JP2012042233A - Correction method of fluorescent signal to be measured by spfs (surface plasmon excitation enhanced fluorescence spectrometry), assay method using the same, structure to be used for the methods and surface plasmon resonance sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a correction method of measurement data which eliminates an influence of variation of electric field enhancement derived from variation of thickness of a metal thin film constituting an SPFS sensor substrate, and thus, enhances measurement accuracy and reliability of SPFS, etc., without introducing complicated configurations to the SPFS sensor substrate and an assay method using the same.SOLUTION: The present invention provides the correction method of a fluorescent signal for calculating electric field enhancement ratio R from signals (Sp) and (Sq) to be measured in an area (P) where a fluorescent molecule layer is formed without interposing the metal thin film and in an area (Q) where the fluorescent molecule layer is formed via the metal thin film for the fluorescent signal (Sm), and converting the fluorescent signal into the corrected signal (Sm1) using the electric field enhancement ratio R, and the assay method using the method.

Description

  The present invention relates to SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) or SPFS-LPFS (Localized surface Plasmon-field enhanced Fluorescence Spectroscopy) used in the fields of medicine, biotechnology, etc. : Localized surface plasmon excitation-enhanced fluorescence spectroscopy), and more specifically, a technique used in the measurement system and a structure of a surface plasmon resonance sensor.

  In SPFS, when the metal thin film formed on the dielectric member is irradiated with excitation light at an angle at which total reflection attenuation (ATR) occurs, the evanescent wave transmitted through the metal thin film is several tens of times due to resonance with the surface plasmon. This is a method of efficiently exciting a fluorescent dye for labeling an analyte (analyte) captured in the vicinity of a metal thin film and measuring the fluorescence signal by using the enhancement of several hundred times. Since such SPFS is extremely sensitive compared to a general fluorescent labeling method, it can be quantified even when only a very small amount of analyte is present in the sample. A basic aspect of SPFS is disclosed in Patent Documents 1 and 2, for example.

  LPFS is a method that can be used in combination with the above SPFS, and when metal colloid particles are positioned in the vicinity of a fluorescent dye by flowing a colloid solution or the like, plasmons are also generated in the metal colloid particles. This is a method in which the fluorescent signal is measured by exciting a fluorescent dye more efficiently by utilizing the fact that a local electric field enhancement effect is obtained.

  According to a measurement method such as SPFS and SPFS-LPFS using the SPFS (hereinafter collectively referred to as “SPFS etc.”), for example, it is detected by palpation by quantifying a very small amount of tumor marker in blood. The presence of preclinical non-invasive cancer (carcinoma in situ) that cannot be performed can be predicted with high accuracy, and is expected to be used in fields such as medicine and biotechnology.

  By the way, the enhanced electric field caused by plasmon resonance has a characteristic of rapidly decaying as the distance from the surface where the plasmon resonance occurs increases, so it is directly observed from a place away from the site where the plasmon resonance occurs. Is difficult. Therefore, several attempts have been made so far to visualize the electric field enhancement due to plasmon resonance.

  For example, in Non-Patent Document 1, a structure formed by forming a lattice on a gold film formed on the surface of a glass substrate, or a plurality of holes arranged in a single or periodic manner in a silver film formed on the surface of a glass substrate. Surface plasmon is detected by irradiating the formed structure with light and mapping the interference between the incident light near the lattice or hole and the light caused by surface plasmon polarization using a photon scanning tunneling microscope. Attempts have been made.

  Further, in Non-Patent Document 2, a laser beam is irradiated through a microscope objective lens to a structure formed by forming a silver film on the surface of a glass substrate having a plurality of silica protrusions arranged in a band shape. Attempts have been made to visualize surface plasmon polarization by observing fluorescence from fluorescent molecules applied to the surface with a microscope.

  In Non-Patent Document 3, light is irradiated from the back side of a structure formed by forming a dielectric thin film and a gold film on a transparent dielectric substrate partially formed with an aluminum opaque film. Measure the scattering of plasmon waves generated at the interface between the part where the opaque film is formed and the part where the aluminum opaque film is not formed as fluorescence from fluorescent molecules or using a photon scanning tunneling microscope. Thus, attempts have been made to visualize surface plasmon polarization.

Japanese Patent No. 3294605 JP 2006-218169 A

William L. Barnes et al., Nature, Vol. 424, p. 824-830 (2003) H. Ditlbacher, et al., Appl. Phys. Lett., Vol. 81 (10), p. 1762-1764 (2002) J. R. Krenn and J.-C. Weeber, Phil. Trans. R. Soc. Lond. A (2004) Vol. 362, p. 739-756

  When SPFS or the like is applied to a clinical test, it is necessary to ensure the reliability of the measurement value of the fluorescence signal. For that purpose, it is necessary to suppress the variation (CV) of the measurement value within 10%. In particular, in a system in which the absolute value of the signal increases in accordance with the analyte concentration, such as a sandwich system in which the fluorescently modified antibody is immobilized on the SPFS substrate on which the antibody is immobilized via the analyte derived from the analyte, the analyte concentration is generally When the value decreases, CV deteriorates, leading to a decrease in detection sensitivity and quantitative sensitivity. Therefore, in order to reduce the variation in the measured value of the fluorescence signal, it is necessary to reduce the variation in the electric field enhancement between the SPFS sensor substrates. Here, in considering the influence on the fluorescence signal due to the variation in the electric field enhancement, the variation in the thickness of the metal thin film formed on the SPFS sensor substrate becomes a problem. However, since it is not realistic to manufacture the SPFS sensor substrate so that the thickness of the metal thin film is completely the same in all SPFS sensor substrates with no difference in size, the influence on the fluorescence signal due to the variation in the electric field enhancement. In order to minimize the electric field, it is necessary to normalize the electric field enhancement generated in the SPFS sensor substrate and correct the measurement value based on the normalized electric field enhancement. On the other hand, the SPFS sensor substrate used for clinical examination is also required to be inexpensively manufactured. Therefore, it is possible to normalize the electric field enhancement without forming a complicated structure on the SPFS sensor substrate, and to correct the measurement value based on this, to minimize the variation of the measurement data. Necessary for application.

  However, in the above prior art, the surface plasmon polarization is visualized through a specially shaped metal structure formed on the substrate surface, and such a complicated metal structure is introduced into the SPFS sensor substrate used for clinical examination. This is not practical because the production process of the SPFS sensor substrate becomes complicated and the manufacturing cost may be significantly increased.

  Therefore, the present invention eliminates the influence of variations in electric field enhancement resulting from variations in the thickness of the metal thin film constituting the SPFS sensor substrate without introducing a complicated structure into the SPFS sensor substrate. It is an object of the present invention to provide a measurement data correction method and an assay method using the same, which can improve measurement accuracy and reliability.

  In the process of completing the present invention, the present inventor irradiates incident light under total reflection conditions from the back side of a substrate formed by forming a fluorescent molecular layer on a substrate having a metal thin film formed on a part of the surface of a dielectric member. Based on the relationship between the fluorescence intensity in the region where the metal thin film is not formed and the fluorescence intensity in the region where the metal thin film is not formed, the influence of the thickness of the metal thin film on the electric field enhancement can be estimated. The knowledge that it was possible was obtained. And when this knowledge was utilized, it discovered that the said subject could be solved and came to complete this invention.

That is, the present invention provides an assay method, a structure, and a surface plasmon resonance sensor shown in the following [1] to [10].
[1]
Fluorescent dye (Dm) measured using surface plasmon fluorescence spectroscopy for a surface plasmon resonance sensor in which a fluorescent dye (Dm) is fixed on a transparent dielectric member (Tm) via a metal thin film (Mm) About the fluorescence signal (Sm) derived from
(K-1) a transparent dielectric member (Ts);
A region (P) in which a fluorescent molecular layer (Fsp) is formed on the transparent dielectric member (Ts) without using a metal thin film;
A region (Q) in which a fluorescent molecular layer (Fsq) is formed on the transparent dielectric member (Ts) via a metal thin film (Msq);
The fluorescent molecular layer (Fsq) and the fluorescent molecular layer (Fsp) are both composed of the same fluorescent dye (Ds),
The transparent dielectric member (Tm) and the transparent dielectric member (Ts) are both made of the same transparent dielectric,
The metal thin film (Mm) and the metal thin film (Msq) are both structures (X) made of the same metal, and the metal thin film (Msq) has the same thickness as the metal thin film (Mm). About the body (Xt)
(I) For the region (P),
In the measurement of the fluorescence signal (Sm), the transparent dielectric member (Ts) is measured in the fluorescence signal (Sm) at an incident angle as a total reflection condition from the surface opposite to the surface where the fluorescent molecule layer (Fsp) exists. By irradiating incident light of the same wavelength as the incident light used to excite Dm),
Detecting the amount of fluorescence emitted in the region (P) as a total reflection fluorescence signal (Sp);
(Ii) Before the step (i), simultaneously with the step (i), or after the step (i), with respect to the region (Q),
Incident light having the same wavelength as that in the step (i) at an incident angle as a total reflection condition from the surface of the transparent dielectric member (Ts) opposite to the surface where the fluorescent molecular layer (Fsq) exists. By irradiating
(Iii) detecting the amount of fluorescence emitted in the region (Q) as an electric field enhanced fluorescence signal (Sq); (iii) the total reflection fluorescence signal (Sp) obtained through the steps (i) and (ii) and Using the electric field enhanced fluorescence signal (Sq), the following formula (1)
R = Sq / Sp (1)
Calculating the electric field enhancement ratio R based on:
Rt, which is the electric field enhancement ratio R obtained via
(K-2) For the reference structure (Xr), which is the structure (X) and is a structure other than the target structure (Xt), incidence of the same wavelength as that of the target structure (Xt) Using light, Rr which is the electric field enhancement ratio R obtained through the above steps (i) to (iii), the following formula (2)
Sm1 = Sm0 x (Rr / Rt) (2)
Based on the above, a fluorescence signal correction method comprising converting an uncorrected fluorescence signal (Sm0), which is a fluorescence signal (Sm) before correction, into a corrected fluorescence signal (Sm1).

[2]
The transformation of the fluorescence signal (Sm) measured for the surface plasmon resonance sensor integrating the target structure (Xt) as a target structure part includes the steps (i) to (i) for the target structure part. The correction method according to [1], wherein the electric field enhancement ratio R obtained through (iii) is used as the Rt.

[3]
(A) In a plasmon sensor structure having a region (Am) in which an immobilized ligand-containing layer containing a first ligand is formed on a transparent dielectric member (Tm) via a metal thin film (Mm), Preparing a surface plasmon resonance sensor by bringing a specimen into contact with a fluorescent modifying ligand comprising a second ligand and a fluorescent molecule (Dm) in the region (Am);
(B) After the step (a), the region (Am) is irradiated with incident light from the surface of the transparent dielectric member (Tm) opposite to the metal thin film (Mm). Irradiating incident light at an incident angle as a reflection condition, thereby detecting the amount of fluorescence emitted from the region (Am) as a fluorescence signal (Sm);
(C) correcting the fluorescence signal (Sm) detected in the step (b);
(D) evaluating the amount of analyte in the specimen based on the fluorescence signal (Sm) corrected in step (c);
An assay method, wherein the correction of the fluorescence signal (Sm) in the step (c) is performed using the correction method described in [1] above.

[4]
The target structure (Xt) used in (k-1) is
The transparent dielectric member (Ts) further includes a region (As) in which an immobilized ligand-containing layer containing a first ligand is formed via a metal thin film (Msa), and the metal thin film ( Msa) and the metal thin film (Msq) are made of the same metal and have the same thickness.
The assay method according to the above [3], wherein the region (As) is used as the region (Am) to serve as the plasmon sensor structure.

[5]
The correction in the step (c) is
By performing the steps (i) to (iii) for the target structure (Xt) immediately before the step (b), simultaneously with the step (b), and immediately after the step (b). The assay method according to the above [4], which is performed using the required Rt.

[6]
The assay method according to [3] or [4] above, wherein the correction in the step (c) is performed using the Rt calculated in advance.

[7]
The assay method according to [5] or [6] above, wherein the correction in the step (c) is performed using the previously calculated Rr.

[8]
The assay method according to any one of [3] to [7], wherein the specimen is at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.

