and measured as a label.
Metallic ultrafine particles are known to increase resonance Raman scattering due to surface signal sensitization effects (Surface Enhanced Raman Scattering, SERRS: Surface Enhanced Resonance Raman Scatching)], and further, it has been found that the above-mentioned signal sensitizing effect is also exhibited in metal colloids. This method is a method of utilizing the characteristics of such a metal colloid. That is, by using a metal colloid that has a surface sensitizing effect on Raman light as a labeling reagent in this way, various objects to be detected that can bind to the metal colloid, such as antibodies or binding molecules, can be detected. it can. Further, the substance to be detected is brought into contact with a substance with a Raman sensitizing substance such as a specific binding site for the substance and a metal colloid, that is, an intervening molecule, and the object to be detected bound to the medium. It can be detected with high sensitivity using a signal from Raman light as an index.

SPR sensors Various biosensors have been developed in order to detect substances in real time without labeling. One of the main biosensors is a surface plasmon resonance (SPR) sensor. SPR is attracting attention as a highly reliable detection technique because it does not require complicated pretreatment, is non-labeled, can detect substances quickly and easily, and has high sensitivity.

SPR is a refractive index measurement method using near-field optics. That is, when the light is totally reflected on the glass chip surface on which gold is deposited, a part where the reflected light intensity is attenuated is generated in a part of the reflected light (SPR signal), and the angle of this resonance attenuation is the mass on the sensor chip. In addition, depending on the refractive index as a parameter, when a substance is bonded to the surface of the chip, the angle of the resonance wave shifts. From the value of the shift of the resonance angle, the amount of binding, strength, binding speed, etc. can be calculated.
Chip surface treatment method Gold surface has the property of easily adsorbing ionic substances such as proteins and nucleic acids by electrostatic interaction, and so-called non-specific adsorption, which is not related to the specificity of the binding molecule, is specified. It becomes a big problem when detecting this substance. Therefore, at present, the mass change in the vicinity of the interface is measured regardless of the presence or absence of specific binding, and there are many cases where doubts remain in the reproducibility and reliability of the detection data. For this reason, the detection method using the gold surface is not widely used in the general clinical diagnostic field, although high-sensitivity measurement is possible in principle.
Regarding such a problem of non-specific adsorption on the gold surface, a method of coating the gold surface with a polymer or a self-assembled monolayer (SAM) has been proposed. Further, chips coated with polysaccharides such as dextran are commercially available, but a sufficient non-specific adsorption suppressing effect has not yet been obtained.
Gold colloid colorimetric method The antigen detection method by the agglutination method using gold colloid is a method of capturing antigen-antibody reaction as a color change as a gold colloid colorimetric method, and can be expected to have higher sensitivity than the immunolatex method. As in-vitro diagnostics, those currently in practical use include colon cancer screening by fecal occult blood test, pregnancy diagnosis by detection of human chorionic gonadotropin (hCG), etc. A method of quantifying in combination with a measuring device is taken.
Moreover, the following literature is mentioned as literature considered to be related to this invention.
JP 2000-55920 A JP 2002-267669 A JP 2003-14765 A

  As described above, an object of the present invention is to provide a means for further increasing sensitivity in a sensor using a metal surface that can be detected simply, safely, and in real time.

  The present inventor is able to detect a target substance sensitively by a signal obtained by supporting a detection target substance on a metal colloidal particle and bringing it into contact with a sensor chip having the metal surface. As a result, the present invention has been completed.

  That is, according to the present invention, the metal colloidal particles carrying the detection target substance are brought into contact with the “sensor chip having a binding molecule for the detection target substance and having the surface of the metal”, and It is an invention that provides a detection method (hereinafter also referred to as the present detection method) in which a detection target substance is detected using the above signal as an index.

  The “detection target substance” to be detected by this detection method is not particularly limited as long as there is a “binding molecule” for the substance. For example, an antigen or hapten or lectin that can use an antibody as a binding molecule is bound. Examples include sugars that can be used as molecules, substrates that can use enzymes as binding molecules, hormones that can use hormone receptors as binding molecules, nucleic acids that can use complementary chains as binding molecules, and proteins that can be used as binding molecules. be able to.

