JPS604843A - Optical measuring method of liquid specimen and stirrer used therein - Google Patents

Abstract

PURPOSE:To make it possible to measure optical characteristics with good accuracy by using a small amount of a specimen in performing optical measurement while stirring the liquid specimen in an optical measuring cell, by using a specific stirrer. CONSTITUTION:A stirrer comprises a stirring part 4 cut into a U-shape having a rotary shaft 1, a columnar part 2 and a hollow part and a translucent or transparent resin or metal is pref. used as the material of said stirrer. The center of the hollow part of the stirrer inserted into a cell is allowed to coincide with an optical axis and light is irradiated to detect transmitted light while the stirrer is rotated. At least a part of light is not blocked by the stirrer and, therefore, passes a liquid specimen over the entire length of the cell. As a result, measurement of optical characteristics is enabled with good accuracy and, because the stirrer is arranged in the cell, measurement is performed by using a small amount of the specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for optically measuring a liquid sample and a stirring bar used therefor. In particular, the present invention relates to a method and a stirrer for accurately measuring the optical properties of a very small amount of sample.

It is known that the progress of the reaction can be determined by conducting a reaction in an optical measurement cell and optically tracking changes in the optical properties of the sample, such as absorbance, as the reaction progresses. It has been applied to various yuzu analysis methods. For example, one example is the production of antigens or antibodies by antigen-antibody reactions using % sensitized latex, which has recently been in the spotlight.

In a typical example of an antigen-antibody reaction, a sensitized latex supported by an insoluble carrier with an average particle size of 7.6 microns or less is loaded into an optical definition cell, and then the antibody and/or antibody is loaded into an optical definition cell. Add sample f containing M'f and mix with stirring.
Generates an antibody response. The cell has a wavelength of 0.1 to 2. Optical properties of latex 1 Usually, changes in the amount of transmitted light are measured by irradiating light with an amount of μ more than 5 times the average particle diameter of the latex, and more than 5 times the average particle diameter of the latex. Measurements are usually made as measurements of the rate of change of absorbance or transmittance, the absorbance of an open bale at a given time, or the time to reach a given absorbance (see U.S. Pat. ).

In measuring such anti-antibody reactions.

In order to make the inside of the cell uniform and to promote the reaction, it is necessary to stir the liquid inside the cell with appropriate strength. Conventionally, stirring in such cases involves placing a stirring bar outside the optical path, or Even when the stirrer is installed in the optical path, the stirrer is taken out of the optical path during measurement to prevent light from being absorbed or scattered by the stirrer and resulting in inaccurate measurements.

Furthermore, there is a method of using a prismatic stirrer that transmits light when the stirrer is placed in the optical path during measurement (% Kaisho!ter/Niotsu θ2za Publication).

1. If the rudder and stirrer are placed outside the optical path, the measurement directly requires an undesirable cell volume and requires a large amount of sample at the same time. 1. The method of taking the stirrer out of the optical path during measurement requires
It is not possible to continuously observe the state of the sample within the cell, and control for measurement becomes complicated. On the other hand, even when using a prismatic stirrer that transmits the above-mentioned light, further improvement in accuracy is desired.

The inventor of the present invention arrived at the present invention after considering a method for measuring optical properties of a liquid sample in a cell with high precision and a stirrer used therein. That is, the gist of the present invention is as follows. , a method in which a liquid sample is placed in an optical measurement cell equipped with a stirrer, and the optical properties of the sample are measured while the stirrer is positioned in the optical path of a measuring wire and rotated. Use a stirrer having a structure such that during rotation, at least a portion of the measuring light beam passes through the liquid sample over the length of the cell without being substantially blocked by the stirrer, and the optical path length is the longest. This will be seen in the optical side scanning method for liquid samples and the stirring bar used therein.

According to the present invention, even though there is a stirrer in the optical path, substantially the entire cell length (inner dimension of the cell in the direction in which light passes) can be used as the measurement optical path length, so Even samples can be optically measured with high accuracy.

Details of the present invention: 1. The measuring device used in the present invention includes an optical measurement cell containing a sample solution, a stirrer inserted into the cell, and a light source unit that irradiates the cell with measurement light &if; and a light receiving section that measures the measurement light beam that has passed through the cell. As the light source section and the light receiving section, any one used in a similar general optical measuring device can be used. The cell also usually has a rectangular or square cross-sectional shape, which is used in general optical measuring devices. On the other hand, a stirrer is different from the one normally used.The part of the stirrer that is immersed in the sample solution and located in the optical path has hollow parts, holes, cuts, etc. for one rotation. The light from the light source passes through the cell containing the sample solution without being intercepted by the stirrer.

You can go to the detector. When a hollow portion is provided, a transparent thin plate may be provided that does not interfere with measurement. The thinner the filling of the thin plate, the better, but the strength is about 0.
The size of the hollow part, which is 2 mm or more and 2 mm or less, is a size that allows at least a part of the measuring light beam to pass through, preferably j% or more, but the size of the hollow part in the lateral direction is It is preferable that the diameter of the luminous flux and #U are approximately the same.