[9]
A transparent dielectric member (Ts);
A region (P) in which a fluorescent molecular layer (Fsp) is formed on the transparent dielectric member (Ts) without using a metal thin film;
A region (Q) in which a fluorescent molecular layer (Fsq) is formed on the transparent dielectric member (Ts) via a metal thin film (Msq);
A region (As) in which an immobilized ligand-containing layer containing a first ligand is formed on the transparent dielectric member (Ts) via a metal thin film (Msa);
The fluorescent molecular layer (Fsp) and the fluorescent molecular layer (Fsq) are both composed of the same fluorescent molecule (Ds), and
A structure in which the metal thin film (Msa) and the metal thin film (Msq) are both made of the same metal and have the same thickness.

[10]
Including the structure according to [9], an analyte, a fluorescent modifying ligand composed of a second ligand and a fluorescent molecule (Dm),
A surface plasmon resonance sensor in which the fluorescent modifying ligand is bound to the first ligand in the region (As) via the analyte.

  According to the present invention, it becomes possible to eliminate the influence of the variation in electric field enhancement resulting from the variation in the thickness of the metal thin film constituting the SPFS substrate, so that the measurement accuracy and reliability of SPFS and the like are further enhanced than before. In addition, it is possible to suppress an increase in the manufacturing cost of the SPFS substrate caused by responding to the application to the clinical test, which greatly contributes to the practical use of the clinical test system using SPFS or the like.

It is a schematic diagram explaining the assay method of this invention. FIG. 2A is a schematic diagram showing the structure used in the present invention together with the plasmon sensor structure to be corrected by this structure, and FIGS. 2B and 2C are used in the present invention. It is a schematic diagram showing the suitable embodiment of the structure obtained. It is a schematic diagram showing the surface plasmon resonance sensor which applied the suitable structure used by this invention.

Hereinafter, the present invention will be specifically described with reference to FIGS. 1 and 2.
[Correction method]
The fluorescent signal correction method according to the present invention includes:
Fluorescent dye (Dm) measured using surface plasmon fluorescence spectroscopy for a surface plasmon resonance sensor in which a fluorescent dye (Dm) is fixed on a transparent dielectric member (Tm) via a metal thin film (Mm) About the fluorescence signal (Sm) derived from
(K-1) For the target structure (Xt), Rt which is an electric field enhancement ratio R obtained through the following steps (i) to (iii);
(K-2) About the reference structure (Xr), the electric field enhancement ratio obtained through the following steps (i) to (iii) using incident light having the same wavelength as that of the target structure (Xt) Using Rr as R, the following formula (2)
Sm1 = Sm0 x (Rr / Rt) (2)
Based on the above, the uncorrected fluorescence signal (Sm0), which is the fluorescence signal (Sm) before correction, is converted into a corrected fluorescence signal (Sm1).

Here, the target structure (Xt) is
A transparent dielectric member (Ts);
A region (P) in which a fluorescent molecular layer (Fsp) is formed on the transparent dielectric member (Ts) without using a metal thin film;
A region (Q) in which a fluorescent molecular layer (Fsq) is formed on the transparent dielectric member (Ts) via a metal thin film (Msq);
The fluorescent molecular layer (Fsq) and the fluorescent molecular layer (Fsp) are both composed of the same fluorescent dye (Ds),
The transparent dielectric member (Tm) and the transparent dielectric member (Ts) are both made of the same transparent dielectric,
The metal thin film (Mm) and the metal thin film (Msq) are both structures (X) made of the same metal, and the metal thin film (Msq) has the same thickness as the metal thin film (Mm). It is.

The reference structure (Xr) is the structure (X) and is a structure other than the target structure (Xt).
Specifically, the structure (X) serving as the target structure (Xt) and the reference structure (Xr) has a structure like the structure 10 shown in FIG.

In the present invention, the electric field enhancement ratio R for the target structure (Xt) and the reference structure (Xr) is as shown in FIG. 1 for each of the target structure (Xt) and the reference structure (Xr). In addition,
(I) For region (P),
In the measurement of the fluorescence signal (Sm) from the surface of the transparent dielectric member (Ts) 11 opposite to the surface on which the fluorescent molecule layer (Fsp) 13a is present, at an incident angle that is a total reflection condition, By irradiating incident light 61a of the same wavelength as the incident light used to excite Dm),
Detecting the amount of fluorescence emitted in the region (P) as a total reflection fluorescence signal (Sp);
(Ii) Before step (i), simultaneously with step (i) or after step (i), for region (Q),
From the surface of the transparent dielectric member (Ts) 11 opposite to the surface on which the fluorescent molecular layer (Fsq) 13b exists, incident light 61b having the same wavelength as that in the step (i) at an incident angle that is a total reflection condition. By irradiating
A step of detecting the amount of fluorescence emitted in the region (Q) as an electric field-enhanced fluorescent signal (Sq); (iii) a total reflection fluorescent signal (Sp) and an electric field-enhanced fluorescent signal obtained through steps (i) and (ii) Using (Sq), the following formula (1)
R = Sq / Sp (1)
Calculating the electric field enhancement ratio R based on:
It is obtained through

  In the correction method of the present invention, the target structure (Xt) is used for estimating the electric field enhancement in the surface plasmon resonance sensor that is to correct the fluorescence signal. In the present invention, the strength of the electric field enhancement in the surface plasmon resonance sensor is evaluated based on the strength of the electric field enhancement generated in the region (Q) of the subject structure (Xt). The constituent metal thin film (Msq) is made of the same metal as the metal thin film (Mm) constituting the surface plasmon resonance sensor and needs to have the same thickness. From this point, in the present invention, it is preferable to use a surface plasmon resonance sensor that is intended to correct a fluorescence signal, in which the target structure (Xt) is integrated as a target structure portion. In this case, the electric field enhancement ratio R obtained through the steps (i) to (iii) for the target structure portion can be used as Rt, and the fluorescence measured for the surface plasmon resonance sensor using this Rt. Correction for the signal (Sm) can be performed. In such a surface plasmon resonance sensor, it is more preferable that the metal thin film (Msq) constituting the target structure portion is integrated with the metal thin film (Mm) and formed as a single metal thin film.

  On the other hand, the reference structure (Xr) is used as a standard for normalizing the electric field enhancement in the surface plasmon resonance sensor represented by the intensity of the electric field enhancement generated in the region (Q) of the target structure (Xt). It is. By using this reference structure (Xr), it is possible to determine the relative relationship of the electric field enhancement strength inherent to each target structure (Xt) between a plurality of different target structures (Xt). Based on this, it is possible to correct variations in electric field enhancement occurring between the surface plasmon resonance sensors.

The calculation of the electric field enhancement ratio R for the target structure (Xt) and the reference structure (Xr) will be described in detail later.
In the present invention, fluorescence detection in a region having a metal thin film such as regions (Am), (As), and (Q) described later is performed under surface plasmon resonance conditions, and a metal thin film as described in region (P) described later. Fluorescence detection in a region that does not have is performed under the total reflection attenuation condition. However, in the present invention, even in fluorescence detection in a region having a metal thin film, irradiation of incident light is performed at an incident angle at which total reflection attenuation (ATR) occurs. Also uses the term “incidence angle as a total reflection condition”.

[Surface plasmon resonance sensor to be applied]
The surface plasmon resonance sensor to which the correction method of the present invention is applied is a surface plasmon resonance sensor in which a fluorescent dye (Dm) is fixed on a transparent dielectric member (Tm) through a metal thin film (Mm). Any surface plasmon resonance sensor that is generally widely used may be used, regardless of its configuration. There are no particular limitations on the manner in which the fluorescent dye (Dm) is immobilized in the surface plasmon resonance sensor to which the correction method of the present invention is applied. For example, the fluorescent dye (Dm) is immobilized using an antigen-antibody reaction. Alternatively, they may be immobilized using a double strand formation reaction or intercalation in nucleic acids having mutually complementary sequences.

  Here, the surface plasmon resonance sensor 50 shown in FIG. 1 will be further described as an example. Usually, the surface plasmon resonance sensor is formed on a transparent dielectric member (Tm) 21 via a metal thin film (Mm) 22. In the plasmon sensor structure 20 having the region (Am) in which the immobilized ligand-containing layer 23 containing one ligand 231 is formed, the first ligand 231 in the region (Am) has the second ligand 411 and fluorescence. A fluorescence-modifying ligand 41 composed of a molecule (Dm) 412 has a form bound via an analyte 42.

  Here, this region (Am) is irradiated with incident light 61c from the surface of the transparent dielectric member (Tm) 21 on the side opposite to the metal thin film (Mm) 22 at an incident angle that is a total reflection condition. As a result, surface plasmons are generated in the metal thin film (Mm) 22, and the fluorescent molecules (Dm) are excited by the electric field enhancement caused by the surface plasmons to emit fluorescence. In the assay method using SPFS or the like, the fluorescence at this time is detected as a fluorescence signal (Sm).

  However, even if the amount of the fluorescent molecule (Dm) 412 fixed to the surface plasmon resonance sensor 50 is the same, the strength of the electric field enhancement increases in proportion to the thickness of the metal thin film (Mm) 22, and as a result, The apparent amount of fluorescence detected as a fluorescence signal (Sm) increases. Here, the point that becomes a problem in applying SPFS or the like to clinical tests is that when a certain value is measured as the fluorescence signal (Sm), how far is the value based on the amount of the analyte contained in the specimen, It is often difficult to evaluate where the apparent increase / decrease value is due to the variation in the thickness of the metal thin film (Mm) 22. Therefore, the variation (CV) in the measurement results allowed for the assay method used for clinical examination is usually within about 10%. In particular, since the problem becomes apparent when used for an inspection in which the difference between the normal value and the abnormal value is small, it is necessary to suppress the CV to 1 to 2% in some cases. Considering this, the plasmon sensor structure 20 used for the surface plasmon resonance sensor 50 can be removed although the influence on the fluorescence signal (Sm) due to the variation in the thickness of the metal thin film (Mm) 22 needs to be removed to the maximum extent. It is difficult to manufacture the metal thin film (Mm) 22 so that the thickness of the metal thin film (Mm) 22 is completely the same throughout the whole body. Therefore, it is necessary to correct the influence of the thickness of the metal thin film (Mm) 22 on the electric field enhancement, and for that purpose, it is necessary to estimate the influence of the thickness of the metal thin film (Mm) 22 on the electric field enhancement. .

  In the correction method of the present invention, the influence of the thickness of the metal thin film (Mm) 22 on the electric field enhancement is estimated using the “structure” described below, and based on this, the fluorescence signal measured for the surface plasmon resonance sensor 50 is measured. (Sm) is corrected, and the influence of the variation in the thickness of the metal thin film (Mm) 22 is removed.

〔Structure〕
In the present invention, the structure used as the structure (X) is as shown in FIG.
A transparent dielectric member (Ts) 11;
A region (P) in which a fluorescent molecular layer (Fsp) 13a is formed on the transparent dielectric member (Ts) 11 without a metal thin film,
A region (Q) in which a fluorescent molecular layer (Fsq) 13b is formed on the transparent dielectric member (Ts) 11 via a metal thin film (Msq) 12;
Both the fluorescent molecular layer (Fsq) 13a and the fluorescent molecular layer (Fsp) 13b are the structure 10 composed of the same fluorescent dye (Ds).

In the present invention, the structure 10 is used for correcting the fluorescence signal of the surface plasmon resonance sensor to which the correction method of the present invention is applied.
In the following description, the structure 10 may be referred to as an “electric field enhancement evaluation structure” to distinguish it from a structure used in a general sense.

  In the preferred embodiment of the present invention, as shown in FIG. 2 (b), the structure 10 includes a first ligand 331 on a transparent dielectric member (Ts) 11 via a metal thin film (Msa) 32. It further includes a region (As) in which an immobilized ligand-containing layer 33 containing is formed. At this time, the metal thin film (Msa) 32 and the metal thin film (Msq) 12 are both made of the same metal and have the same thickness.

≪Region (P) ≫
The electric field enhancement evaluation structure 10 used in the present invention has a region (P) in which a fluorescent molecular layer (Fsp) 13a is formed on a transparent dielectric member (Ts) 11 without using a metal thin film. . Here, the fluorescent molecular layer (Fsp) 13a is a layer composed of fluorescent molecules (Ds). This region (P) is drawn on the left side in the electric field enhancement evaluation structure 10 shown in FIG.

