The invention relates to a method of producing an inspection chip used for a specimen detection device that detects a specimen using localized plasmons and a specimen detecting method of detecting a specimen using localized plasmon.
Among known detection devices for detecting a minute specimen is one using an enhanced electric field created by localized plasmons or an enhanced electric field further intensified by localized plasmon resonance, which is a phenomenon where resonance is produced by coupling between localized plasmons and light.
In a detection device of this type, a specimen is placed on a substrate on a surface of which a number of metallic portions capable of exciting localized plasmons are disposed, with Raman scattered light of the specimen intensified by an enhanced electric field that is generated by localized plasmons. Since Raman scattered light displays a spectrum proper to each specimen under examination, the specimen can be identified or existence of a specific specimen can be determined by detecting Raman scattered light of the specimen.
Substrates that generate an enhanced electric field intensified by localized plasmon resonance include, for example, ones having configurations described in JP 2005-144569 A and JP 2007-240361 A.
JP 2005-144569 A describes a substrate on which particles each coated with a thin metallic film are disposed with a high density. To be more specific, JP 2005-144569 A describes a substrate having a two-dimensional array structure formed by reducing dielectric or semiconductor particles densely arranged on the substrate through anisotropic dry etching process and attaching a metal or a semiconductor onto the particles in a hemispherical form by vapor deposition or sputtering to keep the metallic parts of adjacent particles spaced at a given controlled distance.
JP 2007-240361 A describes a substrate having a number of minute projections formed thereon. The projections have a metallic layer disposed on the surface thereof; the metallic layer has a dielectric layer formed on the surface thereof, and the dielectric layer has secured to the surface thereof specific binding members capable of specifically binding to a substance to be measured that is contained in a test sample to form a specific binding substance.
JP 2005-195440 A describes a specimen detecting method using a microstructure comprising a substrate and first metallic particles. The substrate has micropores formed and distributed on one surface thereof. The first metallic particles each have dimensions permitting excitation of localized plasmon resonance and are disposed in the micropores of the substrate so that the head portions of the first metallic particles project above the surface of the substrate. The microstructure is placed in a solution with a substance capable of specifically binding to the specimen secured to the head portions of the first metallic particles. The solution contains dispersed therein second metallic particles having dimensions permitting excitation of localized plasmon resonance, with a substance capable of specifically binding to the specimen secured to the second metallic particles. The surface of the microstructure from which the head portions of the first metallic particles project is irradiated by light to measure the intensity of light component reflected or transmitted at said surface, thereby detecting the specimen contained in the solution based upon the measured intensity of the light.
According to the method described in JP 2005-195440 A, the specimen binds to the second metallic particles to which a substance capable of specifically binding to the specimen is secured whereas the specimen also binds to the first metallic particles to the head portions of which a substance capable of specifically binding to the specimen is secured, so that the change in plasmon resonance wavelength that varies greatly when the first metallic particles and the second metallic particles approach is detected to achieve detection of the specimen.
In the detection devices using the substrates described in JP 2005-144569 A and JP 2007-240361 A, the metallic particles or metallic projections having dimensions permitting excitation of localized plasmon resonance are allowed to adsorb specimen to detect, for example, the variation in an enhanced electric field, fluorescent light from the specimen excited by the enhanced electric field, and Raman scattered light thereby to achieve detection of the specimen.
According to such a method, however, a great enhancement cannot be achieved because only an enhanced electric field created around metallic particles that lie below the specimen can be used.
Although, according to the method described in JP 2007-240361 A, the efficiency with which the specimen binds to the metallic film may be increased by securing a substance capable of specifically adsorbing the specimen to the surface of the metallic film, a great enhancement cannot be achieved because, also in this case, only an enhanced electric field created around metallic particles that lie below the specimen can be used.
Furthermore, the need to select for each specimen a specific adsorbing substance for attachment to the metallic particles adds to the cumbersome procedure and limits the specimen to which the detection device can be used.
The method proposed in JP 2005-195440 A, which detects the variation of a plasmon resonance wavelength, requires the specific adsorption to be performed twice. Accordingly, two or more molecules of a specific adsorbing substance (referred to as “antibody” below) needs to be bound to the specimen. Thus, where the specimen is a substance having a small molecular mass, two molecules of an antibody cannot be adsorbed onto the specimen, making the detection of the specimen impossible.
Furthermore, because the method described in JP 2005-195440 A achieves detection of the variation in plasmon resonance wavelength caused as the first metallic particles and the second metallic particles come close to each other through the intermediary of the specimen, it is essential that the first metallic particles and the second metallic particles approach each other only through the intermediary of the specimen.