JPS5995440A - Method and device for optical measurement - Google Patents

Abstract

PURPOSE:To perform measurement of absorbance of liq. samples e.g. antibody, antigen, serum, etc. with no need at all for vessels and cells, etc. for contg. sample and without influence of the material quality and uneven shape of the cell, etc. by measuring the spherical liquid drop of the sample liquid formed in a space. CONSTITUTION:A cruciform chamber 14 for measuring a sample is formed in a device. An upper pipe holder 12 and a lower pipe holder 13 are bolted to the body at the top and bottom of the hole in the perpendicular direction of said chamber. A sample dropping nozzle 15 and a sample rest 16 are supported respectively on the holders 12, 13. Both are disposed in an electrically insulated state apart from each other at a specified space (a) so that the nozzle and the rest can be electrically charged with each other. The nozzle 15 is preferably constituted of a pipe having a water repellent inside surface, for which, for example, ''Teflon'', PE, etc. are used. The pipe having about 0.3mm. inside diameter and 0.5-0.6mm. outside diameter is preferred. The space (a) between the nozzle 15 and the rest 16 is satisfactory if it is within the range of the size at which the drop of the sample liquid to be formed is unaffected by gravity.

Description

[Detailed description of the invention] be.

Generally, absorbance values of liquid samples such as antibodies, antigens, blood coagulates, serum, and reaction solutions are measured using an apparatus as shown in FIG.

That is, a liquid sample (2) is placed in an optical measurement cell (1) made of transparent glass, a light source (3) of 10,000 meters emits light (4) with a specific wavelength, and a slide plate (5) is placed in the liquid sample (2). ) and apply it to sample (2).

The absorption rate of light (4) by this sample (2) is detected by a detector (6) such as a phototube on the other side.

Conventionally, in such an optical measurement device, the storage cell □ for measuring the sample generally has a rectangular tube shape as shown in Fig. 1, and the optical path length (6), which is the length through which the light beam passes, is 10zz. is used as standard.

However, in order to optically measure antibodies and other liquid samples (2) as mentioned above, this standard cell (1) is difficult to use.
The volume of the sample storage portion (7) is too large, requiring an extra amount of sample, and since it is difficult for light to pass through a highly concentrated sample, the sample must be diluted before measurement.

Therefore, a device as shown in FIG. 2 is used in which the volume of the sample storage portion is reduced and the optical path length is shortened.

The external shape of the one shown in Figure 2 is the standard cell (1) in Figure 1.
It has a prismatic shape similar to , but the internal optical path length is, for example, 2
~0.5 demand, and this gap is used as the sample storage part (7). In this way, the above-mentioned drawbacks can be solved.

However, simply narrowing the sample storage portion makes it difficult to discharge the sample once placed and to clean the inside of the cell.

As a result, the interior of the cell was contaminated by the previous sample, making it impossible to obtain accurate measured values. Also, to clean the cell, remove it from the photometry path of the measuring device.
The work of resetting must be performed every time a measurement is performed, and therefore it has been impossible or difficult to efficiently measure a large number of specimens one after another.

In addition, in order to analyze samples that flow out continuously, such as samples obtained by continuous reactions or continuous fractions obtained by chromatography, it is necessary to use the shapes shown in Figures 1 and 2. The purpose is not fully achieved with these cells. In such cases, or when a large number of specimen samples are to be continuously aspirated and the absorbance measured efficiently, a sample inlet (a) and an outlet (Q) as shown in Fig. 4 should be provided. A method is used in which a shellfish flow cell (flow cell) (b) is used, a sample is caused to flow into (b) from (a), and the absorbance of the sample passing through (b) is measured.

- 10,000, as shown in Figures 5 and 6, the sample reaction vessel
In order to create an optical measurement cell, a culture or reaction container made of glass or plastic that has been specially processed is used, and the light source and light receiving part or the container are sequentially moved to transmit light. Various efforts have been made, such as making measurements one after another.

However, these methods each have their own drawbacks and do not fully meet their objectives.

In other words, in the case of the flow cell shown in Figure 4, contamination from the previous sample remaining in the cell is washed away with the next sample, so the cell must be filled with enough water to prevent contamination and obtain accurate measurements. 3 to 4 times the amount of sample is required, resulting in a length of 1.5 to 2 m.
1 sample amount is required. Therefore, when measuring a small amount of sample of about 0.1 vessel, operations such as dilution must be added to increase the liquid volume. There is a limit to reducing the container diameter, and as a result,
Since the optical path length cannot be shortened, if the analyte in the container is highly concentrated, the absorbance increases and the light receiving section cannot detect it. In addition, there were drawbacks such as poor measurement accuracy due to unevenness in the material and dimensions of the container.

