JPS5847484A - Analytical device for specimen of living body - Google Patents

Abstract

PURPOSE:To analyze components of a living body in high sensitivity, by mixing a specimen passed from a column for immobilized oxidase with luminol and a solution of red prussiate of potash, sending it to a flow cell, detecting chemical emission. CONSTITUTION:A specimen injected from the specimen inlet 4 together with a buffer is sent to the column 2 for immobilized oxidase by the rotation of the peristaltic pump 9 in an fixed amount, and it and hydrogen peroxide caused by oxidation are sent to the mixing means 10. While a solution of red prussiate of potash and a luminol solution are passed from the feed paths 6 and 7 through the spiral pipe 8, blended with the specimen by the mixing means 10, sent to the flow cell 11, and chemical emission is detected by the receptor 12. By selecting the oxidase, glucose, uric acid, etc. can be analyzed. Continuous analysis can be effected by changing plural columns.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a biological sample analyzer. (More specifically, the present invention relates to a highly sensitive biological sample analyzer that utilizes an immobilized oxidase that produces hydrogen peroxide and chemiluminescence of sensitized hydrogen using a luminescent reagent.)

Life is maintained by a dynamic equilibrium state IIi of a wide variety of substances, but the quantitative and qualitative fluctuations are closely linked to various diseases. Therefore, by analyzing the biological components and knowing the exact changes, it is possible to understand the pathological condition. Conventionally, these analyzes have been carried out by chemical analysis, but recently methods using enzymes, which are more advantageous in terms of specificity, sensitivity, etc., have been developed and are being put into practical use. However, most of these methods use enzymes from horse mackerel (*ii) and have abandoned the possibility of repeated use of p-enzymes. For this reason, an analysis method using a so-called immobilized enzyme tube, in which enzymes are placed on the surface of a support such as glass beads, has been proposed, and various biological sample analysis devices using this have been studied. .

This development was made possible through intensive research into a flow system biological sample analyzer that uses an immobilized oxidase that produces hydrogen peroxide, and has a higher sensitivity than conventional ones. The present invention provides a biological sample analysis device. In order to enable particularly highly sensitive measurements, the inventors of the present invention discovered that the hydrogen peroxide tube produced from the immobilized oxidant, the luminol-red blood salt-based luminescent agent from a specific supply channel, 19 Adopts a flow system that generates chemiluminescence and introduces it into a specific cell [-1] Combines a measurement method that digitally integrates the amount of chemiluminescence from the C cell over a certain period of time using a photo/counter via a light receiver. We are pleased to have achieved the desired purpose (particularly) and to have arrived at this invention.In addition, there has recently been a research report in which glucose is introduced into a hydrogen peroxide chemiluminescent system for analysis, but the inventors of this invention To the best of our knowledge, these methods employ conventional analog methods to detect chemiluminescence, have problems in sensitivity, and are difficult to develop into a multipurpose flow system.

Thus, according to the present invention, the analyte component supply channel supplies the analyte component together with the buffer at a predetermined flow rate through the immobilized oxidase column, and the luminol solution and red blood salt solution are supplied at a predetermined flow rate through the spiral tubular premixing section. A luminescent agent supply flow channel, a test component-luminescent agent mixing means connecting the above-mentioned flow channel, a coplanar spiral flow channel-like flow cell connected to the mixing means, and a constant flow channel on the light transmission surface of the flow cell. The light-receiving surface is surface-coupled with the area, and the large-bone optical device and the light-receiving device O
A biological sample analyzer using chemiluminescence is provided, which includes a photon counter that integrates a photon receiver for a predetermined period of time.

The present invention will be described in detail below with reference to the % figures aiK.

1, the invention is not limited thereby.

