JPH0475462B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved control method for applying an analyte to a moist support used in electrophoresis.

In an electrophoresis apparatus, a support made of a cellulose acetate membrane or the like is once wetted with a buffer solution, a sample is uniformly applied to the wet support, and then an electrophoresis operation is performed. In order to perform accurate electrophoretic analysis, it is required to uniformly apply the specimen to the support and to apply the specimen when the support is at a certain degree of humidity. In particular, if the wettability of the support is non-uniform or fluctuates, the sample absorption capacity of the support will change, causing problems such as the sample not being applied uniformly and distorting the electrophoretic image. The degree of wetting of the support varies greatly depending on the time elapsed after wetting and the temperature and humidity of the surrounding atmosphere. Therefore, if the sample is always applied after a certain period of time after wetting the support with a buffer solution, the degree of wettability of the support at the time of sample application may be affected. The disadvantage is that the wettability varies greatly depending on the environmental conditions. As a method for solving this problem, the applicant has proposed in Japanese Patent Laid-Open No. 70454/1983 a method of measuring and controlling the electrical resistance of the support after it has been wetted with a buffer solution. However, since the electrical resistance of a wet support varies greatly due to slight changes in the pH value of the buffer solution, it is difficult to accurately control the wettability of the support. Furthermore, the method in which the operator controls the temperature while measuring the ambient temperature and humidity not only has problems with reproducibility, but also has the disadvantage that analysis operations cannot be performed automatically.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for applying a specimen, which eliminates the above-mentioned drawbacks and allows automatic application of a specimen when a support always has a constant humidity level, regardless of the ambient temperature and humidity.

The method for applying a sample according to the present invention involves detecting the temperature difference between the temperature of the support after wetting and the ambient temperature of the support when applying the sample to a support that has been wetted with a buffer solution in advance, and comparing this temperature difference with the support. This method is characterized in that the relationship between the evaporation rate and the buffer solution contained in the body is determined in advance, and the sample application timing is determined based on the evaporation end time in this relationship.

The present invention will be described in detail below with reference to the drawings.

FIG. 1 is a diagram showing the relationship between the elapsed time after wetting the support with a buffer solution and the temperature difference between the support and the surrounding atmosphere under various environmental conditions. The vertical axis shows the temperature difference, and the horizontal axis shows the elapsed time, and the curves and characteristics under, for example, high temperature and low humidity, high temperature and normal humidity, and high temperature and high humidity conditions are respectively represented.
The support temperature is equal to the ambient temperature immediately after wetting with the buffer solution, and as time passes, the temperature of the support decreases as the buffer solution evaporates, creating a temperature difference. Then, this temperature difference gradually increases and reaches a peak value. After that, the temperature difference becomes smaller and approaches the ambient temperature. Further, as time passes, evaporation of the buffer solution progresses, and the buffer solution partially evaporates, resulting in white spots on the support. The times at which these white spots occur are indicated by t _p1 , t _p2 and t _p3 , respectively. Under high temperature and low humidity conditions, the evaporation rate is fast, so the peak value of the temperature difference is large as shown in the curve, and white spots appear in a short time, whereas under high temperature and high humidity conditions, the evaporation rate is slow, so the peak value of the temperature difference is large. The value is small and white spots appear after a long period of time. Although there are slight differences in the time at which the peak value occurs depending on the environmental conditions, this is negligible when compared with the time at which white spots occur. White spots that appear on the support are caused by the buffer solution evaporating locally from the support, and if the sample is applied after the white spots have occurred, the sample will become non-uniform and the electrophoretic image will be distorted. This causes a problem in which accurate migration analysis cannot be performed. Therefore, sample application must be performed before these white spots appear.

FIG. 2 is a graph showing the relationship between the time until white spots appear after the support is wetted and the peak value of the temperature difference between the support and the atmosphere under various environmental conditions. The vertical axis shows the time t _p until white spots appear, and the horizontal axis shows the peak value T _p of the temperature difference. As understood from FIG. 2, the relationship between t _p and T _p lies on the same curve regardless of environmental conditions such as temperature and humidity, and the following relationship holds true.

T _p ×t _p = const The fact that the above equation holds true with respect to changes in temperature and humidity of the surrounding atmosphere can be qualitatively analyzed as follows. When the support is wetted with a buffer solution, the buffer impregnated in the support causes evaporation and takes away latent heat from the support. Therefore, under conditions where the evaporation rate is high and the peak value of the temperature difference is large, evaporation progresses in a short time and white spots occur in a short time. On the other hand, under conditions where the evaporation rate is low and the peak value of the temperature difference is small, the evaporation progresses slowly and white spots appear after a long period of time. As a result, by detecting the peak value of the temperature difference between a wet support and its surrounding atmosphere, the state of buffer evaporation from the support can be easily and accurately predicted, which allows for the determination of the analyte for various environmental conditions. It will be possible to predict the optimum time for application. That is, by determining the relationship between the peak value T _p and the white spot generation time t _p according to the type of buffer solution and support, the peak value T _p and the optimal time Δt _p for applying the specimen can be determined.
An ideal curve R representing the relationship between . Therefore, by creating the above ideal curve in advance according to various buffer solutions and supports, it is possible to automatically determine the optimal time for applying the specimen by detecting the peak value T _p . Become. Moreover, since the time at which the peak value occurs is almost the same under various temperature and humidity conditions, the time at which the peak value occurs can also be accurately known in advance, and by detecting and determining the time at which the peak value occurs in advance. By detecting the wet support temperature and the ambient temperature at one point, this detected value can be used as a peak value.