  For a structure composed of a transparent dielectric member having a layer containing a fluorescent dye on the surface, the layer containing the fluorescent dye exists from the opposite side of the surface on which the layer containing the fluorescent dye exists through the transparent dielectric member. When incident light is irradiated toward the surface under total reflection conditions, fluorescence (hereinafter referred to as “total reflection fluorescence”) may be emitted from the fluorescent dye even when no metal is present in the structure. Therefore, in the present invention, the region (P) is used as a total reflection fluorescence reference region for measuring the total reflection fluorescence in a state where there is no influence of the electric field enhancement by the metal thin film.

≪Region (Q) ≫
The electric field enhancement evaluation structure 10 used in the present invention has a region (Q) in which a fluorescent molecular layer (Fsq) 13b is formed on a transparent dielectric member (Ts) 11 via a metal thin film (Msq) 12. Also have. Here, the fluorescent molecular layer (Fsq) 13b is a layer composed of fluorescent molecules (Ds). Here, the fluorescent molecular layer (Fsq) 13a and the fluorescent molecular layer (Fsp) 13b are both composed of the same fluorescent molecule (Ds).

  A surface plasmon resonance sensor used for measuring an analyte in a specimen based on SPFS or the like generally has a first ligand formed on a transparent dielectric member (Tm) 21 via a metal thin film (Mm) 22. In the plasmon sensor structure 20 having the region (Am) in which the immobilized ligand-containing layer 23 containing 231 is formed, the specimen, the second ligand 411, the fluorescent molecule (Dm) 412 and the region (Am) It is prepared by contacting with a fluorescent modifying ligand 41 comprising Here, the electric field enhancement in the metal thin film (Mm) 22 tends to increase in proportion to the film thickness of the metal thin film (Mm) 22. Also, the metal changes the optical properties related to the electric field enhancement.

  In the present invention, this region (Q) is used as an electric field-enhanced fluorescence reference region for evaluating the electric field enhancement in the metal thin film constituting the surface plasmon resonance sensor to be corrected. Therefore, in correcting the fluorescence signal detected for such a surface plasmon resonance sensor using the structure 10 used in the present invention, the metal thin film (Msq) 12 constituting the structure 10 is corrected. It is necessary to be made of the same metal as the metal thin film constituting the surface plasmon resonance sensor to be performed and to have the same thickness. As long as the structure is integrated with the surface plasmon resonance sensor, the surface plasmon resonance sensor may be a separate structure.

<< Area >>
As shown in FIG. 2 (b), the electric field enhancement evaluation structure 10 used in the present invention, in a preferred embodiment, is formed on a transparent dielectric member (Ts) 11 with a metal thin film (Msa) 32 interposed therebetween. It further includes a region (As) in which an immobilized ligand-containing layer 33 containing one ligand 331 is formed. This region (As) is a region used as a surface plasmon resonance sensor, and by having this region (As), the electric field enhancement evaluation structure 10 also has a function as a plasmon sensor structure. In other words, in this embodiment, the electric field enhancement evaluation structure 10 includes the immobilized ligand-containing layer 23 containing the first ligand 231 on the transparent dielectric member (Tm) 21 via the metal thin film (Mm) 22. The plasmon sensor structure 20 having a region (Am) formed with a structure integrated with the plasmon sensor structure 20, and this region (As) has the same function as the region (Am) in the plasmon sensor structure 20. Used as a measurement area.

  In the immobilized ligand-containing layer 33, the first ligand 331 for capturing the analyte 42 may be formed directly on the metal thin film (Msa) 32, or may be formed on the metal thin film (Msa) 32 by a conventional method. A SAM, a spacer layer, etc. (all not shown) may be formed and then formed thereon. In addition, a blocking process is usually performed after the layer made of the first ligand 331 is provided so that the fluorescence-modifying ligand 41 or the like does not bind nonspecifically to the sensor surface or the like.

≪Structure structure≫
The electric field enhancement evaluation structure 10 used in the present invention has a metal thin film (Msq) in a region to be a region (Q) and a region to be a region (As) when applicable on the surface of the transparent dielectric member (Ts) 11. ) And a metal thin film (Msa), respectively, and then formed by forming fluorescent molecular layers (Fsp) and (Fsq) in the region to be the region (P) and the region to be the region (Q), respectively. . Here, in the case of the electric field enhancement evaluation structure 10 further having a region (As), an immobilized ligand-containing layer is used instead of the fluorescent molecular layer in the region where the metal thin film (Msa) to be the region (As) is formed. It is created by forming 33.

Transparent dielectric member (Ts)
In the present invention, the transparent dielectric member (Ts) 11 constituting the electric field enhancement evaluation structure 10 constitutes the skeleton of the structure of the electric field enhancement evaluation structure 10, and in the electric field enhancement evaluation structure 10, a metal thin film (Msq ) It also functions as a medium for guiding incident light to the surface on which 12 is formed.

  In the present invention, the transparent dielectric member used as the transparent dielectric member (Ts) 11 is not particularly limited as long as the object of the present invention is achieved. For example, the transparent dielectric member is made of glass, polycarbonate (PC), cycloolefin A plastic material such as a polymer (COP), preferably a material having a refractive index [nd] in the range of 1.40 to 2.20 at d-line (588 nm) can be used.

  However, in the present invention, the material of the transparent dielectric member used as the transparent dielectric member (Ts) 11 is the transparent dielectric member (Tm) constituting the surface plasmon resonance sensor to which the correction method of the present invention is applied. ) Of the same material.

In the present invention, the transparent dielectric member (Ts) 11 may be a transparent flat substrate, and has a form of an integrated structure block that further includes a prism portion and a sample holding portion in addition to the metal thin film forming plane. It may be. Here, when a transparent flat substrate is used as the transparent dielectric member (Ts) 11, if the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm, the size (vertical x horizontal) is There is no particular limitation.
In addition, before forming the metal thin film (Msq) 12 described later, it is preferable that the surface of the transparent dielectric member (Ts) 11 is cleaned with an acid or plasma.

Metal thin film (Msq) / (Msa)
A general surface plasmon resonance sensor for the metal thin film (Msq) 12 constituting the electric field enhancement evaluation structure 10 used in the present invention and the metal thin film (Msa) 32 further included when the region (As) exists. The same metal as that generally used as the metal thin film constituting the film can be used. Among these, it is preferable to consist of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, and it is more preferable to consist of gold. About these metals, the form of the alloy may be sufficient and the thing which laminated | stacked the metal may be sufficient. Such metal species are preferable because they are stable against oxidation and increase in electric field due to surface plasmons increases.

  Here, when a transparent dielectric member made of glass is used as the transparent dielectric member (Ts) 11, in order to bond the glass and the metal thin film more firmly, a thin film of chromium, nickel chrome alloy or titanium is used in advance. It is preferable to form.

  Here, the metal constituting the metal thin film (Msq) 12 constituting the electric field enhancement evaluation structure 10 used in the present invention is the metal thin film constituting the surface plasmon resonance sensor 50 to which the correction method of the present invention is applied. It is necessary to be the same as the metal constituting (Mm) 22, and when the region (As) is present, it is necessary to be the same as the metal constituting the metal thin film (Msa) 32.

  As a method for forming a metal thin film on a transparent flat substrate, a method generally used for forming the metal thin film (Mm) 22 constituting the surface plasmon resonance sensor 50 to which the correction method of the present invention is applied. Examples thereof include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since it is easy to adjust the thin film formation conditions, it is preferable to form a chromium thin film and a metal thin film by sputtering or vapor deposition.

  The thickness of the metal thin film (Msq) 12 and the metal thin film (Msa) 32 can be the same thickness as that of a metal thin film constituting a generally used surface plasmon resonance sensor, so that surface plasmons are easily generated. The thickness of the metal thin film made of gold, silver, aluminum, copper, platinum, or an alloy thereof is preferably 5 to 500 nm, and the thickness of the chromium thin film is preferably 1 to 20 nm. From the viewpoint of the electric field enhancement effect, gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm are more preferable, The thickness of the chromium thin film is more preferably 1 to 3 nm.

  Here, the thickness of the metal thin film (Msq) 12 constituting the electric field enhancement evaluation structure 10 used in the present invention is the same as the metal thin film (surface plasmon resonance sensor 50 to which the correction method of the present invention is applied). Mm) 22 needs to be the same as the thickness of the metal thin film (Msa) 32 when the region (As) is present.

  Here, in the electric field enhancement evaluation structure 10 having the region (As), the metal thin film (Msq) and the metal thin film (Msa) may be formed separately, but the metal thin film (Msq) 12 and the metal thin film ( As shown in FIG. 2 (c), the metal thin film (Msq) 12 and the metal thin film (Msa) 32 are integrated to form one metal thin film so that the Msa) 32 has the same thickness. It is preferable to form at once in such a manner.

Fluorescent molecule In the present invention, the term “fluorescent molecule” means a molecule that emits fluorescence by irradiating predetermined excitation light in the present invention, or excited by using the electric field effect. Various light emission such as phosphorescence is also included.

  In the present invention, the fluorescent molecule used as the fluorescent molecule (Ds) constituting the fluorescent molecular layer (Fsp) 13a and the fluorescent molecular layer (Fsq) 13b constituting the electric field enhancement evaluation structure 10, and a fluorescent modifying ligand 41 described later Any of the fluorescent molecules used as the fluorescent molecule (Dm) 412 that constitutes is not particularly limited, and may be any known fluorescent dye. In general, fluorescent dyes with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromator and also increase the efficiency of detection are preferred. Here, the fluorescent molecule constituting the fluorescent molecular layer (Fsp) 13a and the fluorescent molecule (Ds) constituting the fluorescent molecular layer (Fsq) 13b need to be the same. However, the fluorescent molecule (Ds) and the fluorescent molecule (Dm) 412 may be the same, or may have the same excitation wavelength and different emission wavelengths.

  Examples of fluorescent dyes that can be used in the present invention include fluorescein family fluorescent dyes (integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), hexachlorofluorescein Family fluorescent dyes (Applied Biosystems Japan), Coumarin family fluorescent dyes (Invitrogen), Rhodamine family fluorescent dyes (GE Healthcare Biosciences), cyanine family Fluorescent dyes, Indocarbocyanine family fluorescent dyes, Oxazine family fluorescent dyes, Thiazine family fluorescent dyes, Squaline family fluorescent dyes, Tanide family fluorescent dye, BODIPY (registered trademark) family fluorescent dye (manufactured by Invitrogen), naphthalenesulfonic acid family fluorescent dye, pyrene family fluorescent dye, triphenylmethane family fluorescent dye, Alexa Fluor (registered trademark) dye series (manufactured by Invitrogen Corp.) and the like, and further, U.S. Patent Nos. 6,406,297, 6,221,604, 5,994,063, The fluorescent dyes described in US Pat. Nos. 5,808,044, 5,880,287, 5,556,959, and 5,135,717 can also be used in the present invention.

  Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.

The fluorescent dye used as the fluorescent molecule is not limited to the organic fluorescent dye. For example, rare earth complex fluorescent dyes such as Eu and Tb can be used as fluorescent molecules in the present invention. In general, rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there. An example of a commercially available rare earth complex-based fluorescent dye is ATBTA-Eu ³⁺ .

  Furthermore, it is represented by blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (DsRed), or allophycocyanin (APC; LyoFlogen (registered trademark)). Fluorescent proteins and fluorescent fine particles such as latex and silica are also included in the fluorescent molecules.

In the SPFS-LPFS measurement system, it is desirable to use, as a labeled ligand, a fluorescent dye having a maximum fluorescence wavelength in a wavelength region where light absorption by the metal colloid involved in LPFS is small. For example, when using gold colloid, in order to minimize the influence of light absorption by the gold colloid, in the near infrared region such as a fluorescent dye having a maximum fluorescence wavelength of 600 nm or more, such as Cy5, Alexa Fluor (registered trademark) 647, etc. It is desirable to use a fluorescent dye having the maximum fluorescence wavelength. The use of a fluorescent dye having the maximum fluorescence wavelength in the near-infrared region can minimize the influence of light absorption by iron derived from blood cell components in the blood. Is also useful. On the other hand, when silver colloid is used, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more.
These fluorescent dyes may be used alone or in combination of two or more.