  Examples of the metal constituting the metal colloid particles and the surface of the sensor chip include gold, platinum, silver, copper, and aluminum. Gold is preferable. The particle diameter of the metal particles is 0.5 nm to 500 μm, preferably about 1 nm to 1 μm.

The material of the sensor chip surface, that is, the material of the support of the sensor chip surface can be the same as the material of the substrate of the chip (that is, the substrate itself forms the sensor chip surface), It is also possible to form a metal thin film on the surface of the substrate and use this metal as a support.
This detection method can be performed using detection means using a sensor chip defined in the present invention, and is preferably used for detection in a biosensor using surface plasmon resonance (SPR). However, the present invention is not limited to the above, and any sensor can be used as long as it can detect any change that occurs on the chip surface due to the formation of the binding pair in the set of the detection target substance and the binding molecule. be able to. Examples of the change to be detected include radioactivity, contact angle, sedimentation, ultraviolet spectroscopy, fluorescence, chemiluminescence, and electrochemiluminescence in addition to the surface plasmon resonance.
[ Supporting target substance and binding molecule ]
The detection target substance for metal colloidal particles (hereinafter also referred to as metal particles) and the loading of binding molecules to the metal surface (hereinafter also referred to as metal surface) of the sensor chip are physically adsorbed on the metal particles or metal surface. It is possible to bond directly using chemical adsorption force, chemical bonding force and the like. In addition, one end of a polymer containing a hydrophilic linear monomer unit (hereinafter also referred to as a hydrophilic polymer) is bonded to a metal particle or metal surface, and a detection target substance or binding molecule is connected to the other end of the hydrophilic polymer molecule. (The hydrophilic polymer molecule bonded to the metal particle or the metal surface in this way is in an upright state based on one end on the surface of the metal particle or the metal surface. Is generally). By performing such binding of the hydrophilic polymer, it is possible to suppress a non-specific reaction when performing this detection method. As described above, the detection target substance or the binding molecule on the metal particle or the metal surface can be directly supported, and one end of the hydrophilic polymer can be bound. In the case of this aspect, the other end of the hydrophilic polymer is in an inactive state (alkoxylation treatment such as methoxylation treatment and ethoxylation treatment, and inactivation treatment such as esterification treatment such as carbonylation treatment). The detection target substance or binding molecule may be bound to the vicinity of the other end of the hydrophilic polymer.
The hydrophilic polymer molecule has a functional group that binds to a metal at one end and a functional group that binds directly or indirectly to a detection target substance or binding molecule at the other end. is required. The “direct bond” mentioned above means that the substance to be detected or the binding molecule is directly bonded to the functional group or atom at the other end of the hydrophilic polymer molecule. "Means that the functional group at the other end of the polymer molecule and the substance to be detected or binding molecule are bonded via a linker molecule.
The following (a) and (b) can be mentioned as a suitable hydrophilic polymer (in a state where the other end is stabilized) that can be used in the present invention.
(A) Brush-like structure constructed by the hydrophilic polymer derivative represented by the formula (1)

ALPPP (1)
[In the formula, A is a functional group capable of coordinating to a metal particle or metal surface, L is a divalent group or valence bond, P is a hydrophilic polymer segment, B is 1 Is a valent group]
Coordination to the metal particle or metal surface is performed by the functional group A, and the hydrophilic polymer derivative (1) is supported on the metal particle or metal surface, whereby the hydrophilic polymer derivative (1) has a desired brush-like structure. It can be a thing.

  Specifically, for example, a mercapto group, an amino group, a carboxyl group, or a sulfide group is preferable as the functional group A.

  Examples of the hydrophilic polymer segment P include polyalkylene oxide, polyacrylamide, polyacrylic acid and the like, and polyalkylene oxide is preferable.

