The present invention relates to a measuring method, a measuring system and a measuring program of intermolecular interactions, in particular, to a measuring method of intermolecular interactions being capable of generating a value as an index indicating a degree of progress in the intermolecular interactions of biomolecules and organic polymers etc., a measurement system for the method, and a program that causes a computer to execute a process for such measurement.
Labeling by radioactive or fluorescent substances has generally been employed for conventional measurements of bonds or links such as intermolecular interactions between biomolecules, e.g., antigen-antibody reactions and intermolecular interactions between organic polymers. This labeling, however, is time-consuming, and especially labeling of proteins requires complicated procedures and causes denaturation of the proteins in some cases. To address this, recently, RIfS (Reflectometric interference spectroscopy) making use of a change in interference color of an optical thin film has been proposed and already put into practical use as simplified means for directly detecting bonds or links between biomolecules and organic polymers without labeling. The principle of the RIfS is disclosed in Patent Document 1 and Non-Patent Document 1, for example.
RIfS will now be briefly described. RIfS uses a substrate 102 provided with an optical thin film 104 as illustrated in
Ligand molecules 120 are provided on the optical thin film 104 in order to detect intermolecular interactions, as illustrated in
In a temporal transition of the variation in the bottom wavelengths, as shown in
Unfortunately, the study of the present inventors demonstrates that observing a temporal transition of the variation in bottom wavelengths, in principle, has a limitation of the accuracy in measurement and thus the method is unsatisfactory for more accurate determination of the degree of progress in intermolecular interactions. This is for the following reasons.
Bonds or links such as intermolecular interactions between biomolecules, e.g., antigen-antibody reactions, and intermolecular interactions between organic polymers generally proceed evenly, albeit microscopic repetitions of binding and leaving. That is, intermolecular interactions proceed over time, as illustrated in
However, actual data output from a detector for determining the spectral intensity of the reflected light has repeated minute fluctuations, for example, like reflectance data 151 shown in
Furthermore, the method of tracing a change in the difference Δλ in bottom wavelengths only traces a shift of a single point on the graph of the spectral characteristics of the reflected light and does not detect a general shift of the graph, which limits accurate detection of variation.
On top of that, the above-described calculation of the approximate curve may need a high-performance arithmetic unit or a complicated arithmetic, or the speed of arithmetic may be insufficient in a high-rate intermolecular interaction.
An object of the present invention, which has been made in view of such a conventional problem, is to provide a measuring method of intermolecular interactions, the method being capable of accurately and efficiently generating a value as an index indicating the degree of progress in intermolecular interactions of biomolecules and organic polymers, a measuring system for the method, and a program for causing a computer to execute a process for such measurement.
The invention described in claim 1 for solving the above problems is a measuring method of an intermolecular interaction for optically measuring an intermolecular interaction between a plurality of substances on a thin film, the method comprising detecting intensity at a first wavelength of reflected light or transmitted light before start of the intermolecular interaction with a detection unit which detects the reflected light from the thin film or the transmitted light transmitted through the thin film; detecting intensity at the first wavelength of the reflected light or the transmitted light after the start of the intermolecular interaction with the detection unit; and calculating a difference in intensity of the reflected light or the transmitted light at the first wavelength between before and after the start of the intermolecular interaction.
The invention described in claim 2 is the measuring method of an intermolecular interaction of claim 1, wherein the intensity of the reflected light is spectral intensity of the reflected light or a value containing the spectral intensity of the reflected light as a variable, or the intensity of the transmitted light is spectral intensity of the transmitted light or a value containing the spectral intensity of the transmitted light as a variable.
The invention described in claim 3 is the measuring method of an intermolecular interaction of claim 1 or 2, further comprising calculating a difference in intensity of the reflected light or the transmitted light at a second wavelength which is different from the first wavelength.
The invention described in claim 4 is the measuring method of an intermolecular interaction of claim 1 or 2, further comprising measuring a plurality of differences in intensity of the reflected light or the transmitted light at different wavelengths within a predetermined wavelength range and calculating a sum of the differences or a sum of absolute values of the differences.
The invention described in claim 5 is a measuring system of an intermolecular interaction for optically measuring an intermolecular interaction between a plurality of substances on a thin film, the system comprising a detection unit which detects reflected light from the thin film or transmitted light transmitted through the thin film, the detection unit detecting intensity at a first wavelength of the reflected light or the transmitted light before start of the intermolecular interaction, and detecting intensity at the first wavelength of the reflected light or the transmitted light after the start of the intermolecular interaction; and an arithmetic unit which calculates a difference in intensity of the reflected light or the transmitted light at the first wavelength between before and after the start of the intermolecular interaction.
