JP3716301B2 - Method and apparatus for measuring biomolecule adsorption layer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention is used in many areas where the adsorption of biomolecules such as proteins is an issue, and is a preventive technique in medical / medicine (including dentistry), an inspection technique area, a food industry, and an environment-related inspection. It is about technology.
[0002]
[Prior art]
The phenomenon that biomolecules such as proteins are strongly adsorbed on the surface of various materials is well known, but the wettability, hydrophobicity / hydrophilicity, and interfacial tension of these biological materials that are easily changed in structure are quasi-nondestructive. The method for measuring the temperature is first disclosed in Japanese Patent No. 27852502 (name: method for measuring dynamic interfacial tension of biomolecule adsorbing layer and apparatus used therefor). On the other hand, various measurement methods have been reported for the amount of biomolecules adsorbed and the process thereof, and ellipsometry, reflectometry, and the like are known as optical methods.
[0003]
[Problems to be solved by the invention]
In the already disclosed measurement methods such as hydrophobicity / hydrophilicity, wettability, interfacial tension, etc. exhibited by the adsorption layer surface such as protein, information can be obtained only as an overall characteristic value of the biomolecule adsorption layer surface. It was not possible to identify whether it was due to the amount of adsorption or due to a change in molecular morphology due to interfacial adsorption.
[0004]
[Means for Solving the Problems]
The present invention can measure the adsorption amount simultaneously by measuring the time change of the adsorption phenomenon, and can clearly distinguish whether the measured hydrophobicity / hydrophilicity indicated by the adsorption layer surface is derived from the adsorption amount or the molecular shape change. Provide a method. As a result, the characteristics of each molecule can be associated with the measured values, and more accurate information than the molecular level can be obtained. Specifically, the invention shown in the above-mentioned Japanese Patent No. 27852502 is combined with an invention for optically observing the amount of biomolecule adsorbed and its process. In other words, the sample plate is immersed in an aqueous solution containing biomolecules, and the time-dependent change of the biomolecule adsorbed on the sample plate due to the change in the polarization state of the reflected light is measured (reflectometry and ellipsometry) simultaneously. After a predetermined time later, a part of the sample plate is instantaneously exposed to the gas phase, and the laser beam is used to change the reflection intensity of the laser beam between two points of the sample or the change of optical signals such as interference fringes. By measuring, the falling speed of the liquid film falling on the surface of the biomolecule adsorption layer is acquired, and the amount of biomolecule adsorption and the wettability, hydrophobicity / hydrophilicity and surface tension of the adsorption layer surface are measured simultaneously. is there.
[0005]
Embodiment 1
FIG. 1 schematically shows an outline of the present invention. In FIG. 1, 1 is a sample plate, 2 is a cell containing a biomolecule solution, 3 is a laser beam, 4 is a laser beam used for polarization analysis, 5 is a biomolecule adsorption layer, and 6 is a liquid thin film.
First, the sample plate 1 is immersed in the cell 2 containing the biomolecule solution, irradiated with the laser beams 3 and 4, and the change in the thickness of the biomolecule adsorption layer is measured. After confirming that the thickness of the adsorption layer has saturated to a certain value, the cell is dropped. Alternatively, the sample plate is pulled out rapidly. Here, two laser beams are irradiated to different points on the sample, and the liquid that falls on the surface of the biomolecule adsorbing layer is correlated with the temporal change of the optical signal such as the reflection intensity of the reflected light or interference fringes. By measuring the falling speed of the membrane, the amount of biomolecule adsorbed and the wettability, hydrophobicity / hydrophilicity, and surface tension of the surface of the adsorption layer are measured simultaneously.
[0006]
Embodiment 2
FIG. 2 shows a conceptual diagram of a measuring apparatus using two light beams of laser light. 7 and 8 are laser light sources, 9 is a depolarizing plate or rotating polarizer, 10 is an aqueous solution cell containing a biomolecule, 11 is a sample plate, 13 is a beam splitter, 12, 14 and 15 are photoelectric conversion elements, and 16 is a voltmeter. (2 channel multimeter), 17 is a computer having a data capturing device.