  In particular, the polyalkylene oxide is preferably an alkylene oxide having 2 to 4 carbon atoms, that is, a polyalkylene oxide having ethylene oxide, propylene oxide, or butylene oxide as monomer units. In addition, the polymerization degree of the alkylene oxide unit in the polyalkylene oxide is preferably 2 to 10,000, and particularly preferably 10 to 3000.

The monovalent group B is preferably a group capable of constituting the end of the hydrophilic polymer segment P corresponding to the tip of the brush-like structure. Specific examples include a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, a hydroxyl group, an acetal group, and the like.
Examples of the divalent group that L can take include, for example, an alkylene group having 1 to 12 carbon atoms, preferably 1 to 3 carbon atoms, and a group (2)

[Wherein L1 represents a divalent group such as —COO—, —O—, —S—, etc., and p is 1 to 12, preferably 1 to 3.]
Etc. can be mentioned.

  The hydrophilic polymer derivative (1) can be produced by a known means depending on its specific structure. For example, when the hydrophilic polymer segment P is a polyalkylene oxide, it can be produced according to the method and conditions described in JP-A-7-316285 or modified.

  Here, an example of the manufacturing process of acetal-PEG-SH is disclosed as this hydrophilic polymer derivative (1). In this manufacturing process, r means a positive integer.

When a brush-like structure corresponding to the hydrophilic polymer segment P is constructed on a metal particle or metal surface, the functional group A of the hydrophilic polymer derivative (1) is a mercapto group, an amino group, a carboxyl group, or In the case of a sulfide group, the hydrophilic polymer derivative (1) is dissolved in an appropriate buffered aqueous solution, and this polymer solution is supported at a suitable temperature, for example, at about 20 to 37 ° C. by immersing the chip. Producing a chip surface provided with a desired brush-like structure by incubating for several tens of minutes to several hours in contact with the surface of a body (metal such as gold, silver, copper, and aluminum). Can do. The concentration of the hydrophilic polymer derivative (1) in this polymer solution varies depending on the molecular weight of the block polymer used, but is usually about 0.1 to 5 mg / ml, preferably about 1 mg / ml of the solution.
(B) Brush-like structure constructed by hydrophilic block polymer derivative As a preferred embodiment of the hydrophilic polymer derivative, the hydrophilic polymer constituting the brush-like structure on the metal particle or the metal surface is: The hydrophilic polymer segment constituting the first block of the block polymer (3) having the characteristics (a) and (b) and the polymer segment constituting the second block are formed on the metal particles or the metal surface. The aspect which is a functional group which has coordination ability can be mentioned.
(A) Let said hydrophilic polymer segment be a 1st block.
(B) A polymer segment having a degree of polymerization of 1 to 200 having a monomer having a functional group having coordination ability on a metal particle or metal surface as a monomer unit is defined as a second block.

  The hydrophilic polymer segment in the first block is the same as the hydrophilic polymer segment P described above.

In addition, as the functional group having a coordination ability to the support on the surface of the sensor chip in the polymer segment in the second block, for example, a mercapto group, a dithiol group, a sulfide group, an amino group, a carboxyl group, a silanol group, or Examples thereof include a trialkylsilyl group having 1 to 5 carbon atoms. Examples of monomer units having these functional groups include methacrylic acid (2-diethylaminoethyl), methacrylic acid (2-dimethylaminoethyl), polycysteine, polylysine, polyglutamine, and methacrylic acid (3-trimethoxysilylpropyl). Etc. A second block having a desired functional group can be formed by subjecting these monomer units to a polymerization reaction in the production process of the block polymer derivative (3) according to a conventional method.
Furthermore, as one of the preferred embodiments of the second block, the formula (4)

[Wherein, m represents an integer of 1 to 10, n represents an integer of 1 to 100, and R4 and R5 each independently represent an alkyl group having 1 to 5 carbon atoms, an amino group, a mercapto group, or And a segment of methacrylic acid polymer represented by].