Fluorescent molecular layer The fluorescent molecular layer (Fsp) 13a and the fluorescent molecular layer (Fsq) 13b constituting the electric field enhancement evaluation structure 10 used in the present invention are composed of the above "fluorescent molecules", and both are the same. It consists of fluorescent molecules (Ds).

  The fluorescent molecular layer (Fsp) 13a and the fluorescent molecular layer (Fsq) 13b are formed by applying or vapor-depositing the “fluorescent molecules” on the surface of the transparent dielectric member (Ts) 11 or the metal thin film (Msq) 12. can do. In addition, the transparent dielectric member (Ts) 11 or the metal thin film (Msq) 12 is formed by laminating a member formed of a light-transmitting material containing a fluorescent dye or a thin layer containing the fluorescent dye on the surface of the metal thin film (Msq) 12. May be.

  What is necessary is just to adjust suitably the thickness etc. of these fluorescent molecular layers in the range which can perform the above measuring methods. However, both the fluorescent molecular layer (Fsp) 13a and the fluorescent molecular layer (Fsq) 13b are usually formed to have a thickness of about 10 to 2000 nm, preferably 20 to 200 nm.

  When preparing the fluorescent molecular layer, an antioxidant may be used in combination in order to improve the temporal stabilizer of the fluorescent dye. Examples of the antioxidant include pentaerythrityl tetrakis [3- (3,5-di-t-butyl-4-hydroxyphenyl)] propionate, 2,6-di-t-butyl-4-methylphenol, 2 , 2′-dioxy-3,3′-di-t-butyl-5,5′-dimethyldiphenylmethane, tetrakis [methylene-3- (3,5-di-t-butyl-4-hydroxyphenyl) propionate] methane Etc.

  Here, in the electric field enhancement evaluation structure 10, the fluorescent molecular layer (Fsp) 13a and the fluorescent molecular layer (Fsq) 13b may be formed separately, or the fluorescent molecular layer (Fsp) 13a and the fluorescent molecular layer (Fsq). It may be formed at a time in such a manner that a single fluorescent molecule layer is formed as a whole by integrating with 13b.

Immobilized Ligand Containing Layer When the region (As) is formed in the electric field enhancement evaluation structure 10 used in the present invention, the immobilized ligand containing layer is formed on the metal thin film (Msa) 32 as shown in FIG. 33 is formed. The immobilized ligand-containing layer 33 contains a ligand 331, and this ligand 331 becomes a base on which the analyte 42 contained in the specimen is bound. This region (As) has a function as a surface plasmon resonance sensor by binding a fluorescent modifying ligand 41 described later to the analyte 42.

  In the present invention, in the immobilized ligand-containing layer 33, the ligand 331 may be directly bonded to the metal thin film (Msa) 32. Usually, at least one of the spacer layer and the SAM described below is used. Are connected through.

Spacer layer In the present invention, when a region (As) is present in the electric field enhancement evaluation structure 10, the electric field enhancement evaluation structure 10 is subjected to metal quenching of the fluorescent molecules 412 by a metal thin film (Msa) as necessary. In order to prevent this, a spacer layer made of a dielectric may be formed between the metal thin film and the immobilized ligand-containing layer 33 (or SAM described later).

As the dielectric, various optically transparent inorganic substances and natural or synthetic polymers can be used. Among these, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) is preferably used because of excellent chemical stability, production stability, and optical transparency.

  The thickness of the spacer layer is usually 10 nm to 1 mm, and preferably 30 nm or less, more preferably 10 to 20 nm from the viewpoint of resonance angle stability. On the other hand, the thickness is preferably 200 nm to 1 mm from the viewpoint of the electric field enhancement effect, and more preferably 400 nm to 1,600 nm from the stability of the electric field enhancement effect. In the SPFS-LPFS measurement system, the thickness of the spacer layer is preferably 10 to 100 nm in order to effectively enhance the electric field generated between the sensor surface (metal thin film) and the metal colloid particles.

  Examples of the method for forming a spacer layer made of a dielectric include a sputtering method, an electron beam evaporation method, a thermal evaporation method, a formation method by a chemical reaction using a material such as polysilazane, or an application by a spin coater.

SAM
In the present invention, when a region (As) is present in the electric field enhancement evaluation structure 10, the electric field enhancement evaluation structure 10 includes a metal thin film (or the spacer layer) and the immobilized ligand-containing layer 33 as necessary. A SAM (Self-Assembled Monolayer) may be formed between the layers.

  The SAM is composed of a functional group (silanol group, thiol group, etc.) capable of binding to a metal thin film at one end of the molecule, and a reactivity capable of binding to the molecule constituting the reaction layer at the other end. A compound having a functional group (amino group, carboxyl group, glycidyl group, etc.) is used. Such a compound can be easily obtained as a silane coupling agent or a SAM-forming reagent. For example, a carboxyalkanethiol having about 4 to 20 carbon atoms (such as 10-carboxy-1-decanethiol) has little optical influence, that is, a SAM having a high transparency, a low refractive index, and a thin film thickness. It is preferable because it can be formed. A SAM can be formed by bringing a solution of an SAM constituent molecule (such as an ethanol solution) into contact with a metal thin film or the like and bonding one functional group of the molecule to the metal thin film or the like.

Molecules that can specifically bind to the ligand analyte are referred to as “ligands”. In the present invention, the “ligand” is a ligand 331 constituting the electric field enhancement evaluation structure 10 having the ligand 231 and the region (As) constituting the surface plasmon resonance sensor 50 to which the correction method of the present invention is applied. It is also used as a ligand 411 that constitutes a fluorescence modifying ligand 41 described later. In particular, ligands (ie, ligand 231 and ligand 331) immobilized on the surface of the electric field enhancement evaluation structure 10 having the surface or region (As) of the surface plasmon resonance sensor 50 for capturing the analyte on the sensor surface. A “first ligand” (or “first antibody” if the ligand is an antibody), a ligand bound to a fluorescent dye (ie, ligand 411) present in the solution to label the analyte. 2 "(or" second antibody "if the ligand is an antibody). Note that the ligand portions of the first ligand and the second ligand may be the same or different. However, when the first ligand is a polyclonal antibody, the second ligand may be a monoclonal antibody or a polyclonal antibody, but when the first ligand is a monoclonal antibody, the second ligand It is preferably a monoclonal antibody that recognizes an epitope that the first ligand does not recognize, or a polyclonal antibody.

  An appropriate ligand may be selected depending on the analyte to be captured, and antibodies, receptors, and other specific molecules (for example, biotinylated analytes) that can specifically bind to a predetermined site of the analyte. Avidin) for trapping can be used as a ligand.

  The ligand 231 can be immobilized on the surface of the surface plasmon resonance sensor 50, and the ligand 331 can be immobilized on the surface of the electric field enhancement evaluation structure 10 having the region (As).

≪Use as surface plasmon resonance sensor≫
In the present invention, the electric field enhancement evaluation structure 10 further including the region (As) includes, in the region (As), a specimen, a fluorescent modified ligand 41 composed of a second ligand 411 and a fluorescent molecule (Dm) 412. By contacting, it can be used as a surface plasmon resonance sensor. Referring to FIG. 3, the electric field enhancement evaluation structure 10 further including a region (As) is used as the surface plasmon resonance sensor 51.

That is, the present invention includes the electric field enhancement evaluation structure 10 further including the region (As), the analyte 42, the fluorescent modification ligand 41 composed of the second ligand 411 and the fluorescent molecule (Dm) 412.
A surface plasmon resonance sensor in which the fluorescent modifying ligand 41 is bound to the first ligand 331 in the region (As) via the analyte 42 is provided.

  Since such a surface plasmon resonance sensor also has the region (P) and the region (Q), it also has a function as the target structure (Xt) and is formed in the region (As). In correcting the fluorescence signal (Sm) measured for each surface plasmon resonance sensor portion, the electric field enhancement ratio R obtained for the region (P) and the region (Q) functioning as the target structure portion can be used as Rt. .

Analyte In the present invention, the “analyte” 42 is the first ligand 231 immobilized on the surface of the transparent dielectric member (Tm) 21 constituting the surface plasmon resonance sensor 50 to which the correction method of the present invention is applied. Or the first ligand 331 immobilized on the surface of the transparent dielectric member (Ts) 11 constituting the electric field enhancement evaluation structure 10 further including the region (As) is specifically recognized (or recognized). Means a molecule or molecular fragment capable of binding; Examples of such “molecule” or “molecular fragment” include, for example, nucleic acid (DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), which may be single-stranded or double-stranded, or the like, or Nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules thereof In particular, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signaling substance, a hormone, or the like, and is not particularly limited.

Specimen A substance used for SPFS or the like containing or possibly containing the “analyte” 42 is referred to as “specimen”. For example, humans, non-human mammals (model animals, pets, etc.), blood (serum / plasma) collected from other animals, urine, nostril, saliva, feces, body cavity fluids (spinal fluid, ascites, pleural effusion, etc.) Etc. are mentioned as specimens. At the time of analysis, the sample may be used by mixing with various solvents such as pure water, physiological saline, buffer solution, reagent solution, etc., if necessary. Such a sample mixture or the sample itself, or a solution containing an analyte prepared for a predetermined purpose, is sent to a predetermined region of the surface plasmon resonance sensor in order to measure a signal by SPFS or the like. Fluids (including solutions, suspensions, sols, and other fluid substances) are collectively referred to as “samples”.

  In addition, it is convenient to use the above solvent when preparing the “labeled ligand solution”. In addition, when measuring a signal in SPFS or the like, the flow path is usually filled with a liquid containing neither an analyte nor a labeled ligand. Such a liquid is used for the preparation of a sample liquid. It is convenient to use the solvent alone.

Fluorescent Modified Ligand The fluorescent modified ligand 41 used in the present invention is composed of a second ligand 411 and a fluorescent molecule (Dm) 412. When a secondary antibody is used as the ligand, an analyte ( It is preferably an antibody that can recognize and bind to a target antigen. This fluorescently modified ligand 41 can be prepared in the same manner as a complex (conjugate) of a ligand and a fluorescent dye that is also used in a general immunostaining method. For example, a complex of ligand and avidin (including streptavidin) and a complex of fluorescent dye and biotin are prepared and reacted to bind the fluorescent dye to the ligand via avidin / biotin. A complex (maximum 4 biotins can bind to 1 avidin) is obtained. In addition to the reaction of biotin and avidin as described above, the primary antibody-secondary antibody reaction mode used in the fluorescent labeling method, carboxyl group and amino group, isothiocyanate and amino group, sulfonyl halide and amino group, Reactions such as iodoacetamide and thiol groups may be used.

  In the present invention, the second ligand is a ligand used for the purpose of labeling the analyte 42 with a fluorescent molecule, and may be the same as or different from the first ligand.

[Correction of fluorescence signal (Sm)]
In the present invention, the fluorescence signal (Sm) measured for the surface plasmon resonance sensor is corrected by the metal thin film (Msq) of the electric field enhancement evaluation structure 10 being the metal thin film (Mm) constituting the surface plasmon resonance sensor. ) Is made of the same metal as that constituting the structure (X).

Obtaining the electric field enhancement ratio R First, as the first stage of correction, the structure (X) in which the metal thin film (Msq) has the same thickness as the metal thin film (Mm) is defined as the target structure (Xt). After using any structure (X) other than the target structure (Xt) as the reference structure (Xr), the electric field enhancement ratio R for the target structure (Xt) and the reference structure (Xr) Are obtained as Rt and Rr through the steps shown in (i) to (iii) below for each of the target structure (Xt) and the reference structure (Xr).

(I) Detection of total reflection fluorescence signal (Sp) In the correction method of the present invention, as a first step for calculating the electric field enhancement ratio R, the transparent dielectric member (Ts) is applied to the region (P). 11, the surface opposite to the surface on which the fluorescent molecular layer (Fsp) 13a is present (refers to the surface on the lower side of the paper in FIG. 1; hereinafter, with respect to the transparent dielectric members (Ts) and (Tm). The side where the fluorescent molecular layer (Fsp) / (Fsq) or the metal thin film (Msq) / (Mm) / (Msa) is present may be referred to as “upper” and the opposite side may be referred to as “lower”. ), The step of detecting the amount of fluorescence emitted in this region (P) as the total reflection fluorescence signal (Sp) is performed by irradiating the incident light 61a with the incident angle that becomes the total reflection condition.