First, a block diagram of a specific example of the biological sample folding apparatus of the present invention is shown in IIIWJ. In FIG. 5, the test component supply channel (1) t! , a buffer supply channel (■), a sample inlet (4) attached to the supply channel (■), an immobilized fermentation enzyme column (
21 and a flow path connected to the mixing means. On the other hand, the luminescent agent supply channel (5th generation), the large runol solution supply channel (61) and the red blood salt supply channel (7), both channels 1
6) and (7), a spiral tubular premixing section (8
) and a flow path connected to the mixing means. Set I (Tuffer supply channel (31, luminol solution supply channel t6) and red blood salt solution supply channel <7) are adjusted by the peristaltic ν take pump 191 so that a predetermined flow rate can be supplied, respectively.

Above, the test component supply channel (1) and the luminescent agent supply channel (!
I>tX It is connected to a 4-diet mixer which is one of the mixing means 00. Figure 2 shows the flow path configuration in the 4-jet mixer. Flow paths (1) and (5) are 4-jet z
Each channel is divided into two channels within the quina, and these channels meet at point A, so that the component to be detected and the luminescent agent are mixed with high efficiency.

The mixing means (2) K has a spiral flow path shape 70 in the same plane.
- A rectangular spiral flow channel-shaped flow cell, which is one of the cell boxes, is integrally connected. The light-receiving surface of the light receiver is surface-coupled to the light-transmitting surface 0 of the flow cell U with a certain area. l [Figure 3 shows a 1 m cross-sectional view of a concrete connection of the mixing means member [channel (5) is omitted in the figure], 70-cell I, and receiver @. 70-cell #! Shown as a plan view in Figure 4,
The outlet channel of the mixing means is connected to the center of a 70-cell spiral channel in the form of a rectangular spiral channel. The outlet of the flow cell is connected to a drain.

A 7-photon counter (2) is connected to the photoreceptor υ mentioned above, and a recorder is attached to the photon counter 03. One example is a configuration in which a phototube is used to digitally display the number of photons and an output voltage corresponding to the number of photons is recorded on a recording needle.

The analysis of a biological sample using the analyzer of the present invention as described above is carried out as follows when measuring a glucose tube. In this case, glucose oxidase (hereinafter referred to as COD) is used as the oxidase of -nitridase.

First, a biological sample is introduced from the sample person [] using a syringe. At the same time, by operating the peristaltic pump (9) at a constant rotation speed, the biological sample is guided to the immobilized COD column together with the buffer at a constant flow rate. Hydrogen peroxide corresponding to the glucose concentration in the biological sample is produced in the column and then introduced at a constant flow rate into the 4-jet Net-, which is a mixing means.

On the other hand, due to the operation of the peristaltic pump, the luminol solution and the red blood salt solution are supplied at a constant flow rate to the spiral tubular prexing part (8) and mixed for a certain period of time.
The jet tank is introduced at a constant flow rate. In this way, by mixing the luminol solution and red blood salt solution for a certain period of time before reacting with the test component, it is possible to attenuate the background light emission caused by the reaction between luminol and red blood salt. Next, by mixing in a four-jet mixer, hydrogen peroxide, which is a component to be tested, reacts with the luminescent agent, thereby producing chemiluminescence corresponding to the hydrogen peroxide concentration.

The mixed liquid undergoing chemiluminescence is guided at a constant flow rate into the aforementioned 70-cell in the form of a coplanar spiral flow path, and is discharged through the drain. Then, by integrating the amount of light emitted from the mixed liquid passing through the flow cell over a certain period of time using a light receiver and a photon counter and comparing it with a standard sample, the hydrogen peroxide concentration and, in turn, the glucose concentration in the sample can be quantified with high sensitivity. I can do it.

The oxidase used in this invention may be any oxidase that produces hydrogen peroxide, and various oxidases can be used depending on the combination with the target of analysis. If you keep it, as mentioned above, when glucose is the subject of analysis, t! If GOD is a numeric value, uricase (UILIC) is used, and if galactose is used, galactose oxidation IH1l (G a L
OD), but in the case of cholesterol O, cholesterol oxidase (COD) is used. Immobilization of these oxidases can be achieved, for example, by activating long-chain alkylene glass beads with bromine cyanide and adding the desired After adding oxidase and reacting under appropriate & pH conditions, washing with buffer is carried out. The immobilized oxidase column of the present invention can be obtained by enclosing the immobilized glass beads in, for example, an acrylic resin chape. - In this invention, the supply of buffer, luminol solution and red blood salt solution may be carried out by one peristaltic pump as described above, or may be carried out by separate peristaltic pumps, t
In addition to this, other liquid feeding pumps that can control the supply amount can be used. Furthermore, instead of attaching the sample inlet to the buffer supply path, the sample may be supplied by connecting an auto sampler (4°) as shown in the IIs diagram.