When setting the ideal curve R, the degree of wettability in the support is taken into account. In other words, a certain degree of uniform moisture is necessary for proper electrodynamics, but
If the amount of buffer solution contained in the support is too large, the sample will be obstructed by the buffer solution when the sample is applied, making it impossible to apply the sample uniformly. Therefore, it is desirable that the degree of wettability of the support at the time of applying the specimen is relatively low, and therefore it is desirable to apply the specimen a little more time than the point at which white spots occur. On the other hand, since there are variations in the local end point of evaporation of the buffer solution in the support, that is, the point at which white spots appear, the ideal curve R is determined taking these variations into account. Specifically, based on the relational expression T _p × t _p = constant that defines the evaporation rate of the buffer solution in the support, these amounts are An ideal curve R is set by plotting a time corresponding to . By defining the ideal curve R in this manner, it is possible to accurately determine the point in time when white spots do not occur and the humidity level is constant even if the environmental conditions change.

FIG. 3 is a diagram showing the configuration of an embodiment of the present invention. The support 1 is immersed in a buffer solution tank 3 filled with a buffer solution via a pair of rollers 2, and then conveyed via the rollers 2 again to the sample application section 4.
This pair of rollers 2 also functions as liquid squeezing rollers, so the support 1 transported by the rollers 2
is almost uniformly wetted with buffer. Sample application section 4
is covered with a cover 5, making it less susceptible to changes in temperature and humidity. A pair of drive rollers 6 are disposed in the cover 5 in correspondence with the positions of the inlet opening 5a and the outlet opening 5b, and two endless belts 7 are hung on each end of the drive roller 6 in the longitudinal direction. It is being Furthermore, a thermistor 8 for measuring the ambient temperature inside the cover 5 and a thermistor 9 for measuring the temperature of the support are provided inside the cover 5, and the connecting wires of these two thermistors are connected to a control circuit to be described later. It is connected. Furthermore, within the cover 5, a sample applicator 10 is disposed above the belt 7, which is movable up and down by a drive device (not shown). A specimen is applied onto 1. The support moistened with the buffer bath 3 has an inlet opening 5.
a, the specimen is conveyed by the belt 7 to a predetermined application position in the specimen application section 4, and then stopped. After wetting the support at the stop position, the temperature after the peak generation time has elapsed (this peak generation time is measured in advance) is detected by thermistor 9, and at the same time, the ambient temperature is detected by thermistor 8, and this temperature difference will be described later. This value is used to calculate the optimal sample application time stored in the control circuit, and after this time elapses, the sample applicator is moved up and down via a sample applicator drive (not shown). A sample is applied onto the support 1. FIG. 4 is a block diagram of the sample application control circuit. The wetting time of the support 1 as the roller 2 rotates is detected by the support wetting detection circuit 11;
This signal is input to the central processing unit 12. The support body temperature and the ambient temperature are detected by thermistors 9 and 8 at a predetermined time when the peak value of the temperature difference occurs, and the detected temperature signal is sent to the comparator 1.
3, and the temperature difference between the support 1 and the surrounding atmosphere is input to the central processing unit 12. The memory 14 stores the relationship between the peak value T _p of the temperature difference and the optimum sample application time Δt, and the optimum sample application time is instructed from the memory 14 to the central processing unit 12 based on the detected value of T _p . After the optimum time has elapsed, the sample applicator 10 is driven via the sample applicator driving device 15, and the sample is applied to the support.

Although two thermistors are used in this example, the support 1 is discharged from the sample application section 4,
Measure the ambient temperature while the next support is being carried in, temporarily store this value, and then detect the temperature of the support being carried into the sample coating section 4 and compare it with the stored ambient temperature. By doing so, it is possible to create a configuration that requires only one thermistor.

As explained above, according to the present invention, even when the environmental conditions vary greatly, it is possible to apply the specimen to the support in an optimally moist state, and it is possible to apply the specimen to the support accurately without being affected by changes in the surrounding environmental conditions. This makes it possible to conduct electrophoretic analysis. Moreover, the optimum coating conditions can be determined simply by detecting the support temperature and ambient temperature after a predetermined period of time after wetting the support.
There is no need for a timer or the like, and it is also possible to simplify the control circuit.

[Brief explanation of drawings]

Figure 1 is a diagram showing the relationship between the temperature difference between the support and the surrounding atmosphere and the elapsed time after wetting the support, and Figure 2 is the peak value of the temperature difference between the support and the surrounding atmosphere and the occurrence of white spots. FIG. 3 is a diagram showing the relationship with time, FIG. 3 is a diagram showing the configuration of an embodiment of the present invention, and FIG. 4 is a block diagram showing the control circuit section of an embodiment of the invention. DESCRIPTION OF SYMBOLS 1...Support body, 2...Roller, 3...Buffer solution tank, 4...Sample application part, 5...Cover, 6...Drive roller, 7...Belt, 8, 9...Thermistor, 10... ...Specimen applicator, 11...Support wetness detection circuit, 12...Central processing unit, 13...Comparator, 14...Memory, 15...Specimen applicator drive circuit.

Claims (1)

[Claims] 1. When applying a specimen to a support that has been wetted with a buffer solution in advance, the temperature difference between the temperature of the support after wetting and the ambient temperature of this support is detected, and the temperature difference and the buffer solution contained in the support are detected. A method for applying a specimen, characterized in that the relationship between the evaporation rate and the evaporation rate is determined in advance, and the timing of applying the specimen is determined based on the evaporation end time in the relationship.