The invention described in claim 6 is the measuring system of an intermolecular interaction of claim 5, wherein the intensity of the reflected light is spectral intensity of the reflected light or a value containing the spectral intensity of the reflected light as a variable, or the intensity of the transmitted light is spectral intensity of the transmitted light or a value containing the spectral intensity of the transmitted light as a variable.
The invention described in claim 7 is the measuring system of an intermolecular interaction of claim 5 or 6, wherein the arithmetic unit calculates a difference in intensity of the reflected light or the transmitted light at a second wavelength which is different from the first wavelength.
The invention described in claim 8 is the measuring system of an intermolecular interaction of claim 5 or 6, wherein the arithmetic unit measures a plurality of differences in intensity of the reflected light or the transmitted light at different wavelengths within a predetermined wavelength range to calculate a sum of the differences or a sum of absolute values of the differences.
The invention described in claim 9 is a program for causing a computer to execute a process for optically measuring an intermolecular interaction between substances on a thin film, the program causing the computer to execute a process of detecting intensity at a first wavelength of reflected light or transmitted light before start of the intermolecular interaction with a detection unit for detecting the reflected light from the thin film or the transmitted light transmitted through the thin film; a process of detecting intensity at the first wavelength of the reflected light or the transmitted light after the start of the intermolecular interaction with the detection unit; and a process of calculating a difference in intensity of the reflected light or the transmitted light at the first wavelength between before and after the start of the intermolecular interaction.
The invention described in claim 10 is the program of claim 9, wherein the intensity of the reflected light is spectral intensity of the reflected light or a value containing the spectral intensity of the reflected light as a variable, or the intensity of the transmitted light is spectral intensity of the transmitted light or a value containing the spectral intensity of the transmitted light as a variable.
The invention described in claim 11 is the program of claim 9 or 10, further causing the computer to execute a process of calculating a difference in intensity of the reflected light or the transmitted light at a second wavelength which is different from the first wavelength.
The invention described in claim 12 is the program of claim 9 or 10, further causing the computer to execute a process of measuring a plurality of differences in intensity of the reflected light or the transmitted light at different wavelengths within a predetermined wavelength range to calculate a sum of the differences or a sum of absolute values of the differences.
According to the present invention, a variation is calculated between the intensity of the reflected light or the transmitted light at a first wavelength before the start of intermolecular interactions and the intensity of the reflected light or the transmitted light at the first wavelength after the start of the intermolecular interactions, so that an exact value as an index indicating the degree of progress in the intermolecular interactions can be efficiently determined by using the difference as an index indicating the degree of progress in the intermolecular interactions.
An embodiment of the present invention will now be described with reference to the accompanying drawings; however, the embodiment shall not limit the scope of the present invention.
With reference to
The arithmetic and control unit 50 in the measuring system 1 serves as a control unit of the measuring mechanisms such as the light source and the spectrometer incorporated in the measuring unit 80, an arithmetic unit for detected information, and an input/output unit (interface) for outputting and inputting a control instruction and the detected information.
The measuring unit 80 includes a lower housing 82 and an upper housing 81 pivotally supported by the lower housing 82. The lower housing 82 is provided with a table 83 for holding the measuring member 10. A connection portion 84 is provided on the inner surface of the upper housing 81. The connection portion 84 has an injection orifice 85 and a suction orifice 87, which are to be connected to the measuring member 10 for providing the flow of a sample, and a detection window 86. The upper housing 81 includes a white-light source 20, a spectrometer 30, and optical fibers 40 and 41 as described later. In the upper housing 81, light is emitted or received through the detection window 86. In the preparation for measurement, the upper housing 81 is pivotally moved upwardly to open the table 83 of the lower housing 82 and then the measuring member 10 is placed on the table 83. The upper housing 81 is then moved downwardly to close the unit, and thereby the injection orifice 85 and the suction orifice 87 are connected to the measuring member 10 and the detection window 86 faces the measuring member 10. The preparation for measurement is thereby completed.
As illustrated in
The flow cell 14 is a transparent member made of silicone rubber. The flow cell 14 has a channel 14a. Close contact of the flow cell 14 to the sensor chip 12 defines a closed fluid passage 14b, as illustrated in
The flow cell 14 is replaceable to the sensor chip 12 in the measuring member 10, namely, the flow cell 14 can be disposable. The sensor chip 12 may be surface-modified, for example, with a silane coupling agent to facilitate replacement of the flow cell 14.