In this invention, the Lukinol solution and red blood salt solution, which serve as luminescent agents, are mixed in advance for a certain period of time in a spiral tubular pre-extrusion section, and then used for reaction with the test component. Therefore, compared to the conventional method of simultaneously mixing luminol solution, red blood salt solution, and test component to emit light, it is possible to greatly reduce the luminescence from luminol and red blood salt, that is, the background. It is possible to accurately measure the amount of luminescence caused only by the reaction with colored hydrogen peroxide.&O, this is especially limited because it is related to the tube diameter and length of the spiral tubular preplexing part, the flow rate, and the concentration of the luminescent agent. The shape of the spiral tube is not limited to that, but may be any spiral tube shape that can sufficiently mix the luminescent agent and sufficiently attenuate background light emission.

As the mixing means used in this invention, the above-mentioned 4-jet mixer is suitable from the viewpoint of combination with the 70-cell. good.

The flow cell according to the present invention has a spiral flow channel in the same plane, which is suitable for uniform mixing of the luminescent agent and the test component and accurate measurement of the amount of luminescence. As the spiral flow path, the above-mentioned rectangular spiral flow path may be mentioned, and in addition to this, a circular spiral flow path or the like may be used. By combining such a flow circuit with a light receiver and further connecting these to a photon counter that electrically counts the amount of light received through an interface, j! Accurate luminescence measurement becomes possible. Note that the light-receiving surface of the light receiver needs to be surface-coupled to the light-transmitting surface of the 7g-cell, but the surface-coupling may be done not on the entire light-transmitting surface of the flow dragon but on the surface.
Further, as the photoreceiver, a known photomultiplier tube or a photomultiplier tube can be used.

'& O, for example, 10 pl of glucose solution (5011F
/d) is used as a sample, the optimal conditions are luminol solution ;L7SX1@ in a spiral cell.
-'M, red king salt solution; 5X1G-"M; flow rate; 711
1//*, pH; 1 G, 5, premixing time: 1 minute. 1, in this case 1G-〇~10-
``Linearity is obtained in the concentration range of M, and the detection sensitivity f is 309mo.
It was l. Therefore, as shown in the tR1 diagram, in the analyzer of this invention, if the luminescent reagent and buffer tube are used in Tybon tubes with the same inner diameter (2 tone), the optimal concentration is luminol solution; Salt solution;! X
When a 20 mM acetate buffer is used as the buffer, the pH of the luminol solution is preferably 11.0.

In the configuration of the present invention, by using different immobilized oxidase columns and applying means for sequentially switching these columns, it is possible to perform multiple analysis from the same sample.

Therefore, from this point -〇 from another point, -tt+-g1+pa^
a analyte component supply channel having a plurality of different immobilized oxidase columns connected in parallel via a valve, and supplying a analyte component together with a buffer in a predetermined amount through any of the columns by opening and closing the valve; A luminescent agent supply channel that supplies a luminol solution and a red blood salt solution at a predetermined flow rate through a spiral tubular premixing section, a test component-luminescent agent 1 combination means that connects both channels, and a means for mixing the luminescent agent. It consists of a spiral flow cell in the same plane, a light receiver whose light-receiving surface is surface-coupled with a certain area on the light-transmitting surface of the armpit cell, and a photon counter that calculates the amount of light received by the light receiver over a predetermined period of time. A multi-item biological sample analyzer using chemiluminescence is provided.

aSS shows a specific example of the multi-item biological sample analyzer of the present invention. In FIG. 6, side, mouse, - and @ respectively indicate electromagnetic valves, (to) is a liquid pump, and @111
4 interface, rBu computer, rIa is printer, (5 is operation panel, (c) is panel display, (
) indicates a recorder. And (2A) is the identified COD
Column (2B) is an immobilized URIC column, and (2B) is a -electrified COD column, respectively.