  Specifically, as shown in FIG. 1, a prism (not shown) such as a laser beam on the lower side of the paper surface is placed in contact with the lower portion of the transparent dielectric member (Ts) 11 ( Through the region (P), the incident light 61a is irradiated through the region (P), so that the fluorescence emitted from the region (P) is placed on the target structure (Xt) or the reference structure (Xr). This is a step of detecting by the light detection unit and digitizing the amount of fluorescence as a total reflection fluorescence signal (Sp).

At this time, water, a buffer solution used for diluting the specimen, a specimen solution, or the like may be present on the fluorescent molecular layer (Fsp) 13a.
Here, the incident light 61a introduced to excite the fluorescent molecules (Ds) constituting the fluorescent molecular layer (Fsp) 13a is a fluorescent signal in the surface plasmon resonance sensor 50 to which the correction method of the present invention is applied. The incident light 61c has the same wavelength as that used to excite the fluorescent molecule (Dm) in the measurement of (Sm).

  In this step (i), since the total reflection fluorescence in the state not affected by the electric field enhancement by the metal thin film is detected, the total reflection fluorescence signal (Sp) is used for evaluating the influence of the variation in the electric field enhancement. Blank reference.

(Ii) Detection of electric field-enhanced fluorescence signal (Sq) In the correction method of the present invention, as a second step for calculating electric field enhancement ratio R, transparent dielectric member (Ts) with respect to region (Q) 11 is irradiated from the surface opposite to the surface where the fluorescent molecular layer (Fsq) 13b is present with the incident light 61b at an incident angle that is a total reflection condition, whereby the amount of fluorescence emitted in this region (Q) Is detected as an electric field enhanced fluorescence signal (Sq).

  Specifically, as shown in FIG. 1, a prism (not shown) such as a laser beam on the lower side of the paper surface is placed in contact with the lower portion of the transparent dielectric member (Ts) 11 ( The region (Q) is irradiated with incident light 61b through an unillustrated region, whereby the fluorescence emitted from the region (Q) is placed on the target structure (Xt) or the reference structure (Xr). This is a step of detecting by the light detection unit and digitizing the amount of fluorescence as an electric field enhanced fluorescence signal (Sq).

  At this time, water, a buffer solution used for diluting the specimen, or a specimen solution may exist on the fluorescent molecular layer (Fsq) 13b. In this case, the fluorescence in (i) above is present. The same medium that was present on the molecular layer (Fsp) 13a is present.

  Here, the incident light 61b introduced to excite the fluorescent molecules (Ds) constituting the fluorescent molecular layer (Fsq) 13b is light having the same wavelength as that in the step (i), that is, the correction method of the present invention. Is the same wavelength as the incident light 61c used to excite the fluorescent molecule (Dm) in the measurement of the fluorescent signal (Sm) in the surface plasmon resonance sensor 50 to be applied. However, the optimum incident angle for the measurement under the total reflection condition in the step (ii) may be different from the incident angle in the step (i).

This step (ii) may be performed simultaneously with the step (i), or may be performed before or after the step (i).
By this step (ii), since the total reflection fluorescence enhanced by the influence of the electric field enhancement by the metal thin film in the target structure (Xt) or the reference structure (Xr) is detected, the electric field enhanced fluorescence signal ( Sq) is a reference value representing the degree of electric field enhancement in the target structure (Xt) or the reference structure (Xr).

(Iii) Calculation of electric field enhancement ratio R In the correction method of the present invention, as a third step for calculating the electric field enhancement ratio R, the total reflection fluorescence signal obtained through the steps (i) and (ii) ( Sp) and the electric field enhanced fluorescence signal (Sq)
R = Sq / Sp (1)
The step of calculating the electric field enhancement ratio R based on the above is performed. The electric field enhancement ratio R is a value indicating how much the total reflection fluorescence is increased by the surface plasmon by the metal thin film (Msq) formed on the target structure (Xt) or the reference structure (Xr), that is, the metal thin film. This is a value indicating the degree of electric field enhancement by (Msq), and the correction method of the present invention evaluates the electric field enhancement specific to each target structure (Xt) and reference structure (Xr) with this R. That is, if Rt which is the electric field enhancement ratio R calculated for the target structure (Xt) and Rr which is the electric field enhancement ratio R calculated for the reference structure (Xr) are obtained, each target structure (Xt) And the electric field enhancement properties inherent in the reference structure (Xr) can be contrasted in the form of Rt or Rr.

  Here, the steps (i) to (iii) may be performed in a series of assay steps for measuring the fluorescence signal (Sm) for the surface plasmon resonance sensor, or may be performed in advance before the assay step.

Moreover, the said process (i) and the said process (ii) may be performed simultaneously, or may be performed at different time.
When the step (i) and the step (ii) are performed at different times, the total reflection fluorescence signal (Sp) and the electric field enhanced fluorescence signal (Sq) are detected using a single light detection unit, and these 1 can be supplied using a single light source, so that the measurement system can be simplified, and the light source and the light detection unit There is an advantage that variations due to individual differences in the performance of the devices constituting the system can be avoided.

  On the other hand, when performing the said process (i) and the said process (ii) simultaneously, in order to detect a total reflection fluorescence signal (Sp) and an electric field enhancement fluorescence signal (Sq), a some light detection part will be used. Alternatively, these signals may be detected at the same time by using an image sensor capable of multipoint measurement such as a CCD image sensor. Each incident light (that is, incident light 61a, 61b in FIG. 1) used for obtaining these signals may be supplied using a plurality of light sources having the same optical characteristics. Alternatively, a single light source may be used, and incident light may be supplied simultaneously to each of the regions (P) and (Q) by dividing the light emitted from this light source by an appropriate beam splitter. However, it is used to measure the incident angle of the incident light 61a used for measuring the total reflection fluorescence signal (Sp) in the step (i) and the electric field enhanced fluorescence signal (Sq) in the step (ii). Since the incident angle may be different from the incident angle of the incident light 61b, a mechanism for appropriately adjusting the direction of the divided light according to the change in the incident angle is required, and the measurement system may be complicated.

  By the way, when manufacturing the plasmon sensor structure 20 as a material of the surface plasmon resonance sensor 50 by cutting a plasmon sensor bulk structure formed by forming a metal thin film on one large transparent dielectric member, It is not always necessary to actually measure Rt for the target structure (Xt) corresponding to all the surface plasmon resonance sensors 50. For example, the surface plasmon resonance sensors 50 obtained from the same plasmon sensor bulk structure are likely to have the same film thickness or have a certain tendency in the film thickness distribution of the metal thin film. Therefore, in this case, Rt of the target structure (Xt) corresponding to some of the surface plasmon resonance sensors 50 is measured and another surface plasmon obtained from the same plasmon sensor bulk structure is obtained. As the virtual Rt for the target structure (Xt) corresponding to the resonance sensor 50, the measured Rt value is used as it is, or the value estimated from the measured Rt value. Can also be used.

As a second step of conversion and correction of the fluorescence signal, Rt and Rr, which are the electric field enhancement ratios R obtained for the target structure (Xt) and the reference structure (Xr), respectively, are used. )
Sm1 = Sm0 x (Rr / Rt) (2)
Based on the above, the fluorescence signal (Sm) is corrected by converting the uncorrected fluorescence signal (Sm0), which is the fluorescence signal (Sm) before correction, to the corrected fluorescence signal (Sm1).

  Even when the fluorescence signal (Sm) is measured using a surface plasmon resonance sensor for a sample solution containing an analyte of the same concentration, a metal having the same film thickness as the metal thin film (Msq) 12 constituting the target structure (Xt) When measured using the surface plasmon resonance sensor 50 having the thin film (Mm) 22, the surface having the metal thin film (Mm) 22 having the same film thickness as the metal thin film (Msq) 12 constituting the comparative structure (Xr). Compared with the case of measuring using the plasmon resonance sensor 50, the intensity of the apparent fluorescence signal (Sm) is (Rt / Rr) times. Therefore, when converted into a measured value by the surface plasmon resonance sensor 50 having the metal thin film (Mm) 22 having the same film thickness as the metal thin film (Msq) 12 constituting the comparative structure (Xr), the original fluorescence signal ( It is necessary to multiply Sm) by its reciprocal.

  Therefore, if the fluorescence signal (Sm) is corrected by the above formula (2), the fluorescence signal (Sm) in the surface plasmon resonance sensor 50 having the different thicknesses of the metal thin film (Mm) 22 is obtained from the comparison structure (Xr). Normalization can be achieved by converting the measured value to the measured value by the surface plasmon resonance sensor 50 having the metal thin film (Mm) 22 having the same film thickness as the metal thin film (Msq) 12 to be formed.

Optical system The light source used in the correction method of the present invention is not particularly limited as long as it can generate total reflection fluorescence in the region (P) and electric field enhanced fluorescence in the region (Q). From the standpoint of unity and intensity of light energy, it is preferable to use laser light as a light source. It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism through the optical filter.

  As the “laser light”, for example, an LD laser having a wavelength of 200 to 900 nm, 0.001 to 1,000 mW, a wavelength of 230 to 800 nm (resonance wavelength depending on the metal species used for the metal thin film (Msq) / (Mm) / (Msa)) For example, a semiconductor laser of 0.01 to 100 mW.

  The “prism” is intended to allow the laser light through various filters to efficiently enter the plasmon resonance sensor, and preferably has the same refractive index as that of the transparent dielectric member 11. In the present invention, various prisms for which total reflection conditions can be set can be selected as appropriate, and therefore, there is no particular limitation on the angle and shape. For example, a 60-degree dispersion prism may be used. The prism may be incorporated in the transparent dielectric member (Ts) 11 as a prism portion of the integrated structure block.

  Examples of the “optical filter” include a neutral density (ND) filter and a diaphragm lens. The “darkening (ND) filter” (or neutral density filter) is intended to adjust the amount of incident laser light. In particular, when a detector with a narrow dynamic range is used, it is preferable to use it for carrying out a highly accurate measurement.

The “polarizing filter” is used to make the laser light P-polarized light that efficiently generates surface plasmons.
“Cut filters” are external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), plasmon scattering light (excitation light originated from plasmon A filter that removes various types of noise light such as scattered light generated by the influence of structures or deposits on the surface of the resonance sensor), autofluorescence of the enzyme fluorescent substrate, and examples thereof include interference filters and color filters. It is done.

  The “collecting lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system. As a simple condensing system, a commercially available objective lens (for example, manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be used. The magnification of the objective lens is preferably 10 to 100 times.

  As the “photodetector”, a photodetection device used for detecting SPFS by an assay method based on SPFS or the like can be used. From the viewpoint of ultra-high sensitivity, a photomultiplier tube (manufactured by Hamamatsu Photonics Co., Ltd.) Photomultiplier) is preferred. In addition, although the sensitivity is lower than these, a CCD image sensor capable of multipoint measurement is also suitable because it can be viewed as an image and noise light can be easily removed. In the present invention, the “light detection unit” also functions as an SPFS detection unit used for detecting a fluorescent signal (Sm) in an assay method described later.

[Assay Method]
The assay method according to the present invention comprises:
(A) In a plasmon sensor structure having a region (Am) in which an immobilized ligand-containing layer containing a first ligand is formed on a transparent dielectric member (Tm) via a metal thin film (Mm), Preparing a surface plasmon resonance sensor by bringing a specimen into contact with a fluorescent modifying ligand comprising a second ligand and a fluorescent molecule (Dm) in the region (Am);
(B) After the step (a), the region (Am) is irradiated with incident light from the surface of the transparent dielectric member (Tm) opposite to the metal thin film (Mm). Irradiating incident light at an incident angle as a reflection condition, thereby detecting the amount of fluorescence emitted from the region (Am) as a fluorescence signal (Sm);
(C) correcting the fluorescence signal (Sm) detected in the step (b);
(D) including a step of evaluating the amount of analyte in the specimen based on the fluorescence signal (Sm) corrected in the step (c).
Here, correction | amendment of the fluorescence signal (Sm) in this process (c) is performed using the correction method mentioned above.

