JPS62129742A - Photoacoustic measurement - Google Patents

Abstract

PURPOSE:To achieve a higher measurement, by a method wherein the distance between a gel plate surface and a cell window is set at a specified value and the gel plate surface with an electrophoresis going on is irradiated with an incident light in a specified range of frequency to measure a photoacoustic signal. CONSTITUTION:A microphone 2 in a cell 1 is stuck on the cell 1 to be communicated through a sample chamber 3 and a waveguide 4. A cell window 5 made of a hard glass adheres to the cell 1 with an epoxy resin. After a gel plate is housed in the cell 1, a cell lid 8 with a rubber packing is inserted and pressed fit on the cell 1. The distance between the gel plate surface and the cell window 5 is set at 0.7-1.0mm to irradiate the gel plate with an electrophoresis going on with an incident light at the frequency of 20-80Hz of a light source consisting of a Ga-Al-As based light emitting diode. Thus, a photoacoustic measurement is done at a high accuracy.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a photoacoustic measurement method, and is particularly applicable to electrophoresis or a plate using a light emitting diode (abbreviated as LED hereinafter) as a light source. The present invention relates to a photoacoustic measurement method using a photoacoustic measurement device.

(Prior art) Electrophoresis is a phenomenon in which when a substance with a surface charge is energized in the coexistence of an appropriate electrolyte, it moves to a pole opposite to the surface charge. Electrophoresis analysis is an analytical method that applies this phenomenon. There is a law. Therefore, all charged substances are subject to this analysis method, and it is also applied to the analysis of substances related to biological phenomena such as proteins.

A typical example of electrophoretic analysis is 5DS (sodium dodecyl sulfate)-2 lyacrylamide electrophoresis. This method is based on the principle that when tannin and SDS, an anionic surfactant, are combined and electrophoresed in an acrylamide picket, the tannin will be separated according to its size, that is, its molecular weight. It is based on

Using a protein of known molecular weight as a standard, the molecular weight of an unknown analyte can be determined from its mobility.

On the other hand, photoacoustic analysis is an analysis method that utilizes heat or strain generated during the process of a sample absorbing light and being deactivated without radiation. This can also be applied to opaque and light-scattering solid samples such as electrophoresed del plates.

However, when measuring using a micro-capacity cell, the preparation of a calibration curve is complicated because the target portion of the cell plate must be cut out and then placed in the cell for measurement. In contrast, open-end photoacoustic cells have been developed, but they are not as sensitive as closed cells in terms of measurement sensitivity.

The present inventors have developed an optical sweeping type photoacoustic analyzer that can directly quantify the stationary phase from electrophoresed glue plates without scraping or cutting them, and a large-sized glue plate (for example, 26 x 76 mM) that can be stored in this device. developed. In this method, intermittent light guided by an optical fiber is irradiated onto a gel plate from above a photoacoustic cell, and the amount is quantified while sweeping xy. As a result, the samples on the same dull plate were fixed in the same state, making it possible to perform repeated measurements and easily create a calibration curve.

In addition, in order to increase the photoacoustic intensity, the light source for photoacoustic measurements usually uses a light intensity sensor. Conventionally, xenon arc lamps, halogen lamps, or lasers are used, but the equipment is It had the disadvantage of being large and expensive. However, the present inventors developed an improved type of device that uses a light emitting diode (hereinafter abbreviated as LED) as a light source, thereby making the device small and capable of turning. I was able to do K.

With LEDs, it is easy to obtain intermittent light through power modulation, and it is also easy to change the intermittent frequency. Therefore, the mechanical vibration of the chopper and the generation of noise, such as when the light source beam is made into intermittent light using a conventional mechanical switch, are avoided. can be reduced to zero, and a remarkable improvement in the accuracy of the photoacoustic signal can be seen.

However, according to the measurement experience in photoacoustic measurements using the large-capacity cell developed by the present inventors, the measurement density is still influenced by the characteristic values, which can be called device characteristics.
In particular, the distance between the electrophoresed gel plate surface and the cell window,
In order to improve the accuracy of photoacoustic measurements, it is strongly desired to find the optimum conditions for the frequency of the input irradiation light.

(Problems to be Solved by the Invention) The present invention aims to find optimal conditions for solving the problem of improving the accuracy of photoacoustic measurements in a new device that is an improvement over the above-mentioned conventional technology.

(Means for Solving the Problems and Effects thereof) As a means for solving the above-mentioned problems, the present invention has a photoacoustic cell that can house a gel plate subjected to electrophoresis, and uses input irradiation light to the plate surface. In a photoacoustic measuring device that outputs the generated sound as a photoacoustic signal, the distance between the electrophoresed dull plate surface and the cell window is 0.771.0111 m.
The photoacoustic measurement method includes measuring the frequency of input irradiation light in the range of 20 to 80 Hz, preferably 30 to 60 Hz.

In explaining the method of the present invention, first, a photoacoustic device according to the method of the present invention will be explained.

FIG. 1 shows a structural diagram of a photoacoustic cell. The cell 1, which can accommodate 26×76 large gel plates, is made of aluminum. As the microphone 2, an electret condenser microphone is used. A microphone to prevent noise from outside the cell is glued to the cell with epoxy resin adhesive. The microphone and the sample chamber 3 are connected by a unibu guide 4 having an inner diameter of about 1 y+m and a length of 15 mm. For example, the sample chamber is 27X77X:3++
If x is the size of the glu plate stored here is 26
This is 76 x 1 pilgrimage. A hard glass of 93×35×1 and 5×m is used as the cell window 5 and is also bonded to the cell 1 with an epoxy resin adhesive. 6 is a cell body and 7 is a Brella Charelise. After storing the guru plate in the cell,
A cell lid 8 with a rubber/4' seal having a thickness of I Im is inserted and crimped onto the cell 1. Cell capacity α is approximately 5.9c
Chill with i3. The microphone output is a homemade LF356.
It was amplified with a preamplifier using H.