  The production process of the block polymer derivative (3) having the methacrylic acid polymer segment (4) as the second block is disclosed in detail in JP-A No. 2002-80903. A block polymer derivative having the acid polymer segment as the second block can be produced.

That is, for example, the formula A-L-P-OH (6)
[The definitions of A, L, and P in the formula are the same as those in the hydrophilic polymer derivative (1).]
Compound (6) [Production method of this compound (6) itself is also known (see, for example, JP-A-7-316285, JP-A-11-322916, JP-A-2001-200050, etc.) In contrast, the block polymer derivative (3) can be obtained by subjecting polydimethylaminoethyl methacrylate to a polymerization reaction so as to obtain a desired degree of polymerization.

  When the block polymer derivative (3) obtained as described above is supported so that the first block becomes a brush-like structure, it is substantially the same as the supporting step for the hydrophilic polymer derivative (1) described above. The same loading process can be performed. That is, by using the block polymer derivative (3) having the second block having a functional group capable of coordinating on the metal particle or metal surface as a segment, the block polymer derivative (3) can be easily converted into the metal particle or metal. Can be fixed on the surface.

  When carrying, the functional group of the second block becomes a metal particle or metal surface by mixing or immersing a sensor chip having a metal particle or metal surface in a solution containing the block polymer derivative (3). The block polymer derivative (3) can be fixed to the chip surface. As a result, metal particles or a metal surface in which the first block side of the block polymer derivative (3) stands up like a brush can be produced. The concentration of the block polymer derivative (3) in the polymer solution varies depending on the molecular weight of the block polymer used, but is usually about 0.1 to 5 mg / ml, preferably about 1 mg / ml of the solution.

(C) Masking treatment A non-specific reaction can be further suppressed by performing a masking treatment on the metal particles or the metal surface supporting the hydrophilic polymer provided as described above. This masking treatment can be performed by bringing the metal particles or metal surface carrying the hydrophilic polymer into contact with a masking agent solution. As a masking agent, 2-mercaptoethanol can be mentioned as one of suitable embodiments. Further, the hydrophilic segment P is shorter than the bulk of the brush-like structure carrying the detection target substance or the binding molecule, and the vicinity of the tip of the standing part is an alkoxylation treatment such as a methoxylation treatment or an ethoxylation treatment, Mention may be made, for example, of hydrophilic polymer derivatives (1) which have been subjected to inactivation treatment such as esterification treatment such as carbonylation treatment. In this case, the hydrophilic segment of the masking agent preferably has a molecular weight that is about 10 to 70% longer than the hydrophilic segment that carries the detection target substance or the binding molecule.

  The concentration of the masking agent in the masking agent solution in these cases varies depending on the type of the masking agent and the like, but is generally about 0.001 to 1000 mM, and preferably about 0.1 to 10 mM. The masking treatment time is preferably about 3 minutes to 1 day. Further, the masking process is preferably performed at a temperature of about 20 to 37 ° C.

(D) Carrying of detection target substance and binding molecule As a premise for carrying this support, the functional group at the other end of the hydrophilic polymer carried on the metal particle or the metal surface is directly or indirectly detected. It is often necessary to activate the binding molecule by converting it to a functional group capable of binding (for example, conversion of an acetal group to an aldehyde group). Desired loading can be performed by directly or indirectly binding the substance to be detected or the binding molecule to the activated functional group.

[ Aspects of this detection method ]
As described above, in this detection method, a metal particle carrying a detection target substance is brought into contact with a sensor chip having a metal surface carrying a binding molecule for the detection target substance, and a signal from the sensor chip is obtained. This is a detection method for detecting a detection target substance as an index.
As a typical embodiment of this detection method, a method for performing the following steps can be mentioned.

  1) A step of creating “a metal surface of a sensor chip on which a metal particle carrying a detection target substance is bonded via a binding molecule”, that is, “a sensor chip carrying a binding molecule for the detection target substance” By bringing the metal particle carrying the detection target substance into contact with the metal surface, the binding force between the detection target substance on the metal particle and the binding molecule on the metal surface is exerted to detect the detection target substance on the metal surface. A step of creating a metal surface in which the metal particles on which is supported are preferably bonded until saturated.