<Process (a)>
In the assay method of the present invention, in the step (a), the region (in which the immobilized ligand-containing layer containing the first ligand is formed on the transparent dielectric member (Tm) via the metal thin film (Mm) ( In the plasmon sensor structure having (Am), a surface plasmon resonance sensor is prepared by bringing a specimen, a fluorescently modified ligand composed of a second ligand and a fluorescent molecule (Dm) into contact with the region (Am). It is a process. In other words, this step (a) is a step of preparing a surface plasmon resonance sensor to which the correction method of the present invention is applied, and in FIG. 1 is a step of preparing the surface plasmon resonance sensor 50.

(Plasmon sensor structure)
The plasmon sensor structure 20 used in the step (a) is a region in which an immobilized ligand-containing layer containing a first ligand is formed on a transparent dielectric member (Tm) via a metal thin film (Mm). A plasmon sensor structure having (Am). Such a plasmon sensor structure can be prepared by a conventionally known method.

Transparent dielectric member (Tm)
In the present invention, the transparent dielectric member (Tm) 21 constituting the plasmon sensor structure 20 constitutes a skeleton of the structure of the plasmon sensor structure 20 and is a surface plasmon resonance sensor 50 prepared from the plasmon sensor structure 20. 2 functions as a medium for guiding the incident light 61c to the surface on which the metal thin film (Mm) 22 is formed.

In the present invention, the transparent dielectric member used as the transparent dielectric member (Tm) 21 may be any of the various materials and forms mentioned in the section “Transparent dielectric member (Ts)”.
Before forming a metal thin film (Mm) 22 to be described later, it is preferable to perform a cleaning treatment with acid or plasma on the surface of the transparent dielectric member (Tm) 21.

Metal thin film (Mm)
In the present invention, the metal thin film (Mm) 22 constituting the plasmon sensor structure 20 is similar to the metal thin film (Msq) 12 and metal thin film (Msa) 32 described above, and the metal thin film constituting a general surface plasmon resonance sensor. The same metals as those generally used can be used, and as the metal species, those of various metal species and forms mentioned in the section of “Metal thin film (Msq) / (Msa)” can be used.

The metal thin film (Mm) 22 is formed on the transparent dielectric member (Tm) 21 with respect to the region to be the region (Am), as described in the above section “Metal thin film (Msq) · (Msa)”. Can be formed.
The absolute value of the thickness of the metal thin film (Mm) 22 can also be set to the range described in the above-mentioned section “Metal thin film (Msq) · (Msa)”.

Immobilized Ligand-Containing Layer In the present invention, the immobilized ligand-containing layer 23 constituting the plasmon sensor structure 20 includes the first ligand 231. Such an immobilized ligand-containing layer 23 can have the same configuration as the immobilized ligand-containing layer 33 constituting the electric field enhancement evaluation structure 10 further including the region (As), and is formed by the same method. Is done.

(Surface plasmon resonance sensor)
In the present invention, the surface plasmon resonance sensor 50 brings the specimen, the fluorescent ligand 41 composed of the second ligand 411 and the fluorescent molecule (Dm) 412 into contact with the region (Am) of the plasmon sensor structure 20. It is prepared by.

Contact In the present invention, “contact” means that the surface of the surface plasmon resonance sensor on which a ligand or the like is immobilized is immersed in the liquid supply, and the object contained in the liquid supply is transferred to the surface plasmon. Contacting a resonance sensor. Here, the “surface plasmon resonance sensor” referred to in this section and the following “channel” section includes the surface plasmon resonance sensor 50 shown in FIG. 1 and the surface plasmon resonance sensor 51 shown in FIG. It is a concept to do.

  In the step (a), the “contact” between the specimen and the surface plasmon resonance sensor means that the specimen is included in the liquid feed circulating in the flow path, and only the one surface on which the ligand of the surface plasmon resonance sensor is immobilized A mode in which the surface plasmon resonance sensor and the specimen are brought into contact with each other in a state of being immersed in the liquid feeding is preferable.

The “liquid feeding” used in the present invention is preferably the same as the solvent or buffer in which the “sample” described below is diluted. For example, phosphate buffered saline (PBS), Tris buffered saline ( TBS) and the like, but are not particularly limited.

In the present invention, the term “flow path” refers to a rectangular parallelepiped that can efficiently deliver a small amount of a chemical solution and can change or circulate the liquid feeding speed in order to promote the reaction. It is tubular. As the shape of the flow path, the vicinity of the location where the surface plasmon resonance sensor is installed preferably has a rectangular parallelepiped structure, and the vicinity of the location where the chemical solution is delivered preferably has a tubular shape.

  The material is a homopolymer or copolymer containing methyl methacrylate, styrene or the like as a raw material in the surface plasmon resonance sensor part; a light-transmitting material such as polyolefin such as polyethylene, and a silicone rubber or Teflon in the chemical solution delivery part. (Registered trademark), a polymer such as polyethylene or polypropylene is used. Here, the “surface plasmon resonance sensor portion” in the section of “channel” means a portion in contact with the assay measurement region of the surface plasmon resonance sensor in the channel, and specifically, the surface plasmon shown in FIG. This is a portion in contact with the region (Am) in the resonance sensor 50 or the region (As) in the surface plasmon resonance sensor 51 shown in FIG.

  However, for the surface plasmon resonance sensor part, all of the flow path structure is not necessarily transparent unless the flow path is kept in a constant shape during fluorescence measurement and the detection of fluorescence generated by plasmon excitation is not hindered. It does not have to be composed of only materials. That is, a part of the flow path of the surface plasmon resonance sensor part that is necessary for transmitting the fluorescence generated by plasmon excitation and guiding it to the detection part, specifically, a part that includes a light transmission window necessary for collecting the fluorescence. However, other parts may be partially or entirely made of a chemically stable material other than the light transmissive material. When the flow path of the surface plasmon resonance sensor unit has a rectangular parallelepiped structure, when the surface of the surface plasmon resonance sensor where the metal thin film and the SAM are present on the surface is the bottom surface, for example, the ceiling surface at a position facing the bottom surface The light transmitting material may be used, and the side surface may be formed of a chemically stable material other than the light transmitting material.

  Here, the other part, for example, the side surface does not necessarily need to be a rigid body as long as a certain shape is maintained at the time of fluorescence measurement, and may have an appropriate elasticity to ensure a sealing property. For example, regarding the flow path of the surface plasmon resonance sensor unit, the ceiling surface may be made of polymethyl methacrylate (PMMA) and the side surface may be made of silicone rubber.

  In the surface plasmon resonance sensor unit, from the viewpoint of increasing the contact efficiency with the specimen and shortening the diffusion distance, the cross section of the flow path of the surface plasmon resonance sensor unit is preferably about 100 nm to 1 mm in length and width.

In the flow path, the position of the liquid feeding inlet for introducing the liquid feeding from the drug delivery section to the surface plasmon resonance sensor section and the position of the liquid feeding outlet for discharging the liquid feeding from the surface plasmon resonance sensor section are both measured by fluorescence. There is no particular limitation as long as it does not hinder. For example, when the flow path of the surface plasmon resonance sensor unit has a rectangular parallelepiped structure, it is convenient in creating the flow path to provide both the liquid feeding inlet and the liquid feeding outlet on the ceiling surface. Either one or both of the liquid delivery outlets may be provided on the side surface.
The method for fixing the surface plasmon resonance sensor to the flow path is not particularly limited as long as the flow path is maintained in a fixed shape and fluorescence measurement is not hindered.

  As an example of such a fixing method, in a small lot (laboratory level), first, a silicone rubber sheet or O having a certain thickness is formed on the surface on which the metal thin film of the surface plasmon resonance sensor is formed. A side structure of the flow path is formed by placing a ring, and then a light transmitting top plate (for example, a PMMA substrate) provided with a liquid feeding inlet and a liquid feeding outlet is disposed thereon. Examples include a method of forming a ceiling surface of a road, and then crimping and fixing them with an appropriate fastener. At this time, if a silicone rubber sheet having an appropriate thickness, with a hole having an arbitrary shape and size, is used as the material constituting the side structure, the inner periphery of the hole is the surface. Since the side surface structure of the flow path of the plasmon resonance sensor unit is obtained, it is preferable because a flow path having a required shape and size can be easily formed. For example, first, a perforated polydimethylsiloxane (PDMS) sheet having a channel height of 0.5 mm is formed on the surface on which the metal thin film of the surface plasmon resonance sensor is formed. Then, a PMMA substrate provided with a liquid feeding inlet and a liquid feeding outlet in advance is placed on the polydimethylsiloxane (PDMS) sheet, and then the PMMA substrate is placed. It is preferable to press the polydimethylsiloxane (PDMS) sheet and the surface plasmon resonance sensor and fix them with a fastener such as a screw. In addition, when a silicone rubber sheet or O-ring and a light-transmitting top plate are pressure-bonded and fixed to the surface plasmon resonance sensor, an appropriate spacer made of a material such as silicone rubber or stainless steel is used as necessary. May be used in combination.

  In a large lot (factory level) manufactured industrially, as a method of fixing the surface plasmon resonance sensor to the flow path, a gold substrate is directly formed on a plastic integrally molded product or a separately prepared gold substrate is used. After fixing, the dielectric layer, the SAM layer, and the ligand are fixed on the gold surface, and then the lid is covered with a plastic integrally molded product corresponding to the top plate of the flow path. If necessary, the prism can be integrated into the flow path.

  In the present invention, in the surface plasmon resonance sensor including the region (P) and the region (Q) like the surface plasmon resonance sensor 51 shown in FIG. 3, the region (P) and the region (Q) are also in contact with each other. The “surface plasmon resonance sensor unit” may be configured as described above. Alternatively, in order to introduce a medium, such as water, that is different from the above liquid feeding to the surface of the region (P) and the region (Q), a second flow path is provided so as to be in contact with the region (P) and the region (Q). In the second flow path, the same structure as that of the “surface plasmon resonance sensor unit” may be formed in a portion in contact with the region (P) and the region (Q).

Samples In the present invention, as the sample used for preparing the surface plasmon resonance sensor, those mentioned in the above-mentioned section “<< Use as surface plasmon resonance sensor >>” can be used. Here, the “surface plasmon resonance sensor” referred to in this section is a concept including the surface plasmon resonance sensor 50 shown in FIG. 1 and the surface plasmon resonance sensor 51 shown in FIG.

  In the assay method of the present invention, the contact of the specimen with the region (Am) of the plasmon sensor structure 20 usually involves sending a liquid containing the specimen through the flow path connected to the region (Am). ). Similarly, as shown in FIG. 2B, the contact of the specimen with the structure 10 further having the region (As) is similarly sent through the flow path connected to the region (As). This is done by bringing the liquid into contact with the region (As).

  The temperature and time for circulating the liquid supply vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. × 1 to 60 minutes, preferably 37 ° C. × 5 to 15 minutes.

The initial concentration of the analyte 42 contained in the specimen being sent may be 100 μg / mL to 0.0001 pg / mL.
The total amount of liquid feeding, that is, the volume of the flow path is usually 0.0001 to 20 mL, preferably 0.01 to 1 mL.
The flow rate of liquid feeding is usually 1 to 2,000 μL / min, preferably 5 to 500 μL / min.

Fluorescence-modifying ligand In the present invention, as the fluorescence-modifying ligand 41 used for preparing the surface plasmon resonance sensor, those mentioned in the above section “<< Use as surface plasmon resonance sensor >>” can be used. Here, the “surface plasmon resonance sensor” referred to in this section is a concept including the surface plasmon resonance sensor 50 shown in FIG. 1 and the surface plasmon resonance sensor 51 shown in FIG.

In the assay method of the present invention, the fluorescence modifying ligand 41 is brought into contact with the region (Am) of the plasmon sensor structure 20 through the flow path to the region (Am) of the plasmon sensor structure 20 brought into contact with the specimen. It is carried out by bringing a liquid feeding containing the modified ligand 41 into contact. Similarly, as shown in FIG. 2 (b), with respect to the contact of the fluorescent modification ligand 41 with the structure 10 further having the region (As), similarly, the fluorescent modification ligand 41 is brought into the region (As) through the channel. It is carried out by bringing a liquid feed containing
The concentration of the fluorescent modifying ligand 41 in the liquid feeding is preferably 0.001 to 10,000 μg / mL, and more preferably 1 to 1,000 μg / mL.
The temperature, time, and flow rate at which the liquid is circulated are the same as in the case of the liquid that contains the specimen.