[0007]
In the measurement operation, first, the sample plate is immersed in a cell containing the biomolecule solution for an arbitrary time. For this purpose, the cell is raised so that the sample plate hung from above is immersed by about half. The amount of adsorbed molecules formed on the sample plate is measured with the lower laser light source and its detection system. For this purpose, the incident light is adjusted so that the light intensity component parallel to the laser light incident surface and the perpendicular component are substantially equal, and the reflected light from the sample surface is divided by a beam splitter that divides the light into two orthogonal components, The intensity of each reflected light is converted into an electrical signal by an optical detector. The intensity ratio of each electric signal and the change over time in the intensity sum are measured using a voltmeter (2-channel multimeter). The time change of the adsorption amount is calculated from the time change of the intensity ratio. In order to measure the hydrophobicity / hydrophilicity of a sample plate having an adsorption amount at a certain time, the sample plate is instantaneously exposed to the gas phase at that time, and the movement of the liquid thin film generated from that moment is observed. For this, the solution cell is instantaneously dropped to a specific position. In order to measure the falling speed of the liquid film, the second laser light source in the upper part and the first laser light in the lower part are used. The intensity of the reflected light reflected from the sample having the liquid thin film is detected by an optical detector, and the time variation of the reflected intensity of the first laser beam (the sum of the converted voltage intensity) and the second reflected light intensity is detected. And the relative time interval is measured for the deviation of the change point of the signal intensity. If the distance between the two points on which the laser beam hits and the time required for the fall can be obtained, the liquid thin film can be formed according to the already reported theory (J. Colloid & Interface Sci., 233 (2001) 136-141). After calculating the falling speed, it is possible to calculate the wettability (contact angle) of the adsorption layer surface, and also the hydrophobicity / hydrophilicity and surface tension.
[0008]
Embodiment 3
FIG. 3 is a conceptual diagram of an optical interferometer used for liquid film falling speed measurement. 18 and 19 are laser light sources, 20 is a depolarizing plate or rotating polarizer, 21 and 27 are reflecting mirrors, 22 is a spatial filter, 23 and 30 are beam splitters, 24 is a sample plate, 25 is a beam splitter, 26 and 28 Is a photoelectric conversion element, 29 is a voltmeter (2-channel multimeter), 31 is a lens system, 32 is a video camera, 33 is a monitor television and recording device, and 34 is a computer.
[0009]
In the measurement operation, first, the sample plate is immersed in a cell containing the biomolecule solution for an arbitrary time. For this purpose, the cell is raised so that the sample plate hung from above is immersed by about half. The adsorbed molecular weight formed on the sample plate is measured with the lower laser light source and its detection system. For this purpose, the incident light is adjusted so that the light intensity component parallel to the laser light incident surface and the perpendicular component are substantially equal, and the reflected light from the sample surface is divided by a beam splitter that divides the light into two orthogonal components, The intensity of each reflected light is converted into an electrical signal by an optical detector. The change with time of the intensity ratio of each electric signal is measured using a voltmeter (2-channel multimeter). The time change of the adsorption amount is calculated from the time change of the intensity ratio. In order to measure the wettability, hydrophobicity, hydrophilicity, etc. of a sample plate with an adsorption amount at a certain point in time, the sample plate is exposed to the gas phase instantaneously at that point, and the movement of the liquid thin film generated from that moment is observed. To do. For this purpose, the solution cell is instantaneously dropped to a predetermined position. In order to measure the falling speed of the liquid film, the light beam from the laser light source 19 is split into two beams by a beam splitter through a spatial filter (including a collimator lens), one of which is applied to the sample, By using a reference beam, mixing with another beam splitter, capturing the interference fringes due to the shearing mode as a video image, and measuring the moving speed of the interference fringes on the screen, the previously reported theory (J. Colloid & Interface Sci., 233 (2001) 136-141), it is possible to calculate the wettability (contact angle) of the adsorption layer surface, as well as hydrophobicity / hydrophilicity and surface tension after calculating the falling speed of the liquid thin film.
[0010]
【Example】
An example of an experiment conducted using albumin, which is a typical serum protein, using the measurement apparatus of FIG. First, prepare a measurement cell filled with distilled water. In this cell, a platinum plate is lowered from above the air / water surface in advance and immersed in the middle of the plate. Laser light is irradiated to the part immersed in the aqueous solution. At this time, the polarizer and the depolarizing plate are adjusted so that the component (Ip0) parallel to the incident surface of the light intensity is equal to the vertical component (Is0). The reflected light is divided into each component (Ip, Is) by a beam splitter, each is converted into a voltage by a photoelectric conversion element having a linear amplifier, and the time change of the voltage ratio is converted by a computer with a 2-channel voltmeter. Record it. This voltage ratio corresponds to Ip / Is. At some point, a concentrated aqueous solution of albumin is added to the cell to form an aqueous albumin solution of a predetermined concentration. The aqueous solution in the cell is always stirred with a magnetic stirrer. The subsequent Ip / Is is continuously measured and recorded by a computer.