  2) According to the above-mentioned step 1), the detection target [so-called specimen (blood, urine, lymph, cerebrospinal fluid and other body fluid specimens, of course, is applied to the metal surface on which the metal particles carrying the detection target substance are bound). (Including samples derived from agricultural products, soil, seawater, lake water, etc.), and by detecting (quantitative or qualitative) specific substances to be detected in these samples, medical diagnostic fields, environmental tests The usefulness of the present invention is recognized in the field of inspection such as field, quality inspection field, etc.] to bring the detection target substance in the detection target and the metal particles into the binding molecules on the metal surface. The process of competing.

3) A step of detecting the dissociation of the metal particles from the metal surface as a signal by the substitution of the detection target substance and the metal particles by the competition in the above step 2.

  As described above, this dissociation signal is detected by various sensors such as radioactivity, contact angle, sedimentation, ultraviolet spectroscopy, fluorescence, chemiluminescence, and electrochemiluminescence in addition to the surface plasmon resonance that can be detected by the SPR sensor. It can be a possible signal.

  Depending on the presence or absence or strength of the dissociation signal, the qualitative or quantitative determination of the detection target substance in the detection target can be performed with reference to a calibration curve or the like.

  In addition, a small amount of detection is possible by bringing a metal particle carrying the detection target substance into contact with the metal surface carrying the detection target substance as a detection target and inducing a sensitization reaction due to the enhancement effect of plasmon resonance. It is also possible to detect the target substance with high sensitivity.

  In order to perform this detection method performed in such a manner, for example, 1) metal colloidal particles carrying a hydrophilic polymer not carrying a specific substance, and 2) carrying the same hydrophilic polymer. A set of sensor chips having a metal surface (hereinafter also referred to as an unsupported set).

Or
1) a) a metal colloidal particle carrying a hydrophilic polymer carrying a specific substance, or b) a hydrophilic substance carrying the specific substance directly on the particle surface and carrying or not carrying the specific substance. Colloidal metal particles carrying a functional polymer, and 2) a) a sensor chip having a metal surface carrying a hydrophilic polymer carrying a binding molecule for the specific substance on the metal surface of the sensor chip, or b) A set of sensor chips (hereinafter also referred to as a support set) having a metal surface on which a specific substance is directly supported on a metal surface and a hydrophilic polymer on which the specific substance is supported or not supported.

  Etc.

  The unsupported set is suitable for the user of the set to select the detection target, the detection target substance, and the binding molecule for the substance, and individually perform the detection method according to the above-described procedure. It is a set for detection.

  The carrier set has a sensor chip having a metal surface carrying a predetermined detection target substance and a metal surface carrying a binding molecule for the substance in advance, and has a versatile inspection item according to the above procedure. It is a set for inspection that is suitable for performing efficiently.

The present invention provides a means for further increasing sensitivity in a sensor using a metal surface that can be detected simply, safely, and in real time.

  The present invention will be described more specifically below with reference to examples of the present invention, but this description is not intended to limit the scope of the present invention.

1. Detection Method of Streptavidin Using Biotin as Binding Molecule 1) Preparation of PEGylated Gold Colloid Having Terminal Aldehyde Group Acetal-PEG-SH (molecular weight 10,000 ) Was added and allowed to react at room temperature for 3 hours. Furthermore, after adding methoxy-PEG-SH (molecular weight 2,000) and reacting at 50 ° C. for 3 hours, the acetal group is hydrolyzed by lowering to pH 2 with HCl and incubating for 2 hours, and aldehydes are deposited on the colloidal gold surface. A group was introduced.