Cleaning Process The cleaning process is a process of cleaning at least one of the surface of the plasmon sensor structure 20 and the surface of the surface plasmon resonance sensor 50. It is preferable that at least one of the washing steps is included in the step (a).

  As the washing solution used in the washing step, for example, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer used in the reaction of step (a), preferably 0.00001 to 1 weight. % Content is desirable.

It is preferable that the temperature and flow rate at which the liquid is circulated are the same as in the case of the liquid that contains the sample.
The time for circulating the cleaning liquid is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

<Step (b)>
In the assay method of the present invention, step (b) is opposite to the metal thin film (Mm) of the transparent dielectric member (Tm) with respect to the region (Am) after the step (a). This is a step of irradiating incident light from the surface on the side and irradiating incident light at an incident angle that is a total reflection condition, thereby detecting the amount of fluorescence emitted from the region (Am) as a fluorescent signal (Sm). .

  Specifically, for the assay measurement region in the surface plasmon resonance sensor (that is, the region (Am) in the surface plasmon resonance sensor 50 shown in FIG. 1 and the region (As) in the surface plasmon resonance sensor 51 shown in FIG. 3), As shown in FIG. 1, the incident light 61c is irradiated, whereby the fluorescence emitted from the assay measurement region is detected by the SPFS detection unit installed above the surface plasmon resonance sensor, and the amount of fluorescence is detected by the fluorescence signal (Sm). It is the process of quantifying as.

  Here, as the light source, prism, optical filter, polarizing filter, and condenser lens used in the assay method of the present invention, those mentioned in the section of “optical system” in the above description of the correction method of the present invention are used. It is done. In addition, for the “SPFS detection unit”, a photodetection device which is also cited as the “photodetection unit” described above in the “optical system” section can be used.

  As a surface plasmon resonance sensor, a surface plasmon resonance sensor having both a region (P) and a region (Q) as in the surface plasmon resonance sensor 51 shown in FIG. 3, that is, the target structure (Xt) is a target structure. When a surface plasmon resonance sensor integrated as a part is used, the above steps (i) to (iii) for obtaining the electric field enhancement ratio Rt used in the above correction method are performed together with this step (b). be able to.

  These steps (i) to (iii) may be performed immediately before the step (b) or may be performed immediately after the step (b). In these embodiments, the fluorescence signal (Sm), the total reflection fluorescence signal (Sp), and the electric field-enhanced fluorescence signal (Sq) are detected at different times using a single SPFS detector, and these signals are obtained. Since each incident light used (that is, incident light 61c, 61a, 61b in FIG. 1) can be supplied using a single light source, the measurement system can be simplified, and the light source and the SPFS detection unit are configured. There is an advantage that variation due to individual differences in performance can be avoided.

  Further, these steps (i) to (iii) may be performed simultaneously with the step (b). In this case, a plurality of SPFS detectors are used to detect the fluorescence signal (Sm) and at least one of the total reflection fluorescence signal (Sp) and the electric field enhanced fluorescence signal (Sq). Alternatively, these signals may be detected at the same time by using an image sensor capable of multipoint measurement such as a CCD image sensor. When the steps (i) to (iii) are performed simultaneously with the step (b), each incident light used for obtaining these signals (that is, the incident light 61c, 61a, 61b in FIG. 1) has an optical characteristic. You may supply using the same some light source. Alternatively, by using a single light source and dividing the light emitted from this light source by an appropriate beam splitter, incident light can be simultaneously supplied to each of the region (As), region (P), and region (Q). May be. However, it is used to measure the incident angle of the incident light 61a used for measuring the total reflection fluorescence signal (Sp) in the step (i) and the electric field enhanced fluorescence signal (Sq) in the step (ii). Since the incident angle may be different from the incident angle of the incident light 61b, a mechanism for appropriately adjusting the direction of the divided light according to the change in the incident angle is required, and the measurement system may be complicated.

  Further, by performing the steps (i) to (iii) prior to a series of assay steps performed in the assay method of the present invention, the electric field enhancement ratio Rt applied to the surface plasmon resonance sensor is calculated in advance. Also good. The calculation of the electric field enhancement ratio Rt in such an embodiment is also performed when the correction method of the present invention is applied to the fluorescence signal (Sm) of the surface plasmon resonance sensor not having the region (P) and the region (Q). Useful.

<Step (c)>
In the assay method of the present invention, step (c) is a step of correcting the fluorescence signal (Sm) detected in step (b) described above using the correction method described above. By this step, the uncorrected fluorescence signal (Sm0), which is the fluorescence signal (Sm) just detected by the SPFS detection unit, is converted into the corrected fluorescence signal (Sm1), thereby forming a metal thin film (ie, a surface plasmon resonance sensor) 1 to remove the variation in electric field enhancement in the metal thin film (Mm) 22 constituting the surface plasmon resonance sensor 50 shown in FIG. 1 or the metal thin film (Msa) 32 constituting the surface plasmon resonance sensor 51 shown in FIG. it can.

  Here, the electric field enhancement ratio Rr for the reference structure (Xr), which is used in the correction in the step (c), is usually calculated in advance prior to a series of assay steps performed in the assay method of the present invention. Things are used. However, in parallel with the series of assay steps performed in the assay method of the present invention, the steps (i) to (iii) for calculating the electric field enhancement ratio Rr for the reference structure (Xr) are performed. Is not to be excluded.

<Step (d)>
In the assay method of the present invention, step (d) is a step of evaluating the amount of analyte in the specimen based on the fluorescence signal (Sm) corrected in the above-mentioned step (c), that is, the corrected fluorescence signal (Sm1). is there. Specifically, a calibration curve is created based on the fluorescence signal (Sm) corrected in the step (c), and the amount of target antigen or target antibody in the sample to be measured based on the created calibration curve Is calculated from the measurement signal.

In the assay S / N ratio step (d), the “blank fluorescence signal” measured before the step (b), the “assay fluorescence signal” obtained by the measurement in the step (b), and any modification A non-metal substrate (in the present invention, including a transparent dielectric substrate that is only formed with a metal thin film on the surface and is not further modified) is fixed to the flow path, and the SPFS is flowed while flowing ultrapure water. When the signal obtained by measurement is “initial noise”, the assay S / N ratio represented by the following formula (3a) can be calculated:
Assay S / N ratio = | Ia / Io | / In (3a)
(In the above formula (3a), Ia is the assay fluorescence signal, Io is the blank fluorescence signal, and In is the initial noise).

However, in calculating the assay S / N ratio, the following formula is used on the basis of “assay noise signal” when the concentration of the analyte contained in the sample is 0 instead of the above formula (3a) in practice. You may calculate according to (1b):
Assay S / N ratio = | Ia | / | Ian | (3b)
(In the above formula (3b), Ian is an assay noise signal and Ia is an assay fluorescence signal as in the above formula (3a)).

  When SPR is detected through resonance angle and reflected light intensity instead of SPFS, the corresponding assay signal and plank signal are used instead of assay fluorescence signal and blank fluorescence signal as Ia and Io, respectively. Can do.

[Analyte detector]
The analyte detection apparatus used in the present invention is used for the assay method of the present invention using the surface plasmon resonance sensor as well as the correction method of the present invention.

  The “analyte detection device” includes at least a light source, various optical filters, a prism, a cut filter, a condensing lens, and an SPFS detection unit. This “analyte detection device” can be used for carrying out the correction method of the present invention, and may be provided with light separation means such as a beam splitter. Note that it is preferable to have a liquid feeding system combined with a surface plasmon resonance sensor when handling a sample liquid, a washing liquid, a labeled antibody liquid, and the like. The type of liquid feeding system is not limited as long as the object of the present invention can be achieved. For example, a microchannel device connected to a liquid pump may be used.

  In addition, a surface plasmon resonance (SPR) detection unit, that is, a photodiode as a light receiving sensor dedicated to SPR, an angle variable unit for adjusting the optimum angle of SPR and SPFS (in order to obtain total reflection attenuation (ATR) conditions with a servomotor) The angle of 45 to 85 ° can be changed by synchronizing the photodiode and the light source with a resolution of preferably 0.01 ° or more.), A computer for processing information input to the SPFS detector May also be included.

  Preferred embodiments of the light source, the optical filter, the cut filter, the condensing lens, and the SPFS detection unit are the same as those described above. In the present invention, the SPFS detector can be used as a “light detector” when the correction method of the present invention described above is performed.

  As the “liquid feed pump”, for example, a micro pump suitable for a small amount of liquid feed, a syringe pump with high feed accuracy and low pulsation, which is preferable but cannot be circulated, a simple and excellent handleability but a small amount of liquid feed For example, a tube pump may be difficult.

<Labeled antibody: “Alexa Fluor 647” labeled anti-AFP monoclonal antibody preparation>
Anti-α-fetoprotein (AFP) monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Nippon Medical Laboratory, Inc.) is biotinylated using a commercially available biotinylation kit (manufactured by Dojindo Laboratories) did. The procedure followed the protocol attached to the kit.

  Next, the solution of the obtained biotinylated anti-AFP monoclonal antibody and the solution of “Alexa Fluor 647” streptavidin conjugate (Molecular Probes) were mixed and reacted by stirring at 4 ° C. for 60 minutes.

  Finally, in order to remove unreacted antibodies and unreacted enzymes, the reaction product was purified using a molecular weight cut filter (manufactured by Nippon Millipore) to obtain an Alexa Fluor 647-labeled anti-AFP monoclonal antibody solution. The obtained antibody solution was stored at 4 ° C. after protein quantification.

<Production of substrate>
Substrates A, B, C, D, E, and F forming the assay measurement region (A), the total reflection fluorescence reference region (P), and the electric field enhanced fluorescence reference region (Q) were prepared as follows.

First, 6 sheets of 1 mm thick transparent glass substrate “S-LAL 10” (manufactured by OHARA INC., Refractive index [n _d ] = 1.72) were prepared, and plasma dry cleaner “PDC200” (Yamato) Plasma cleaning was performed by Kagaku Corporation.

  A chromium thin film is first formed by sputtering on the surface of the plasma-cleaned substrate where the assay measurement region (A) and the electric field-enhanced fluorescence reference region (Q) are to be formed. It formed by sputtering method. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm. On the other hand, the chromium thin film and the gold thin film were not formed in the portion where the total reflection fluorescent reference region (P) was to be formed.

Next, the substrate obtained in the above step was immersed in 10 mL of an ethanol solution of 10-carboxy-1-decanethiol adjusted to 25 mg / mL for 24 hours to form a SAM on the surface of the gold thin film. The substrate was taken out of the ethanol solution, washed sequentially with ethanol and isopropanol, and then dried using an air gun.
By performing the above process on each of the six substrates, substrates A, B, C, D, E, and F on which the metal thin film and the SAM were formed (the reaction layer was not formed) were manufactured.

<Production of sensor chip>
For each of the substrates A, B, C, D, E, and F produced as described above, the sensor chip is configured by the following step [1], and then the assay measurement region (A) is defined by the following step [2]. Then, the total reflection fluorescence reference region (P) is produced by the following step [3], and the electric field enhanced fluorescence reference region (Q) is produced by the following step [4], and the sensor chips A, B, C, D, E, F, did.

[1] Configuration of sensor chip To form an assay measurement region (A), a total reflection fluorescence reference region (P), and an electric field-enhanced fluorescence reference region (Q) on the surface of the substrate on which the metal thin film and SAM are formed A sheet made of polydimethylsiloxane (PDMS) having a thickness of 0.5 mm and having three holes each having a flow path length of 10 mm and a width of 5 mm was placed thereon. Furthermore, a silicone rubber spacer was disposed around the PDMS sheet (the silicone rubber spacer is not in contact with the liquid feed). On the PDMS sheet and the silicone rubber spacer, a hole for feeding a liquid (liquid feeding inlet) and a liquid feeding discharge so as to be located inside each of the regions (A), (P) and (Q). A top plate made of polymethylmethacrylate (PMMA) in which a hole (liquid feed outlet) was formed was placed. A laminate of these sensor substrate, PDMS sheet, and PMMA top plate was crimped at the outer periphery and fixed with screws to form a sensor chip.

