JPH0219900B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to an electrophoresis measuring device.

Electrophoresis measurement devices that observe the movement of charged particles are well known in the field of handling microscopic surface charged substances. FIGS. 1 and 2 schematically show an example of a commercially available microscope electrophoresis measuring device. When performing measurements using this device, a sample is supplied into the electrophoresis chamber 2 from the sample injection port 5, and the electrophoresis chamber 2 is equipped with an external temperature control mechanism 4 to maintain a constant temperature. It is placed in a constant temperature bath 1 and kept at a constant temperature. After supplying the sample, the injection port 5 is closed, and an electric field is applied by the DC power supply 7 to a pair of electrodes 3 and 3' provided at both ends of the electrophoresis chamber 2. The applied electric field causes the sample in the electrophoresis chamber 2 to move, and the speed of this movement is observed and measured through the observation window 9 using the microscope 10 and the light source 11. After measurement,
The sample is discharged from the discharge port 6. This electrophoresis device measures the migration speed of particles in a sample. The required electrophoretic mobility is expressed as the velocity of each particle per electric field strength (microns/second per volt/centimeter).

Conventional electrophoresis devices of this type use a two-terminal constant current source device as a power supply device, and when determining the degree of electrophoresis, the strength of the electric field is calculated from the product of the current at that time and the electrical resistance of the liquid. I am calculating and asking for it.
Further, as an improvement on this, a method has been adopted in which the strength of the electric field is determined by measuring the voltage over a certain distance within the electrophoresis chamber.

However, although the above device can obtain correct measured values if the electrical resistance of the liquid in the electrophoresis chamber is constant during measurement, the temperature of the liquid in the electrophoresis chamber may change due to the heat generated by the electric current. If this occurs and the electrical resistance of the liquid changes, correct measurement cannot be performed and is inappropriate. That is, the conventional method has the disadvantage that it cannot cope with changes in the resistance of the liquid in the electrophoresis chamber.

In order to eliminate the above-mentioned drawbacks, it has been proposed to use a regular two-terminal constant voltage power supply for the power supply 7. However, when using a constant voltage power supply, these two terminals are provided on the electrodes, so the voltage between the pair of electrodes 3 and 3' of the electrophoresis apparatus can be kept constant; This is because no compensation is made for the potential gradient (strength of the electric field), for example, the voltage drop V ₁ + V ₂ between the electrodes 3, 3' and the liquid 21 in Figure 3, and a constant voltage is not actually applied. However, the problem still remains that accurate measurements cannot be obtained.

In addition, when the two terminals of the constant voltage power supply are provided at the electrodes 3 and 3' as described above, the movement of the sample specimen due to causes other than electrophoresis, that is, the electrodes 3 and 3'
It is not possible to prevent the occurrence of convection of the sample specimen within the electrophoresis chamber 2 due to the generation of air bubbles.

The present invention was made in view of these problems, and it is possible to prevent the movement of the sample specimen due to causes other than electrophoresis, and to reduce the potential gradient between two points in the sample specimen, which is directly related to the mobility of particles in the sample specimen. An object of the present invention is to provide an electrophoresis measuring device that can measure the electrophoretic mobility of particles, etc. in a sample specimen with high accuracy by maintaining the electrophoresis constant.

According to the present invention, the object is an electrophoretic measuring device for measuring the mobility of particles in a sample specimen made of a liquid in which particles are suspended, an electrophoresis chamber; a pair of electrode chambers each connected to both ends of the electrophoresis chamber to communicate with the electrophoresis chamber; and an electrode chamber for applying a voltage from the power source to the sample specimen in the electrophoresis chamber. a pair of electrodes electrically connected to the power source and a sample specimen placed in the electrophoresis chamber to detect the voltage between the two points. A pair of voltage detection terminals and a pair of diaphragms respectively disposed between the electrophoresis chamber and the pair of electrode chambers are electrically connected to the pair of voltage detection terminals, and the voltage detection terminals are electrically connected to the pair of voltage detection terminals. This is achieved by an electrophoresis measuring device equipped with a circuit that controls the power supply based on the detected voltage in order to maintain the detected voltage between the terminals at a set value.