2) Introduction of streptavidin into PEGylated gold colloid Streptavidin and heterolinker N-succinimidyl 3- (2-pyridyldithio) propionate (hereinafter referred to as SPDP) were mixed at a molar ratio of 1: 3, and pH 7.5 The reaction was carried out at room temperature for 60 minutes to introduce a pyridyl disulfide group into streptavidin.
Next, a reducing agent dithiothreitol (hereinafter referred to as DTT) was added, and the disulfide was cleaved at pH 7.5 at room temperature to prepare SH-streptavidin. By adding N- (maleimidocaproic acid) hydrazide (hereinafter referred to as EMCH), a heterolinker, to this SH-streptavidin at a molar ratio of 1: 1 and incubating overnight at pH 6.5 and 4 ° C. Hydrazide was introduced into streptavidin.
In this way, the synthesized streptavidin hydrazide is reacted with the aldehyde group on the gold colloid surface (PEG: SA = 10: 1, pH 5.5, overnight), and the reductive amination method is performed on the surface of the gold colloid particles. Was introduced.
3) Modification of PEG on the surface of the sensor chip and the surface of the sensor chip using biotin introduced as a support were modified with acetal-PEG-SH (molecular weight 5, OOO) and methoxy-PEG-SH (molecular weight 2,000) (room temperature, 30 minutes x 2). Next, the acetal was hydrolyzed with 0.1N HCl and converted to aldehyde (room temperature, 3 hours), and then biotin was introduced onto the sensor chip surface using biocytin hydrazide (pH 5.5, 4 ° C., overnight ). In addition, as a control, modification was performed using dextran gel (CM5: manufactured by Biacore) instead of PEG. 4) Interaction measurement using a surface plasmon resonance (SPR) sensor
SPR was performed under the following conditions.
Measuring device: BIACORE1000 (Nippon Biacore)
Running buffer: 10 mM phosphate buffer, pH 7.4, 0.15 M NaCl
Analyte: a) Streptavidin-PEG-Gold colloid
b) CHO-PEG-gold colloid
c) 1 μM streptavidin sensor chip surface: a) Biotin-PEG
b) Biotin-CM5 (Biacore)
Measurement conditions: Flow rate 5μL / 1min, 25 ℃
Results The results of the above tests are shown in Table 1-1 (example using biotin-PEG: unit Δθ [°]), -2 (example using biotin-CM5: unit Δθ [°]), and FIG. It describes.

As is clear from these results, it was found that the detection sensitivity was remarkably increased by combining streptavidin with gold colloid. Moreover, this effect was not observed at all in the polymer (dextran) gel-modified sensor chip that is currently widely used. From this, it is clear that the effect of increasing the surface plasmon resonance caused by the binding and dissociation of the metal surface material is exerted by bonding a hydrophilic polymer such as PEG upright on the sensor chip surface. became. Therefore, it can be seen that selecting PEG having a brush structure as the hydrophilic polymer to be bonded to the sensor chip surface is extremely useful.

2. Detection of antigen using antigen-antibody reaction (binding and dissociation of FITC-PEG-gold colloid to SPR chip)
Method 1) PEG modification of SPR gold substrate [PEG5000 (acetal), PEG2000 (Methoxy)] and introduction of antibody Place sensor chip with ozone-washed gold as support in petri dish, pH7.4, 50 mM phosphoric acid containing 1M NaCl Acetal-PEG-SH (1 mM / ml) having a molecular weight of 5000 dissolved in a buffer solution was added, and the mixture was shaken at room temperature for 30 minutes. After completion, the PEG solution was discarded, washed with 50 mM NaOH for 30 seconds and three times with phosphate buffer, and this series of operations was performed twice. Next, Methoxy-PEG-SH (0.2 mg / ml) having a molecular weight of 2000 dissolved in a phosphate buffer was added and shaken at room temperature for 30 minutes. After completion, the PEG solution was discarded and the same washing operation as described above was repeated three times.

  Next, the PEG-modified substrate obtained by the above method was placed in a petri dish, immersed in an O.1N HCl (pH 2) solution and shaken for 3 hours to hydrolyze acetal groups, and aldehyde groups were introduced onto the chip surface. After completion of the reaction, it was washed with distilled water and dried.