[2] Production of region (A) A silicone rubber tube and a peristaltic pump were connected to the liquid feeding inlet and the liquid feeding outlet of the sensor chip region (A) (liquid feeding and circulation of various fluids described below) All were performed using similar tubes and peristaltic pumps).

  After 5 mL of phosphate buffered saline (PBS) containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM water-soluble carbodiimide (WSC) was fed and circulated for 20 minutes, anti-AFP monoclonal antibody (1D5 2.5 mg / mL, 2.5 mL / mL (manufactured by Japan Medical Laboratory), and the solution was circulated for 30 minutes to immobilize the antibody on SAM. Thereafter, PBS containing 1% by weight of bovine serum albumin (BSA) and 1M aminoethanol was circulated for 30 minutes for blocking treatment.

[3] Production of regions (P) and (Q) A fluorescent molecular layer was formed on the surfaces of regions (P) and (Q) by the following procedure.
First, a hot water bath is attached to a round bottom flask, 100 g of ethyl acetate is heated to reflux, and further 20 g of stearyl methacrylate, 50 g of methyl methacrylate and 30 g of 2-acetoacetoxyethyl methacrylate, 0.1 g of N, N′-azobisisovaleronitrile. A mixed solution in which each was dissolved was added dropwise over 2 hours and reacted at the same temperature for 10 hours to obtain an ethyl acetate solution of Resin R-1. 1 ml of this resin solution R-1 was weighed, and 1 ml of an ethyl acetate solution containing 20 mM / ml of an amide compound (F-1) obtained by reacting the fluorescent dye Cy5 (fluorescent dye) mono NHS with the same mole of laurylamine was added. The mixture was stirred to prepare a fluorescent agent coating solution.

  The prepared fluorescent agent coating solution is applied to the region (P) in the region where the regions (P) and (Q) are to be formed on the surface of the sensor substrate obtained in [2] on which the SAM and the like are formed. And it apply | coated with the spin coater so that the dry film thickness in both (Q) might be set to 100 nm, and it dried for 5 minutes at the drying temperature of 100 degreeC, and formed the fluorescent molecule layer.

<Measurement of signal>
For each of the sensor chips A, B, C, D, E, and F manufactured as described above, the measurement of the signal (Sa) in the measurement region (A) by the following step [4] is performed by the following step [5]. The signal (Sp) in the measurement region (P) was measured, and the signal (Sq) in the region (Q) was measured by the following step [6]. Here, the signal (Sa) in the measurement region (A) and the signal (Sq) in the region (Q) are measured under surface plasmon resonance conditions, and the signal (Sp) in the region (P) is measured in total reflection attenuation (ATR). ) Each done under conditions.

[4] Measurement of signal (Sa) First, in the measurement region (A), a solution diluted with PBS buffer (pH 7.4) so that AFP (2.0 mg / mL solution, Acris Antibodies GmbH) becomes 10 ng / mL. Was allowed to flow at 10 μL / min for 20 minutes. Subsequently, a labeled antibody prepared as described above: 10 μL of a solution diluted with 1% BSA-PBS buffer (pH 7.4) so that the “Alexa Fluor 647” -labeled anti-AFP monoclonal antibody was 2.5 μg / mL. For 20 minutes. As a cleaning step, a TBS solution (pH 7.4) containing 0.005% Tween 20 was allowed to flow at 10 μL / min for 10 minutes to obtain a surface plasmon resonance sensor. Then, with the flow path filled with PBS buffer (pH 7.4), laser light (635 nm, 40 μW) is irradiated from the back side of the surface plasmon resonance sensor via the prism, and the amount of fluorescence emitted from the sensor surface is measured. Measured with CCD. This measured value was defined as a signal (Sa).

[5] Measurement of signal (Sp) PBS buffer (pH 7.4) is fed to the region (P), the flow path is filled with this buffer, and the back surface of the surface plasmon resonance sensor is passed through the prism. Laser light (635 nm, 40 μW) was irradiated, and the amount of fluorescence emitted from the sensor surface was measured with a CCD.
This measured value was defined as a signal (Sp).

[6] Measurement of signal (Sq) PBS buffer (pH 7.4) is fed to the region (Q), the flow path is filled with this buffer, and the back surface of the surface plasmon resonance sensor is passed through the prism. Laser light (635 nm, 40 μW) was irradiated, and the amount of fluorescence emitted from the sensor surface was measured with a CCD.
This measured value was defined as a signal (Sq).

<Calculation of electric field enhancement ratio R and corrected signal (Sa ')>
For each of the sensor chips A, B, C, D, E, and F, a corrected signal (Sa ′) was calculated as follows.

Based on the following formula (1), the electric field enhancement ratio R was calculated for each of the sensor chips A, B, C, D, E, and F.
R = Sq / Sp (1)
Next, for the electric field enhancement ratio R calculated for the sensor chips A, B, C, D, E, and F, a correction coefficient k, which is a value obtained by dividing the electric field enhancement ratio R calculated for the sensor chip A, was calculated.

  For the sensor chips A, B, C, D, E, and F, the corrected signal (Sa ′) is obtained by multiplying the measured value of the signal (Sa) measured in the region (A) by 1 / k. Was calculated.

  The results are as shown in Table 2. Here, in the signals (Sa) and (Sa ′), the “substrate A coincidence ratio” represents a relative signal intensity in each sensor chip when the signal intensity in the sensor chip A is 100, and the sensor chip A , B, C, D, E, and F are closer to 100, indicating that the variation in signal is smaller.

10 ... Electric field enhancement evaluation structure 11 ... Transparent dielectric member (Ts)
12 ... Metal thin film (Msq)
13a: Fluorescent molecular layer (Fsp)
13b: Fluorescent molecular layer (Fsq)
20 ... plasmon sensor structure 21 ... transparent dielectric member (Tm)
22 ... Metal thin film (Mm)
32 ... Metal thin film (Msa)
23,33 ... Immobilized ligand-containing layer 231,331 ... Ligand (first ligand)
41 ... Fluorescent modified ligand 411 ... Second ligand 412 ... Fluorescent dye (Dm)
42 ... Analyte 50, 51 ... Surface plasmon resonance sensor 61a, 61b, 61c ... Incident light

Claims (10)

Fluorescent dye (Dm) measured using surface plasmon fluorescence spectroscopy for a surface plasmon resonance sensor in which a fluorescent dye (Dm) is fixed on a transparent dielectric member (Tm) via a metal thin film (Mm) About the fluorescence signal (Sm) derived from
(K-1) a transparent dielectric member (Ts);
A region (P) in which a fluorescent molecular layer (Fsp) is formed on the transparent dielectric member (Ts) without using a metal thin film;
A region (Q) in which a fluorescent molecular layer (Fsq) is formed on the transparent dielectric member (Ts) via a metal thin film (Msq);
The fluorescent molecular layer (Fsq) and the fluorescent molecular layer (Fsp) are both composed of the same fluorescent dye (Ds),
The transparent dielectric member (Tm) and the transparent dielectric member (Ts) are both made of the same transparent dielectric,
The metal thin film (Mm) and the metal thin film (Msq) are both structures (X) made of the same metal, and the metal thin film (Msq) has the same thickness as the metal thin film (Mm). About the body (Xt)
(I) For the region (P),
In the measurement of the fluorescence signal (Sm), the transparent dielectric member (Ts) is measured in the fluorescence signal (Sm) at an incident angle as a total reflection condition from the surface opposite to the surface where the fluorescent molecule layer (Fsp) exists. By irradiating incident light of the same wavelength as the incident light used to excite Dm),
Detecting the amount of fluorescence emitted in the region (P) as a total reflection fluorescence signal (Sp);
(Ii) Before the step (i), simultaneously with the step (i), or after the step (i), with respect to the region (Q),
Incident light having the same wavelength as that in the step (i) at an incident angle as a total reflection condition from the surface of the transparent dielectric member (Ts) opposite to the surface where the fluorescent molecular layer (Fsq) exists. By irradiating
(Iii) detecting the amount of fluorescence emitted in the region (Q) as an electric field enhanced fluorescence signal (Sq); (iii) the total reflection fluorescence signal (Sp) obtained through the steps (i) and (ii) and Using the electric field enhanced fluorescence signal (Sq), the following formula (1)
R = Sq / Sp (1)
Calculating the electric field enhancement ratio R based on:
Rt, which is the electric field enhancement ratio R obtained via
(K-2) For the reference structure (Xr), which is the structure (X) and is a structure other than the target structure (Xt), incidence of the same wavelength as that of the target structure (Xt) Using light, Rr which is the electric field enhancement ratio R obtained through the above steps (i) to (iii), the following formula (2)
Sm1 = Sm0 x (Rr / Rt) (2)
Based on the above, a fluorescence signal correction method comprising converting an uncorrected fluorescence signal (Sm0), which is a fluorescence signal (Sm) before correction, into a corrected fluorescence signal (Sm1).   The transformation of the fluorescence signal (Sm) measured for the surface plasmon resonance sensor integrating the target structure (Xt) as a target structure part includes the steps (i) to (i) for the target structure part. The correction method according to claim 1, wherein the correction is performed by using the electric field enhancement ratio R obtained through (iii) as the Rt. (A) In a plasmon sensor structure having a region (Am) in which an immobilized ligand-containing layer containing a first ligand is formed on a transparent dielectric member (Tm) via a metal thin film (Mm), Preparing a surface plasmon resonance sensor by bringing a specimen into contact with a fluorescent modifying ligand comprising a second ligand and a fluorescent molecule (Dm) in the region (Am);
(B) After the step (a), the region (Am) is irradiated with incident light from the surface of the transparent dielectric member (Tm) opposite to the metal thin film (Mm). Irradiating incident light at an incident angle as a reflection condition, thereby detecting the amount of fluorescence emitted from the region (Am) as a fluorescence signal (Sm);
(C) correcting the fluorescence signal (Sm) detected in the step (b);
(D) evaluating the amount of analyte in the specimen based on the fluorescence signal (Sm) corrected in step (c);
The assay method according to claim 1, wherein the correction of the fluorescence signal (Sm) in the step (c) is performed using the correction method according to claim 1. The target structure (Xt) used in (k-1) is
The transparent dielectric member (Ts) further includes a region (As) in which an immobilized ligand-containing layer containing a first ligand is formed via a metal thin film (Msa), and the metal thin film ( Msa) and the metal thin film (Msq) are made of the same metal and have the same thickness.
The assay method according to claim 3, which is used as the plasmon sensor structure by using the region (As) as the region (Am). The correction in the step (c) is
By performing the steps (i) to (iii) for the target structure (Xt) immediately before the step (b), simultaneously with the step (b), and immediately after the step (b). The assay method according to claim 4, wherein the assay is performed using the required Rt.   The assay method according to claim 3 or 4, wherein the correction in the step (c) is performed using the Rt calculated in advance.   The assay method according to claim 5 or 6, wherein the correction in the step (c) is performed using the previously calculated Rr. The assay method according to any one of claims 3 to 7, wherein the specimen is at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva. A transparent dielectric member (Ts);
A region (P) in which a fluorescent molecular layer (Fsp) is formed on the transparent dielectric member (Ts) without using a metal thin film;
A region (Q) in which a fluorescent molecular layer (Fsq) is formed on the transparent dielectric member (Ts) via a metal thin film (Msq);
A region (As) in which an immobilized ligand-containing layer containing a first ligand is formed on the transparent dielectric member (Ts) via a metal thin film (Msa);
The fluorescent molecular layer (Fsp) and the fluorescent molecular layer (Fsq) are both composed of the same fluorescent molecule (Ds), and
A structure in which the metal thin film (Msa) and the metal thin film (Msq) are both made of the same metal and have the same thickness. A structure according to claim 9, an analyte, a fluorescent modifying ligand comprising a second ligand and a fluorescent molecule (Dm),
A surface plasmon resonance sensor in which the fluorescent modifying ligand is bound to the first ligand in the region (As) via the analyte.