As described above, after the measuring member 10 is placed, the upper housing 81 is pivotally moved downwardly to close the unit, and thereby the detection window 86 faces the flow cell 14 and the optical fiber 40 lies over the closed fluid passage 14b in the flow cell 14, as illustrated in FIG. 3. One end of the optical fiber 40 is connected to the white-light source 20. The white-light source 20 is, for example, a halogen light source. The other end of the optical fiber 40 faces the detection window 86. The optical fiber 41 is connected to the spectrometer 30 at one end, and faces the detection window 86 at the other end. When the white-light source 20 is turned on, the white light enters the closed fluid passage 14b via the optical fiber 40 and the reflected light is detected by the spectrometer 30 via the optical fiber 41. The white-light source 20 and the spectrometer 30 are connected to the arithmetic and control unit 50, which controls the operations of these modules. The white-light source 20, the spectrometer 30, and the optical fibers 40 and 41 etc. configure a detection unit for detecting the intensity of the light reflected by the optical thin film where intermolecular interactions proceed. Note that the transmitted light may also be measured instead of the reflected light measured in the present embodiment. In the measurement of the transmitted light, the optical fiber for guiding the light from the light source and the optical fiber connected to the spectrometer are opposite to each other across the measuring member.
The arithmetic and control unit 50 receives data indicating the spectral characteristics of the reflected light through an interface at a predetermined timing associated with the control of the detection operation in response to the execution of a program stored in a storage device described later. The arithmetic and control unit 50 also serves as an arithmetic unit for calculating a variety of values, described later, on the basis of the received data.
The operation of the measuring system 1 and a measuring method will now be described.
A sample solution 60 containing an analyte 62 is run from the inlet 14c through the closed fluid passage 14b to the outlet 14d, as illustrated in
The arithmetic and control unit 50 turns on the white-light source 20 before the flow of the sample solution 60 into the measuring member 10 and receives, from the spectrometer 30, spectral characteristics data that includes data indicating the intensity of the reflected light from the optical thin film (the SiN film 12b) before the start of intermolecular interactions.
The arithmetic and control unit 50 maintains the lighting of the white-light source 20 also during the flow of the sample solution 60 through the closed fluid passage 14b. White light travels through the flow cell 14 and reaches the sensor chip 12, and the reflected light is detected by the spectrometer 30. Information on the detected intensity of the reflected light detected by the spectrometer 30 is transmitted to the arithmetic and control unit 50.
As shown in
The arithmetic and control unit 50, which receives the data on the intensity of the reflected light after the start of the intermolecular interactions, calculates a difference between the intensity of the reflected light before the start of the intermolecular interactions and the intensity during or after the progress. The arithmetic and control unit 50 then outputs the calculated value as an index indicating the degree of progress in the intermolecular interactions, and the value is displayed on the display 91 or stored in a storage medium with the reader/writer 505.
The intensity of the reflected light for use in the arithmetic is, for example, the spectral intensity of the reflected light, and a variation in spectral intensity of the reflected light between before and after the start of intermolecular interactions at a predetermined wavelength is defined as ΔI. As illustrated in
The intensity of the reflected light used in the arithmetic is the spectral reflectance of the reflected light, for example, and the variation in spectral reflectance of the reflected light between before and after the start of intermolecular interactions at a predetermined wavelength is defined as ΔR. As illustrated in
The principle of measuring the degree of progress in intermolecular interactions in the present embodiment will now be described. With reference to
An interference film is composed of the substance I, the substance II, and the optical thin film III. The non-uniform layer consisting of the substance I and the substance II is now regarded as an effective uniform layer as shown in
While the intermolecular interactions are proceeding from a state A in
R(λ,α)=(1−α)A(λ)+αB(λ) Expression (1)
where A(λ) is the reflectance curve in the state A, B(λ) is the reflectance curve in the state B, and α is the degree of progress in the intermolecular interactions.
For example, at a degree of progress α of 60%, the value R(λ1, 0.6) of the reflectance at a wavelength λ1 can be calculated from Expression (1): 0.4×A(λ1)+0.6B(λ1).
In the graph of the spectral reflectance, the linearity of the variation Δλ in the bottom wavelengths versus the degree of progress α is not necessarily ensured in this case; however, the linearity of the difference ΔR in the reflectance at a predetermined wavelength (e.g., λ1) versus the degree of progress α is ensured as is apparent from the results of a simulation described later. It is noted that ΔR may be measured at any wavelength at which reflectance varies depending on the degree of progress.