  Next, dimethylformamide (DMF) is added so that the substrate is immersed, N- (ε-Maleimidocaproic acid) hydrazide (EMCH) is added so that the final concentration is 2 mM, and the mixture is shaken at room temperature for 2 hours. Dimethylamine borane solution (solvent; DMF) was added every minute and shaking was continued for 2 hours. After completion of the reaction, it was washed with DMF, then washed with distilled water and dried to prepare a surface maleimide gold chip.

  This chip was mounted on an SPR device, 3 μM anti-FITC (ab ′) 2 was injected into the SPR, and an antibody was introduced onto the chip surface. Unreacted maleimide groups were blocked by injecting 100 nM mercaptoethanol.

2) Preparation of FITC-PEG-gold colloid
Acetal-PEG-SH was added to 0.86 mg (100 times the gold concentration) and NaBH4 was added 10 times the amount of PEG, followed by stirring for 1 hour. Adjust the pH of the aqueous solution to 6.5, add 10 mL of colloidal gold solution, stir for 1 hour at room temperature, 2 hours at 75 ° C, and perform ultracentrifugation (100,000 × g, 4 ° C, 20 minutes) 4 times to obtain PEGylation Gold colloidal particles were recovered. The colloidal particles were redispersed in 10 mL of pure water, adjusted to pH 2 and stirred for 2 hours to hydrolyze acetal and introduce aldehyde groups on the surface.
The pH of the aqueous solution was adjusted to 6.5 again, fluorescein hydrazide was added 10 times the amount of PEG and stirred for 6 hours, and NaBH4 was added 10 times the amount of PEG and stirred for 4 hours. Purification was performed 10 times under conditions of 100,000 × g, 4 ° C., and 20 minutes.
Results (SPR measurement and observation of dissociation behavior after FITC addition)
The colloidal gold prepared by the above method was injected into an SPR sensor [BIACORE1000 (manufactured by Nihon Biacore)] and the response behavior was examined. As a control, acetal-PEG-gold colloid was used. The gold concentrations were 11.2 pmol / ml and 11.6 pmol / ml, respectively. The state of a series of SPR responses from the flow of antibody to the last flow of FITC is shown in Table 2 (units other than pmol / ml Δθ [°]) and FIG.

When almost the same concentration of acetal-PEG-gold colloid was used as a control, as shown in Table 2 and FIG. 2, it was not confirmed as non-specific adsorption, and there was almost no response to gold particle binding. Observed. On the other hand, FITC-PEG-gold colloid obtained a large value of 0.4979 ° for the binding response of gold particles. Since no response was observed in the control, this value is considered to be due to antibody binding. Further, after adding 50 mM FITC, a value of −0.1399 ° was obtained as a response considered to be dissociation of gold particles from the antibody.

  From this result, it was confirmed that the antigen can be detected with high sensitivity by the present detection method using an antigen supported on gold particles.

3. Detection of sugar using lectin as a binding molecule Lactose-specific RCA lectin is immobilized on the surface of a sensor chip using gold that has been washed with ozone as a support, and then PEG5000 according to the method described in 2 (1) above. (Methoxy) was supported on the surface in a standing state, and a sensor chip was prepared in which lectin was supported on the surface and blocked with PEG5000.

  Further, in accordance with the above method 2 (2), 0.5 mM p-aminophenyl lactose was added to a gold colloidal dispersion composed of gold fine particles (particle size 20 nm) supported in a standing state with PEG6000 (acetal). After adjusting the pH to 7, it was mixed with a 0.7 mM dimethylaminoborane solution, and lactose was introduced onto the gold colloid surface by a reductive amination reaction. The obtained particles were purified by ultracentrifugation (360.6 × g) three times. The obtained gold fine particles supported lactose molecules at a rate of about 200 particles / 1 gold particle.