A block diagram of the power supply device 7 used in the present invention is shown in FIG. This device 7 includes an AC source 4
1 is rectified by a rectifier circuit 42 to obtain direct current, and based on information set in a voltage setting circuit 43, a current is outputted through a driver 44. This output is applied to the electrodes 3, 3' of the electrophoresis measuring device on the load side. On the other hand, the voltage between the voltage detection needles is fed back to the power supply device through the terminals of the power supply device connected to the voltage detection needles 12, 12' as voltage detection terminals provided in the electrophoresis chamber 2. This feedback voltage is amplified by a high input impedance amplifier 45. Width is increased. The amplified voltage Va is compared with the voltage Vb set in the voltage setting circuit 43 by a designation circuit 47 consisting of, for example, a potentiometer, in a comparator circuit 46, and the result is output to the driver 44.
When Va>Vb, the current is reduced and Va<Vb
When this is the case, the output current by the driver 44 is controlled in the direction of increasing the current, and the voltage between the voltage detection needles is kept constant. This makes it possible to maintain a constant electric field strength by following changes in the electrical resistance of the liquid. Furthermore, it becomes possible to directly adjust the strength of the electric field within the electrophoresis chamber, resulting in a significant improvement in the accuracy of electrophoresis measurement.

The rectifier circuit 42 and driver 44 constitute a power supply, and the comparison circuit 46, voltage setting circuit 43, and designation circuit 47 constitute a circuit that controls the power supply.

Further, according to the present invention, it is no longer necessary to increase the electrode area to reduce the impedance between the electrode and the liquid phase, and this has the advantage of creating a degree of freedom in the structure of the electrophoresis measuring device. By providing a diaphragm between the electrophoresis chamber and the electrode, it is possible to eliminate the influence on migration caused by gas generated due to electrolysis at the electrode.

Furthermore, in the device of the present invention, the voltage detection needles 12, 1 provided in the electrophoresis 2 of the electrophoresis measuring device
By using a material of minute size such as platinum for 2', and using an amplifier circuit with a high input impedance of ¹⁰⁷ ohms or more as the terminal circuit of the power supply device connected to the voltage detection needles 12, 12', without being affected by high impedance between electrodes and samples.
It is also possible to measure samples with very high resistance.

Hereinafter, an embodiment of an electrophoresis measurement apparatus using a microscope based on the present invention will be described with reference to FIG. This device mainly consists of an electrophoresis chamber 2 placed in a constant temperature bath 1, and electrodes 3, 3' that supply current to the electrophoresis chamber from a power supply device 7 connected to both ends of the electrophoresis chamber. It is something.

The constant temperature chamber 1 is equipped with a temperature control device 4 and is adjusted to maintain the electrophoresis chamber at a constant temperature. have glass tubes at the sample inlet 5 and sample outlet 6, respectively.
Further, the open ends of each glass tube are connected to gaskets 14, 14' and diaphragms 13, 1.
3', an electrolytic solution tank 1 serving as an electrode chamber having the electrodes 3, 3' and accommodating a phosphate buffer solution.
5, 15'. This diaphragm 13,1
3' is for preventing the influence of the generated gas on electrophoresis due to electrolysis at the electrodes 3, 3'. Furthermore, the power supply device 7 includes a DC power supply 7' configured to be able to set a voltage Vb, and a pair of platinum voltage detection needles as a pair of voltage detection terminals installed in the flow path of the electrophoresis chamber 2. 12, 12',
The detected voltage Va between the voltage detection needles is fed back, and the current flowing from the DC power supply 7' to the electrodes 3, 3' is adjusted so that the voltage Va is kept constant.
A device that controls in the direction of decreasing Va when Vb and increasing when Va<Vb, and a polarity converter 1 for performing electrophoresis measurement under both positive and negative polarities.
6,17.

Next, the operation and method for measuring mobility using this electrophoresis measuring device will be described.

First, the electrophoresis chamber 2 is placed in a constant temperature bath 1 equipped with a temperature control device 4, and is maintained at a constant temperature. The sample inlet 5 and outlet 6 are automatically opened and closed to supply the sample into the electrophoresis chamber 2. When the sample is stabilized, the voltage between the voltage detection needles 12 and 12' is adjusted by the DC power supply 7 of the present invention. Electrode 3,
3' is supplied with direct current. This DC power supply 7' operates with AC 100V input, maximum 20mA,
It has a 200V output.

Before entering the DC power supply 7', the signals from the voltage detection needles 12, 12' enter a one-way polarity converter 17 so that the polarity is only in one direction, and then are transmitted to the DC power supply 7'.