Specifically, when the cross-sectional shape of the cell is 71 mm square and the diameter of the luminous flux is 3 mm, the following is true. The length in the vertical direction is equal to or greater than the surface diameter of the light beam, and the longer the better to reduce entrainment of bubbles. In addition, the horizontal length of the surface of the stirrer that blocks the measuring beam should be approximately the same as the length of the light beam, or preferably longer than this. and when using a luminous flux, it is approximately co~s mm. The part of the frame that forms the hollow part is 0 in terms of strength.
．． It is preferable to make it more than J.

If the minimum gap between the stirrer and the inner wall of the cell is 0.5 to 2 mm, the stirrer can rotate freely and the sample can be sufficiently stirred.

The stirring bars are arranged in the cell so that the hollow portion thereof passes at least a portion of the measuring light beam, but it is preferable that the center of the hollow portion of the stirring bar coincides with the center of the light beam. Furthermore, the upper part of the hollow part.

It is preferable to position a part of the liquid outside the liquid surface, since the air bubbles will be in the hollow part.

The cross-sectional shape of the portion where the stirrer is located in the optical path (the shape before forming the hollow portion) is preferably rectangular, square, circular, diamond-shaped, or the like.

Even when the stirrer has holes or notches, its shape can be determined using the above-mentioned reference KJ.

Below, the stirrer of the present invention will be described in detail with reference to drawing VC. The stirrer shown in FIG. 2 is a cylindrical portion. Making the entire upper part of the stirrer part cylindrical is clever because it reduces entrainment of air bubbles on the liquid surface. ′=! 1. The stirrer of the present invention is as follows.

(5) It may have a part of the taper for allowing air bubbles f to escape.

The stirrer shown in Figures 4 to 3 has a stirring part (6) with holes, and the width of the stirrer in the figures is approximately 3 mm and 3 mm wide, respectively. The stirring shown in Figure Z is a hollow-shaped stirring part (having force, and its width is approximately 0, S ~, 2 dragons in the diagram, and N is about 3 ~. Specifically, % For example, K is 3111n, L is '1mm, and M is /H1m.

N is Gu silk.

Materials for the stirrer of the present invention include transparent materials such as acrylic resin, glass, vinyl chloride, Teflon, etc. translucent materials such as polypropylene, nylon, polycarbonate, etc., and opaque materials such as Bakelite% Kanahashi, etc. However, a translucent or opaque material is preferable.Even if a transparent material is used, the inner surface of the hollow or hole portion or the notched surface may be cut to match the surface. It may be semitransparent.

In order to measure the optical characteristics of the melt in the cell using the method of the present invention, a stirrer as described above is inserted into the cell, and the hollow part of the stirrer is
The hole or cut portion is positioned within the optical path of the measuring wire, and the cell is irradiated with light while rotating the stirrer. The rotation speed is 2oθ~, preferably 200Orpm%
~/! The light i! which is θOrpm! The distance that 81 travels through the solution, that is, the optical path length, changes periodically as the stirrer rotates. Accordingly, the light intensity entering the detector also changes periodically, and the detector output signal also changes periodically. Measure the target amount at least 7 times.

Of optical quantities? 1411 is listed below! As is clear from the figures and FIG. 6, it is convenient to use a peak hold circuit to extract the optical signal. No.! The figure is a block diagram of the measurement circuit when using a peak hold circuit.
No. g1 is a diagram of wave No. 8 (vertical axis V: voltage, horizontal axis one hour) of (■ ~ 燵) shown in the figure, t8) is a stirring bar,
(9) indicates the luminous flux. To control the peak hold circuit, a pulse synchronized with the rotation is used, but this pulse can be obtained by an internal trigger method. Here is an example of using the internal trigger method: Obtain the pulse of ■ that is synchronized and periodically emitted, and use this to reset the signal of ■ between IN and input to the peak hold circuit. It is possible to hold the peak value of .The pulse of ■, for example, is reset by the stirrer pulse from the time when it reaches a value of about 0% of the peak value from the upsoil of the mountain in ■ of the figure. It is.

According to the present invention, by using a small amount of sample,
Even if the liquid sample N'a-T L rolls unevenly, it can be continuously and easily measured without being affected by this.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples unless it goes beyond the gist thereof.

In the following length examples, a transparent glass cell having a cross-sectional shape of a 2M square and a height of /mm was used as the optical measurement cell.

As shown in Figure 1, the stirrer had a rectangular cross-section with a square cross section, with a notch 3 mm wide and a beveled taper at the top to make it easier to remove air cells. For comparison, a transparent stirrer (B) shown in Figure 7 has a square shape with rounded sides, a rounded bottom, a cylindrical top with a length of am, and many notches and small holes. f was used.