  A sensor chip having a gold surface on which a lectin prepared as described above is fixed and blocked with PEG is mounted on an SPR sensor [BIACORE1000 (manufactured by Nihon Biacore)], and the lactose-carrying gold having a specific concentration is attached to this sensor chip. The colloid is allowed to flow at 10 μl / min for 5 minutes, and then the phosphate buffer is allowed to flow for 2 minutes, and then a galactose aqueous solution having a concentration of 0 to 40 μg / ml is allowed to flow for 5 minutes. The exchange reaction was examined using the difference in SPR values before and after flowing galactose in the SPR sensor as an index.

  As a control, an example was used in which a hydrophilic polymer supported on gold particles was replaced with PEG6000 and dextran gel (CM5: manufactured by Biacore) was passed through a 40 μg / ml galactose aqueous solution.

  The results are shown in Table 3.

As a result, when galactose is brought into contact with the gold surface to which gold particles carrying galactose are bonded via PEG, the gold colloid is detached depending on the amount of galactose, and the initial gold colloid is supported. It was revealed that the amount of colloidal gold released was greater than that of the control using dextran gel, which was significantly higher. From this, it became clear that the detection sensitivity becomes remarkably sharp when using gold particles carrying PEG.

2 is a drawing showing the results of detecting streptavidin using biotin as a binding molecule according to the present invention. 1 is a diagram showing a result of using an antigen-antibody reaction in the present invention.

Claims (13)

The metal colloidal particles carrying the detection target substance are brought into contact with the “sensor chip having a binding molecule for the detection target substance and having the surface of the metal”, and the signal from the sensor chip is detected as an index. A detection method for detecting a target substance. The detection target is brought into contact with the “metal surface of the sensor chip on which the metal colloid particles carrying the detection target are bound via binding molecules”, and the detection target substance in the detection target and the metal colloid A detection method for detecting, as a signal, dissociation of the metal colloidal particles from the metal surface of the sensor chip by substitution with particles. The detection method according to claim 1 or 2, wherein a polymer containing a hydrophilic linear monomer unit is supported so as to stand on the metal colloidal particles and the metal surface of the sensor chip. A substance to be detected is supported near the tip of a polymer chain containing a hydrophilic linear monomer unit supported on metal colloidal particles, and a substance to be detected near the tip of the polymer chain supported on the metal surface of a sensor chip The detection method according to claim 3, wherein a binding molecule is supported. The detection method according to claim 3 or 4, wherein the polymer containing a hydrophilic linear monomer unit is a polyalkylene oxide having a polymerization number of 2 to 10,000 and having an alkylene oxide having 2 to 3 carbon atoms as a monomer unit. . The detection method according to claim 3, wherein the polyalkylene oxide is polyethylene oxide. The detection method according to claim 1, wherein masking treatment is performed on the metal surface and / or metal colloid particle surface of the sensor chip. The masking process on the metal surface and / or metal colloidal particle surface of the sensor chip is such that the bulk of the standing part is shorter than the bulk of the polymer carrying the detection target substance or the binding molecule for the detection target substance, and the standing part The detection method of Claim 7 performed by carrying | support of the said polymer by which the front-end | tip vicinity of inactivation processing was carried out. The detection method according to claim 1, wherein the metal colloid particles are gold colloid particles and the metal of the sensor chip is gold. The detection target substance and binding molecule pair is antigen or hapten and antibody pair, sugar and lectin pair, substrate and enzyme pair, hormone and its receptor pair, nucleic acid and its complementary chain pair, or bound The detection method according to any one of claims 1 to 9, which is a set of a protein and a binding protein. The detection method according to any one of claims 1 to 10, wherein detection of a signal from the sensor chip is performed by a device capable of detecting the state of the sensor chip surface using surface plasmon resonance. 5. A set of metal colloidal particles on which a polymer containing a hydrophilic linear monomer unit is supported and a sensor chip having the surface of the supported metal for performing the detection method according to claim 3 or 4. The set according to claim 12, wherein a detection target substance is supported on a polymer of metal colloidal particles, and a binding molecule for the detection target substance is supported on a polymer on a metal surface of a sensor chip.