1. Field of the Invention
The present invention relates to a sensor unit and assay method of assay in utilizing attenuated total reflection. More particularly, the present invention relates to a sensor unit and assay method of assay in utilizing attenuated total reflection, in which a flow channel is constructed for assay with high precision.
2. Description of the Related Art
An assay apparatus for assay in utilizing attenuated total reflection is used for various kinds of studies in a biochemical field or the like, for example to study interaction of protein, DNA and various biomaterials, and to select candidate drugs by screening. Also, the technique is useful in the fields of the clinical medicine, food industries and the like.
A surface plasmon resonance (SPR) sensor is known as an assay apparatus in utilizing attenuated total reflection. A thin film/dielectric interface of a metal film is fitted on a dielectric block. Light is directed to the thin film/dielectric interface in a manner conditioned for total reflection. Surface plasmon is a term to mean the compressional wave created on the surface of the metal and included in plasmon as quantized expression of the compressional wave. Free electrons in a metal vibrate to generate the compressional wave.
In the assay apparatus, the sensing surface is positioned opposite to the interface where the metal thin film is connected with the dielectric block. The sensing surface is caused to create surface plasmon resonance. Reaction of samples is assayed by detecting the SPR on the sensing surface.
Illuminating light is applied to an interface between the thin film and the prism or a surface back to the sensing surface at an angle of incidence equal to or more than a critical angle to satisfy a condition of total reflection. Then total reflection of the illuminating light occurs. Upon the total reflection created on the metal/dielectric interface, a small component of the light passes through the metal film without reflection, and penetrates to the sensing surface. A wave of the penetrating component is called an evanescent wave. Surface plasmon resonance (SPR) is created when frequency of the evanescent wave coincides with that of the surface plasmon. In response to this, intensity of the reflected light attenuates remarkably. In the assay apparatus, the attenuation in the reflected light reflected by the metal/dielectric interface is detected, to recognize creation of the SPR on the sensing surface.
A resonance angle or an angle of incidence of light for creation of surface plasmon resonance depends upon a refractive index of a medium of transmission of evanescent waves and surface plasmon. In other words, a change in the refractive index of the medium of transmission causes a change in the resonance angle of creation of SPR. The substance or sample in contact with the sensing surface is the medium for transmitting the evanescent waves and surface plasmon. When binding, dissociation or other reaction occurs on the sensing surface between two molecules or samples, the resonance angle changes because of a change in the refractive index of the medium of transmission. The SPR assay apparatus finds the changes in the resonance angle, to assay the interaction between the molecules or samples.
An assay apparatus for assay in utilizing attenuated total reflection is used for various kinds of studies in a biochemical field or the like, for example to study interaction of protein, DNA and various biomaterials, and to select candidate drugs by screening. Also, the technique is useful in the fields of the clinical medicine, food industries and the like. A sample or biomaterial, such as protein, is handled as sample fluid for the purpose of preventing deactivation or modification due to drying. The sample fluid contains biomaterial and fluid medium, examples of which include pure water, physiological saline water, liquid buffer and the like.
JP-A 6-167443 discloses an SPR assay apparatus in which an optical system of Kretschmann configuration is used for incidence of light to the metal film. According to the Kretschmann configuration, the thin film/dielectric interface of the metal film is fitted on a prism, which condenses light and directs the light to the thin film/dielectric interface in a manner conditioned for total reflection. A sensing surface is overlaid inside the flow channel, for immobilizing the sample. Ligand fluid is introduced to the flow channel for immobilizing the ligand on the sensing surface. After this, analyte fluid is introduced for contact of the analyte and the ligand, to assay the interaction between those.
In JP-A 6-167443, a body of the assay apparatus has a prism and an assay stage loadable with a flow cell block having a flow channel. A chip type of sensor unit is set on the assay stage, the chip type having a transparent glass substrate of a dielectric material, and a thin film overlaid thereon. The chip type of the sensor unit is removable from the body of the apparatus, to set the flow channel at the sensing surface and to fit a surface of the thin film on the prism as an interface. Prior to the assay, there is a process of immobilizing a ligand on the thin film in the sensor unit of the chip type. In JP-A 6-167443, the sensor unit of the chip type is kept located on the assay stage also in the sample immobilizing flow process.
In the flow cell block is formed a flow cell recess, which is set opposite to the sensing surface, and causes a flow of fluid on the sensing surface by contacting ligand or analyte in the fluid with the sensing surface. A confronting portion or retraction portion of the flow channel is constituted by the flow cell recess. In relation to a chip type of sensor unit, the sensing surface externally appears. When the sensor unit is loaded on an assay stage, the retraction portion is hermetically closed by closing an open portion of the flow channel with the sensing surface. Then delivery of the sample fluid to the sensing surface is enabled.
Then the ligand is introduced to the flow channel for the sample immobilizing flow. After this, cleaning liquid is introduced to the flow channel for washing. Before introducing the cleaning liquid, fluid of the ligand has been filled in the flow channel. The cleaning liquid is forcibly delivered despite the ligand. The fluid of the ligand is pushed by the cleaning liquid and flows out of the flow channel. This is substitution of fluids in the flow channel by changing over the content.
After washing, the flow channel is supplied with the buffer liquid and then the analyte fluid, for conducting an assay. At a lapse of a predetermined time, the buffer liquid is introduced again to complete the assay. The buffer liquid is introduced for the purpose of detecting a base line of the output of the SPR. Acquisition of the output is started when the flow channel is filled with the buffer liquid, and is ended upon draining the analyte fluid by flowing again of the buffer liquid. Then the interaction of the analyte and ligand from the association until the dissociation can be detected.
To raise the sample amount of an immobilized ligand to the sensing surface, it is effective to increase the sample amount of an introduced ligand to the flow channel. A volume of the flow channel must be greater by enlarging an area of a section of the flow channel at the retraction portion before the sample amount of the introduced ligand can be greater. Assuming that a channel width of the flow channel is constant, the area of the section is determined by the height of depth of the flow channel from the sensing surface to the upper surface of the flow channel. Consequently, a greater height or depth of the flow channel is preferable.
For the purpose of detecting a reaction speed of samples by the surface plasmon resonance system, the height of the flow channel should be small because of short time of reaction of binding or dissociation of the ligand with the analyte. In general, a fluid flows in the flow channel in such a manner that a gradient in the speed occurs from the center of the flow channel in its section toward the inner surface of the flow channel due to viscosity of the fluid. The speed is lower according to closeness to the inner surface. In operation of substitution of the buffer for the analyte, there is a delay in the sensing surface for the substitution in comparison with the center of the flow channel. A small portion of the analyte remains on the sensing surface. Note that a quantitative level of the residual analyte is represented by a parameter of a ratio of substitution of the flow channel. Note that the ratio of substitution is such of an amount of substituting fluid to the preceding fluid amount of the flow channel. The ratio of substitution rises if the introduction of fluid is repeated for a number of times, or if considerable time is taken for waiting. However, the ratio of substitution of a high level at a short time by introduction at one time is required for accurately measuring the reaction speed. If a height of the flow channel is lowered, a distance from the center of the flow channel to the sensing surface is shortened. The ratio of substitution at a high ratio of substitution in a short time can be obtained by reducing a gradient in the speed due to the fluid viscosity.
The channel height of the flow channel should be conditioned differently between the processes of the sample immobilizing flow and assay. No known technique suggests an optimization of the channel height determined suitably for both of the sample immobilizing flow and assay.