The present invention overcomes these drawbacks and difficulties in the conventional methods, and has the following features that cannot be obtained with the conventional methods.In other words, the present invention solves the problems of the sample liquid formed in the space in a true spherical shape. By measuring droplets, there is no need for containers or cells for storing samples, and therefore, it is possible to save the labor of storing samples in cells and cleaning and replacing cells. Furthermore, there are many effects such as the influence of unevenness in the material and shape of cells, etc., and no contamination caused by previous samples.

Furthermore, by being able to perform measurements with a very small amount of sample and, therefore, being able to perform multiple measurements on the same sample, it is possible to apply the method to a wide range of samples to be measured and to improve measurement accuracy.

In addition, since it is possible to measure continuously, it is possible to obtain an extremely compact measuring device that can efficiently measure a large number of samples one after another, and by adjusting the nozzle diameter and the gap between them, Since up to four spheres can be created, even highly concentrated samples can be continuously measured without dilution.

For this purpose, the present invention employs the following configuration. First, the method involves installing a sample dripping nozzle, a sample receiver, and a metal plate with a certain gap between them, and dripping the sample from the sample dripping nozzle into the gap to form a spherical droplet formed in the gap. This is an optical measurement method that involves projecting light with a specific wavelength toward a sample cylinder from one side, and detecting and recording the absorption rate of the light by the sample using a detector on the other side.

The apparatus for carrying out the above method is as follows: ◎ The sample dripping nozzle and the sample receiver are arranged with a certain gap between them, and the light from the light source is focused on one side in the direction perpendicular to this gap. A lens is provided that emits light toward a spherical sample cylinder formed in the gap through a lens, and a light-receiving lens that condenses the transmitted light is arranged on the other side, and the light from the light-receiving lens is further detected.
This is an optical measuring device equipped with a recording detector.

The embodiments shown in the drawings will be described below.

In FIG. 1, (1) is the main body, in which a cross-shaped sample measurement chamber (14) is formed.

Above and below the vertical hole, an upper pipe holder 04 and a lower pipe holder (13) are fastened to the main body with bolts.

In addition, each holder aac'3) has a sample drip nozzle a.
51 and the sample receiver (!!
They can charge each other.

The lens holder (5a) that supports the projection lens (5) is attached to the adjustment flange (3) in the horizontal hole.
The light bulb holder (4a) is further attached to the adjusting flange (3).
Following a), a contact holder (8) is fitted.

(4) is a light bulb supported by a light bulb holder (4a), and contacts 19) of a contact slide aω movably attached to the contact holder (8) come into contact with this light bulb.

Next, (7) is an insulator through which the contact is attached to the contact slide (10). Also, (6) is adjustment 7
This is a diaphragm located in the lunge (3) and interposed between the lens holder (5a) and the light bulb (4).

As mentioned above, the light emitting device is arranged on the - side of the main body (1), and the lens aυ that receives this light is arranged on one side of the horizontal hole, and (lla) indicates the Solns holder. .

(2a) is a photodiode cover that supports the photodiode (2) tube.

Among the devices described above, the sample dripping nozzle Q51 is preferably constructed of a water-repellent pipe inner surface, for example, Teflon, polyethylene, etc. are used. Preferably, the inner diameter is about 0.3π and the outer diameter is about 0.5 to 0145 am. The gap (a) between the sample dripping nozzle α1 and the sample receiver (16) may be within a size range where the sample droplet to be created is not affected by gravity, and this is determined by the size of the surface tension of the liquid. The larger the diameter, the more stable a sphere with a larger diameter can be made, and specifically, about 0.4 to 2 mrn is preferable.

The light bulb is a halogen lamp of about 20 watts, and the projection lens (5) has a focal point that allows the projection light to converge on the center of the sample sphere.For example, it has a diameter of 4 mm and a focal length of F5, or Alternatively, a lens such as an aspherical lens which corrects parallel light by eliminating spherical aberration, such as a lens having a hyperbolic curve, is used, and the light receiving lens has a diameter of 6 mm and a focal length of the same F5.

Now, for the sample receiver, connect the tube to the suction device, depressurize the inside of the sample measurement chamber, and insert the sample into the sample drip nozzle (151). A sample cylinder is formed.

On the other hand, the light from the light source (4) passes through the aperture (6) and the lens (5).
), the light is projected toward the spherical sample tube.The light that has passed through the sample is received by the lens aυ, and is stopped by the photodiode (2) of the detector.

However, since the distance between the sample dripping nozzle and the sample receiver is a constant value that corresponds to the diameter when the sample liquid grows into a true spherical part, when a true spherical part is formed, , the drip nozzle and the sample receiver become electrically connected through the sample liquid bulb, and in response to this command, the light that passes through the bulb at that time, that is, when the bulb grows to its maximum size, is By electrically recording and displaying only the signal of the absorbance value of the sample, the absorbance value of the subject at a constant optical path length l is measured.

Thereafter, the bulb is sucked into the sample holder and discharged from the a leak.