The sample in the electrophoresis chamber 2 is moved by the electric field between the electrodes 3 and 3', and the moving speed in the positive and negative electric fields is measured by the microscope 10 using the light source 11. Although this microscope 10 is not shown in FIG. 5, it is placed in the same position as in FIG. 1, and the movement of particles within the electrophoresis chamber 2 can be observed. The average value of the electrophoretic mobility obtained by dividing the moving speed of each particle in the positive and negative electric fields by the strength of the electric field was defined as the electrophoretic mobility of each particle.

The electrophoresis experimental conditions are as follows.

Electrophoresis sample: Sheep red blood cell suspension solution: Phosphate buffer (15 x 10 ^-3 /cm) Electrophoresis temperature: 20℃ Voltage detection needle voltage: 70V Number of measurement samples: 50 Measurement time: 2 seconds As a result of electrophoresis measurement, electrophoresis degree 0.93 (μ/sec)/
(V/cm) (average value of 50 samples) was obtained. In addition, the voltage detection needle voltage during the above measurement was within 70V±0.5V, and the error due to voltage fluctuation was suppressed to below ±0.7. In addition, when measuring under the same conditions with a constant current electrophoresis measuring device, the voltage change is 67.5 ~
Since the error of 70.0V was ±2%, the effectiveness of the present invention was verified.

In the embodiments, only an electrophoresis measuring device using a microscope has been described, but it goes without saying that the invention is not limited to this and can be applied to an electrophoretic measuring device using a detector Doppler method or the like, or an electroosmotic phenomenon measuring device.

As explained above, in the electrophoresis measuring device of the present invention, a diaphragm is disposed between an electrophoresis chamber and a pair of electrode chambers connected to both ends of the electrophoresis chamber, and a voltage is applied to the electrophoresis chamber through the diaphragm. Since the voltage is applied, it is possible to completely eliminate the influence of movement of the sample specimen due to causes other than electrophoresis, that is, the flow of the sample specimen due to gas generation at the electrodes, on the migration of particles. Furthermore, since the voltage detection terminal is provided between two points on the sample, even if there is a voltage drop due to the diaphragms placed between the electrophoresis chamber and the pair of electrode chambers, particles in the sample will not move. It is possible to reliably detect the voltage between two points on the sample, which is the basis of the measurement, and a voltage control circuit that maintains the detected voltage between the two points at the set value is electrically connected to the voltage detection terminal. Therefore, measurement of particle mobility in a sample specimen can always be performed under a constant voltage. Therefore, according to the present invention, the mobility of particles in a sample specimen can be measured with extremely high accuracy.

[Brief explanation of drawings]

FIG. 1 is a schematic front view of a conventional electrophoresis measuring device, and FIG. 2 is a schematic side view of the device shown in FIG.
FIG. 3 is a characteristic diagram showing the voltage distribution state in each part of the power supply device of the present invention, FIG. 4 is a block diagram of the power supply device of the present invention, and FIG. 5 is a diagram of the electrophoresis measuring device of the embodiment of the present invention. It is a schematic diagram. 1... Constant temperature bath with temperature control device, 2... Electrophoresis chamber, 3, 3'... Electrode, 4... Temperature control device, 5
...Sample inlet, 6...Sample outlet, 7
...Power supply device, 7'...DC power supply, 8...Temperature sensor, 9...Window for microscope, 10...Microscope, 11...Light source for microscope, 12, 12'...Electrophoresis room voltage detection needle , 13, 13'... diaphragm,
14, 14'...gasket, 15, 15'...electrolyte tank, 16...polarity converter, 17...unidirectional polarity converter, _V1 , _V2 ...voltage drop between electrode and liquid.

Claims (1)

[Claims] 1. An electrophoresis measuring device for measuring the mobility of particles in a sample specimen consisting of a liquid in which particles are suspended, which comprises a power source, an electrophoresis chamber for accommodating the sample specimen, and the electrophoresis chamber. A pair of electrode chambers are respectively connected to both ends of the electrophoresis chamber in order to communicate with the electrophoresis chamber, and a pair of electrode chambers are respectively arranged within the electrode chamber to apply a voltage from the power supply to the sample specimen in the electrophoresis chamber. a pair of electrodes electrically connected to a power source; a pair of voltage detection terminals arranged in the electrophoresis chamber to detect a voltage between two points of a sample specimen housed in the electrophoresis chamber;
A pair of diaphragms respectively disposed between the electrophoresis chamber and the pair of electrode chambers are electrically connected to the pair of voltage detection terminals, and detect voltage between the voltage detection terminals. An electrophoresis measuring device comprising: a circuit that controls the power source based on the detected voltage so as to maintain the voltage at a set value.