Its power source is a battery, which is controlled by a regulator. The microphone is used with a bias voltage of 4.5V applied. The light source used was Toshiba's Ga-At-As TLRA150C, but its peak wavelength was 66.
Approximately 3Cd at 0nm, half-width 34nm, Lp=20mA
A luminous output of For modulation, a home-made voltage modulator using μA355 was used, which has a r-neity cycle of 20 to 90% and a frequency of 12 to 100 Hz.
It is variable within the range of .

In order to prevent the LED beam from spreading, the parts except the LED head were covered with aluminum foil. As a result, the beam diameter on the kelp plate becomes 3 mm. This was attached to a sweeping device and swept in the direction of spot development at a speed of 10.6 h/min. The obtained photoacoustic signal is guided to a self-made lock-in amplifier and recorded on a recorder (Model EPR-100A manufactured by Toa Denpa Co., Ltd.).

Although the photoacoustic measuring device described above is an outline of a typical device that uses an LED as a light source, the dimensions of each part of the device are not limited to the above.

Now, in photoacoustic spectroscopy, the photoacoustic signal is inversely proportional to the cell volume. When the del plate is housed in the cell, the cell volume is the value obtained by subtracting the volume of the del plate from the total cell volume. In order to finely adjust the cell volume, spacers of different thicknesses (made of aluminum, for example) are placed under the cell plate.
is inserted, but the air volume through which the acoustic waves propagate inside the cell is almost the same as the cell except for the thickness of the del plate and spacer, and the side spacing is only large enough to allow storage and removal from the cell. Therefore, the distance between the gel plate surface (with or without a cover) and the cell window can be considered.

As a result of determining the intensity of the photoacoustic signal by changing the distance (-) from the del plate surface to the cell window by inserting various smoothers with different thicknesses as described above, the second A relationship diagram as shown in the figure was obtained. Here, the frequency is 35 Hz.

Note that the gel plate sample used for electrophoresis used in this measurement was prepared as follows.

Transferrin was electrophoresed using polyacrylamide 10% gel, stained with amide blank IOB, and vacuum dried on filter paper (Toyo Roshi A 4 A).

Using double-sided tape, attach the glue on the filter paper to 26x76x
It's glued to the glass plate of 1 Dragon.

As is clear from this figure, the photoacoustic signal intensity is high when the distance between the surface of the glue plate and the cell window is 0.7 mm to 1.0 mm. When the distance is less than 0.7 mm, the cell volume decreases, but the generation of the photoacoustic signal sent out in a piston-like manner from the heat diffusion surface of the del plate is hindered, so the signal intensity decreases rapidly, making it unsuitable for measurement purposes. Also, the above distance is 1
．． When it exceeds 0, the signal strength decreases and again the purpose of the measurement is not achieved.

From the above, the optimal range of the distance between the gel plate surface and the cell window to achieve this purpose is Q, 7 van to l, Q t
You can see that it is off with iyx.

In addition, in the above measurements, the microphone and cell window were fixed to the cell body with 22x resin as described above, so the removal of external noise was good, and the above results were obtained under conditions where noise did not adversely affect sensitivity. This is what was obtained.

Next, we examined the photoacoustic signal strength and the intermittent frequency of the light source. That is, as a result of intensive investigation into the relationship between the frequency of the input irradiation light and the signal intensity, we were able to discover the relationship between the two as shown in FIG. In this figure, the distance between the cell plate surface and the cell window is constant1. Q am. The distance of the sample from the gel plate surface to the cell window,
The same one used when determining the relationship with signal strength was used. Also shown in the figure are numerical values determined regarding changes in the S/N ratio.

From this figure, it can be seen that the frequency of both the signal strength and S/N ratio is low for the joists. However, the frequency is 20 I(z
In the following, noise increases rapidly and problems arise in reproducibility, making it impractical. Moreover, at 80 Hz or higher, both the signal strength and the S/N ratio decrease, making it unsuitable for measurement.

As a result of finding the relationship between the frequency, signal strength, and S/N ratio, if we combine these results, the frequency of the input irradiation light is 20.
A range of Hz to 80 Hz has been found to be suitable, more preferably a range of 30 Hz to 60 Hz is optimal.

To summarize the above, setting the distance between the gel plate surface and the cell window to 0.7 to 0, and setting the input optical frequency to 20 to 80 Hz enables photoacoustic measurement with high accuracy and within the above range. It is clear that the measurement method is inappropriate if it deviates from the above.

[Brief explanation of drawings]

Fig. 1 is a structural diagram of a photoacoustic measuring device according to the method of the present invention, Fig. 2 is a diagram showing the relationship between the distance between the cell plate surface and the cell window and the photoacoustic signal intensity, and Fig. 3 is a diagram showing the relationship between the frequency of input irradiation light and the optical A relationship diagram between acoustic signal strength and S/N ratio is shown. ■...Cell, 2...Microphone, 3...Sample chamber, 4...Unibu guide, 5...Cell window, 6...
・Seldeday, 7...Pressure release, 8...
・Cellulid.

Claims (1)

[Claims] In a photoacoustic measurement device that has a photoacoustic cell that can accommodate an electrophoresed gel plate and outputs the sound generated by the input irradiation light to the plate surface as a photoacoustic signal, the distance between the gel plate surface and the cell window is set to 0. .7~1.0mm
A photoacoustic measurement method characterized in that the frequency of input irradiation light is measured within a range of 20 to 80 Hz.