Such an electric circuit is shown in FIG. 8, and its operation will be explained based on FIG. 9. When a droplet becomes a perfectly spherical part and is held in the sample dripping nozzle (151) and the sample receiver (L6), The light bulb (4) irradiates the droplet with light of a constant luminous intensity using the running voltage power source αη, and this transmitted light is received by the photodiode (2) and converted into an electrical signal.This is amplified by the amplifier II! The signal shown in Fig. 9 (a) from
Output for.

On the other hand, the sample drip nozzle (one and sample receiver αQ)
is conductive, and the signal based on this is sent to the comparator (25
) is compared with the reference voltage VREF @This output is shown in Figure 9, and an enlarged version of this is shown in Figure 9 (C). The output signal from the comparator (251) generates a pulse as shown in the monomulti (2υ is shown in Figure 9 while this signal is being input), and this pulse causes the sample and hold circuit e7G to perform sampling, and the pulse rises. A constant output is generated and maintained until the drop.

With the fall of this pulse, the A/D conversion if (c) starts operating, and according to the pulse shown in FIG. 9 (g) generated by the clock generator (2), the Depending on the timing, the top pit (MSB)
) to the least significant bit (LSB) into a 14-bit digital signal. When the value of the least significant bit (LSB) is output, the A/D conversion ends and the data becomes valid from now on. This state is notified to a microcomputer (not shown). This data is stored in a buffer (24
This is held at I and is output to the microcomputer by inputting the enable signal shown in FIG. 9(g) from the microcomputer. In addition, comparator c! The output signal 51 is outputted to the microcombinator to notify that the droplet has contacted the sample receiver U mark.

Generally, the spherical droplet formed in the gap (a) sways, so as mentioned above, the diameter of the light-receiving lens aυ is the diameter of the light-emitting lens (
It is better to have a diameter larger than 5). Also, even though droplets are formed in the gap (a), some of the sample holder will flow along the inner surface of the tube, so it is better to hold the holder below. . In order to guide the droplets toward the receiver, the sample receiver may preferably have a plurality of longitudinal grooves formed on the outer surface of the tube.

Since absorbance values can be measured by forming droplets as described above, the volume of the sample is smaller than that of a cell. Therefore, compared to the amount of analyte in a cell, the present invention can test several drops, so the average value As described above, the present invention not only allows absorbance values to be obtained without error compared to cells, but also has a water-repellent sample dripping nozzle. Then, there is no need for cleaning, and by depressurizing the sample measurement chamber with a suction pump and suctioning the sample from the sample drip nozzle side, and making it into the sample sphere, the entire sample inside the nozzle can be used for measurement. The remaining amount of the previous sample is almost gone and there is almost no risk of contamination by the previous sample.

゛ Also, according to the present invention, it is possible to arrange the analyte samples of 2.13.1 in a large number of arranged small containers and continuously and efficiently measure them while moving the measuring device of the present invention. In the embodiment, a light bulb was directly used as a light source, but a glass fiber may be used to guide the light t-9, and if an aperture is provided, the aperture diameter should be t-0.1 to 0.2 bars.

Furthermore, in the above embodiment, when conduction occurs between the sample dripping nozzle and the sample receiver, it is recorded in the memory circuit of the detector. Parallel light of a different wavelength is applied to the sample droplet, the light that passes through it is received and detected by a linear array, and when the sample diameter reaches a predetermined diameter, the absorbance value output from the photodetector is memorized. You can also do this.

In this case, there is no need to charge the sample dripping nozzle and the sample receiver.

[Brief explanation of the drawings] Figure M1 is a four-part illustration of a conventional optical measuring device using a cell. 2 and 3 are explanatory diagrams of an optical measuring device using the improved cell of FIG. 1, and the slope factor of the cell. FIG. 4.5.6 is an explanatory diagram of another conventional optical measurement device. FIG. 7 is a cross-sectional view of the optical measuring device of the present invention. Figure 8.9 is an electrical circuit diagram and an operational diagram of the present invention. (2)・・・・・・・・・Detector (5)・・・
・・・・・・・・・Light projection lens αυ・・・・・・・・・
・・Light receiving lens a51・・・・Sample drip nozzle αe・・・Sample receiver is patent depositor Eisai Co., Ltd. Figure 1 Figure 3, L

Claims (2)

[Claims] (1) A sample dripping nozzle and a sample receiver are provided with a certain gap between them, and the sample is dripped from the sample dripping nozzle into the gap, and the sample is placed on one side toward the spherical sample droplet formed in the gap. An optical measurement method that consists of emitting light with a specific wavelength from one side, and detecting and recording the absorption rate of that light by the sample using a detector on the other side. (2) The sample drip nozzle and the sample receiver are arranged with a certain gap between them, and the light from the light source is directed to one side in the direction orthogonal to the gap, through the aperture, to the spherical sample droplet formed in the gap. An optical measuring device comprising: a lens that emits light; a light receiving lens that collects the transmitted light; and a detector that detects and records the light from the light receiving lens.