Since the spectral reflectance R and the spectral intensity I have common variables, the same theory holds true even if the spectral reflectance R is replaced with the spectral intensity I.
In this manner, ΔI or ΔR is used as an index indicating the degree of progress in intermolecular interactions. ΔR is a value obtained by dividing the spectral intensity of a white-light source by the spectral intensity of the reflected light, and the spectral intensity of the white-light source is constant based on the device. Thus, the variation characteristics of ΔI and ΔR are the same, and either of them can be used as an index indicating the degree of progress in intermolecular interactions. Alternatively, a value containing the variable ΔI, for example, ΔT=1−ΔR may be used as an index indicating the degree of progress in intermolecular interactions.
ΔI, ΔR, and ΔT each linearly vary with the degree of progress α in intermolecular interactions, as seen from
Furthermore, each of ΔI, ΔR, and ΔT can be calculated without obtaining an approximate curve used for determining the bottom position, and thus the values can be easily calculated. Since these values reflect general shifts of the spectral characteristics of the reflected light, variations can be accurately detected.
ΔI, ΔR, or ΔT may be calculated at only one predetermined wavelength or two or more different wavelengths as seen from
Alternatively, the sum of ΔIs, ΔRs, or ΔTs measured at a plurality of wavelengths within a predetermined wavelength range or the sum of the absolute values of ΔIs, ΔRs, or ΔTs may be calculated. For example, as illustrated in
Such a larger number of values acquired from multiple wavelengths can lead to higher accuracy of measurement. Furthermore, a general shift of the curve of spectral characteristics can be correctly detected, which also results in noise canceling. Consequently, a variation in intermolecular interactions can be advantageously measured with higher accuracy.
In the calculation of values at a single predetermined wavelength, applicability of a monochromatic-light source eliminates the need for an expensive spectrometer that detects spectral intensity. Furthermore, availability of a simply configured monochromatic-light source can lower the costs of the measuring system.
The arithmetic and control unit 50 turns on the white-light source 20 to start detection by the spectrometer 30 and acquires, from the spectrometer 30, data on the spectral characteristics of the reflected light before the start of intermolecular interactions (step S1).
The arithmetic and control unit 50 then determines wavelength for the measurement of the difference in the spectral intensity of the reflected light (measurement wavelength) on the basis of the acquired data on the spectral characteristics of the reflected light (step S2). For example, wavelength corresponding to a range with a steep incline on a spectrum curve, such as wavelength between two adjacent extreme values, is selected as the measurement wavelength such that the progree of intermolecular interactions causes a great variation in the spectral intensity. If measurement wavelength is determined in advance, step S2 is unnecessary. Alternatively, after acquisition of data on the spectral characteristics after the start of intermolecular interactions, the measurement wavelength may be determined on the basis of spectral characteristics before and after the start of the intermolecular interactions.
The arithmetic and control unit 50 then controls the solution feeder 35 for the sample solution 60 to provide the flow of the sample solution 60 in the closed fluid passage 14b and acquires, from the spectrometer 30, data on the intensity of the reflected light during and/or after the progress of intermolecular interactions (step S3).
The arithmetic and control unit 50, which acquires the data on the intensity of the reflected light after the start of the intermolecular interactions, calculates a difference between the intensity of the reflected light before the start of the intermolecular interactions and the acquired intensity of the reflected light during or after the progress (step S4). The arithmetic and control unit 50 then outputs the calculated value as an index indicating the degree of progress in the intermolecular interactions, and the value is displayed on the display 91 or stored in the storage medium (step S5).
In place of or independently of step S4, the arithmetic and control unit 50 may calculate the sum of differences within a predetermined wavelength range or the sum of absolute values of the differences to output the resultant value.
Note that the program can be updated as appropriate via the communication device 504 connected through, for example, a LAN to public lines such as the Internet.
Following is the results of the simulation intended to support that the measurement of a difference in intensity of the reflected light or transmitted light between before and after the start of intermolecular interactions at a predetermined wavelength results in the generation of an exact index indicating the degree of progress in the intermolecular interactions.
The graphs in
With reference to
The present invention can be applied to measurement of the degree of progress in intermolecular interactions of biomolecules and organic polymers, etc.
Number | Date | Country | Kind |
---|---|---|---|
2010-279000 | Dec 2010 | JP | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
---|---|---|---|---|
PCT/JP2011/078123 | 12/6/2011 | WO | 00 | 6/17/2013 |