[0011]
The data of FIG. 4 was observed in this way, and the final concentration of albumin in this case is 2.2 × 10 −2 mg / ml. At the final point of observation, the solution cell is instantaneously dropped to about 10 mm below, the platinum sample plate is exposed to the gas phase, and from this moment, the movement of the liquid thin film generated on the protein layer adsorbed on the plate is observed. . The reflected light intensity reflected from the sample plane of the first laser beam and the second laser beam 4 mm above it is detected by an optical detector, and the reflected intensity of the first laser beam (the intensity of the converted voltage) is detected. Sum) and the reflected light intensity of the second laser are measured over time and stored in a computer. The relative time interval (fall time) is measured for the deviation of the signal intensity change point (FIG. 5).
[0012]
FIG. 6 shows the relationship between the drop time and the concentration in the solution for the two types of proteins (albumin and lysozyme) thus measured.
[0013]
【The invention's effect】
According to the present invention, how the adsorption of biomolecules such as proteins progresses over time, and the wettability (hydrophobic / hydrophilicity), which is an important characteristic of the resulting adsorption film, is simultaneously measured in the same sample. It becomes possible to observe.
[Brief description of the drawings]
[Fig. 1] Conceptual diagram of the measurement method [Fig. 2] Schematic diagram of the apparatus using the two-beam reflection method of the laser [Fig. 3] Schematic diagram of the apparatus using the optical interferometry [Fig. 4] Polarization of the light reflection intensity Change in component ratio over time (adsorption amount measurement)
[Figure 5] Temporal change in reflected light intensity of two laser beams (measurement of hydrophobicity and hydrophilicity)
[Fig. 6] Relationship between drop time of liquid thin film and protein concentration in solution [Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Sample plate 2 Cell containing biomolecule solution 3 Laser beam 4 Laser beam 5 used for polarization analysis Biomolecule adsorption layer 6 Liquid thin film 7, 8, 18, 19 Laser light source 9, 20 Depolarization plate or rotating polarizer 10 Aqueous solution cells 11 and 24 containing biomolecules Sample plates 13, 23, 25, 30 Beam splitters 12, 14, 15, 26, 28 Photoelectric conversion elements 16, 29 Voltmeter (2-channel multimeter)
17, 34 Computers 21 and 27 having a data capturing device Reflector 22 Spatial filter 31 Lens system 32 Video camera 33 Monitor TV and recording device

Claims (3)

In the method of measuring a biomolecule adsorption layer, a sample plate is immersed in an aqueous solution containing biomolecules, the amount of biomolecule adsorbed on the sample plate is measured by optical analysis, and then a part of the sample plate is instantly removed. By exposing to the phase, measuring the optical signal between two points of the sample, and measuring the falling speed of the liquid film falling on the surface of the biomolecule adsorption layer, the amount of the biomolecule adsorption and the surface of the biomolecule adsorption layer A method for measuring a biomolecule adsorbed layer, characterized by measuring characteristics. In an apparatus for measuring a biomolecule adsorption layer, a cell in which an aqueous solution containing a biomolecule is placed, a sample plate immersed in the cell, a first laser light source, and a polarization plane of laser light emitted from the first laser light source An optical element that adjusts the reflected light, a beam splitter that divides the reflected light reflected by the sample plate, an optical detector that converts the intensity of each of the divided reflected light into an electrical signal, and an electrical signal converted by the optical detector. A voltmeter for measuring a signal, a second laser light source for observing the movement of the liquid thin film generated from the moment when the sample plate is exposed to the gas phase, and a sample plate in which the laser light emitted from the second light source has a liquid thin film A raw detector comprising: an optical detector for detecting the intensity of reflected light reflected from the light; and a signal processing device for processing the intensity of reflection of the first laser light and the intensity of the second reflected light. Device for measuring properties of molecular adsorption and biomolecules adsorbed layer surface. In an apparatus for measuring a biomolecule adsorption layer, a cell in which an aqueous solution containing a biomolecule is placed, a sample plate immersed in the cell, a first laser light source, and a polarization plane of laser light emitted from the first laser light source An optical element that adjusts the reflected light, a beam splitter that divides the reflected light reflected by the sample plate, an optical detector that converts the intensity of each of the divided reflected light into an electrical signal, and an electrical signal converted by the optical detector. A voltmeter for measuring a signal, a second laser light source for observing the movement of the liquid thin film generated from the moment when the sample plate is exposed to the gas phase, a beam splitter for splitting the laser light emitted from the second laser light source, and splitting One of the laser beams transmitted through or reflected by the liquid thin film is combined with the other of the divided laser beams to form an interference fringe and sampling. Biomolecule adsorption characterized by comprising the image analyzer to observe the falling speed of the liquid film from the interference fringes and apparatus for measuring the characteristics of the biomolecule adsorption layer surface.

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