When inserting the stirrer into the cell, align the vertical center line of the stirrer with the vertical center line of the cell, and make sure that the bottom of the stirrer is at the position of the dragon's sieve from the bottom of the cell. did.

The light source is an infrared light emitting diode (Monsanto ME7/
, 2g, center wavelength 0.9μ, wavelength half width Q, θ! μ)
was used. The beam had a diameter of 31 mm and its center was located at a height of 9 g mm from the bottom of the cell. A silicon photodetector (309-/θ manufactured by Verno 1 Well Co., Ltd.) was used to detect the transmitted light.

The measurement circuit is first! The block diagram shown in the figure was used.

Example/Measurement of agglutination reaction of anti-AIt'P antibody-sensitized latex (AFP=α-fetoprotein) (1)
Preparation of anti-AFP antibody sensitized latex Glycine buffer bath solution of anti-AFP antibody (mk': 2■/go)/θm/K% Polystyrene latex with an average particle size of 0.23 ￥μ (manufactured by Dow Chemical Company, solid content Concentration: /θ weight) / ml was added, stirred at ice temperature for 30 minutes, then heated to /-2θθOrpm). Decant the precipitate; g L, add the separated anti-AF/P antibody-sensitized latex to bovine blood groove albumin solution (concentration 20, 2% by weight) K
Suspend the sensitized latex particles to a concentration of 9% anti-A by weight.
A FP sensitized latex reagent was obtained.

(2) Transmittance measurement cell K O02rnl standard AFP solution and θ, θ ir
n1' anti-AFP antibody sensitized latex and 0. ．． 21m
1 glycine buffer solution was added. 100θrp with stirrer
While stirring at nn, from -0 seconds to 30 seconds after the start of stirring.
The average transmittance (T, ) from 0 seconds to 20 seconds and the average transmittance (T2) from 0 seconds to 20 seconds were measured. The average rate of change in absorbance, V, was calculated as 717 V = log (T+/T2). The measurement was repeated 77 times and the results were
Shown below.

Table / Above table / It is clear that when using the stirrer (A), the value of the change rate of absorbance is larger than when using the stirrer (B), and the accuracy is better. It is.

[Brief explanation of the drawings] Figure 7 shows stirrer B (comparison) used in Examples/
Figure 2 shows a four-wheel stirrer A (8A). In the figures, /-a and 2-a are front views, and /-b and 2-a are plan views. -10 is a side view. FIG. 3 and FIG. 9 are views showing other actual embodiments of the stirring bar of the present invention, respectively. 3-a and p-a are front views, 3-b and goo-b are plan views, and %goo-C is a side view. FIG. 1 is a block diagram of a measuring circuit when a peak hold circuit is used in the present invention. FIG. 6 is a diagram of signal waves ① to ② shown in FIG. 5. Applicant Mitsubishi Kashin Industries Co., Ltd. Agent Patent Attorney Hase Yo - et al. -ct 3-b 4-1゜

Claims (1)

[Claims] A method for measuring the optical properties of a sample while rotating the sample, in which at least part of the measuring light beam is not substantially blocked by the stirring bar while the stirring bar is rotating. An optical measurement method for a liquid sample, characterized by using a stirrer having a structure that passes through the liquid sample over the cell length and has the longest optical path length. (2) The method according to claim 7, wherein the stirrer has a hollow portion in a portion located within the optical path. (3) The method according to claim 1, wherein the stirrer has a hole or a notch in a portion located within the optical path. (4) The method according to claim 1, characterized in that the stirrer is opaque or translucent; (5) at least the measuring light beam! 2. A method according to claim 1, characterized in that % of the light passes through the length of the cell unobstructed by the stirrer. (6) The method according to claim 7, characterized in that a peak hold circuit that extracts an optical signal is used to measure the optical quantity. (7) A complete pulse is generated from an alternating current component of an optical signal that changes with the rotation of the stirrer in synchronization with the rotation, and a peak hold circuit is controlled by the pulse. the method of. (8) The method according to claim 6, characterized in that a rotational position sensor is provided on the stirrer, and the sensor generates a pulse synchronized with the rotation, and the pulse is used to control a peak hold circuit. (9) The liquid sample according to any one of claims 1 to 7, characterized in that the liquid sample contains inert carrier particles sensitized with an antigen or an antibody, and an antibody size or an antigen against the inert carrier particles. Method. 00 The method according to any one of claims 1 to 2, characterized in that the transmittance of light in a liquid sample is measured. Uυ A stirrer that stirs the liquid sample in the optical definition cell by rotation, and during rotation, at least a portion of the measuring light beam is substantially blocked by the stirrer (passing through the liquid sample over the length of the cell) A stirrer with a structure that allows (121) The stirrer according to claim 1, which has a hollow part in the part located in the optical path. (13) A patent claim characterized in that the part located in the optical path has a hole or a cut Range 1/A soft stirrer,