The electromagnetic valves are all operated by a combiator, and are configured so that the number of samples, the position of the standard sample, and the amount of each sample can be input into the combinator and adjusted by operating the operation panel. The amount of sample is controlled by the opening/closing time of the solenoid valve. The analytical operations are as described above, except that the p column can be selected by switching the solenoid valve. Therefore, with the above configuration, it is possible to analyze glucose, uric acid, and cholesterol with high sensitivity from 10 samples. Furthermore, by applying another immobilized oxidase column, it becomes possible to perform multi-item biological sample analysis of the above three or more items.

The biological sample analyzer of the present invention uses a specific and highly sensitive analysis method that fully utilizes the catalytic ability of enzymes, is easy to operate, and is suitable for multi-item analysis.

Due to its high sensitivity, it is particularly useful in the field of pediatrics, etc., where analysis of minute amounts of samples is required.

Furthermore, it is easy to connect to a computer system.

Below, nine embodiments of the biological sample analyzer according to the present invention will be shown.

Example 1 A biological sample analyzer having the configuration shown in Fig. 1 was prepared,
Glucose or uric acid measurements were performed.

Enzyme insolubilization U 5ebnapp, 8hal it
It was carried out according to the method of in. [ was coupled with the long-chain acylamine glass beads KpHILO activated with bromic cyanide. Enzymes include glucose oxidase (COD, Toyobo) and urikar-t' (URIC).

Toyo Jojo) was used, and the immobilization rate was
Chi, 71*f1h. The enzymes were immobilized and the glass beads were incorporated into a ceataryl resin column (3X301111) into a flow system.

The liquid was fed using a peristaltic tube using a tiebon tube with an inner diameter of 2 mm.

Inject the sample using an autonumerator or 1JV Italo'°
It was performed using a syringe.

(2) Quantitative determination of glucose Glucose was determined using the apparatus 1 using immobilized darcose oxidase. Glucose solution (Zhang 5shou f
/ll1-S Opt/d) Measurement was performed by injecting 10 pl using a microsyringe. μz as buffer
@mM acetate buffer (pH II) was used, and the other optimal conditions described above were used. Figure 7 shows the relationship between glucose concentration and luminescence amount. Linearity was obtained in the concentration range of , and the detection sensitivity was 3 (lpmol).
I made it lzs. ), and under these measurement conditions, it was necessary to dilute the serum 't-5e to ioo times to quantify glucose in normal and abnormal serum. In order to measure the serum without diluting it, the luminescence intensity was lowered to f' (concentrated liter of Akako salt solution [;5XiO-
', tz u y p -11 &''/u) The results are shown in the tms diagram.

In this case as well, the glucose concentration is 1j2s ~ ZSOq/dl (
Almost straightness is obtained in the range of 10-"M-10-"M).

■ Quantification of uric acid Next, add the uric acid constant 'jl' using an immobilized uricase tube, and add uric acid solution (1111/1it-10M1i/
dl) was measured by injecting LOpl with a microsyringe.

Buffer and [2: 10mM Borate buff
er (pH&O) and under the same optimal conditions as in the case of U-glucose O. As shown in Figures 11i and e, uric acid concentration @: (1~/d7-10at/d1)
A linear relationship was obtained between the amount of light and the amount of light emitted (note that the counter sensitivity was [ll1.]).

■ Quantification of blood darcose and uric acid -S in human serum measured using an autoanalyzer in a clinical laboratory was measured using the above device, and the correlation between the two was examined. Clarification enzyme
Uric acid was determined by the peroxidase method (Hitachi 10@Katsu) and by the phosphotungstic acid reduction method (Technicon & -6ol). Calibration was performed using standard serum (~1 an
d Q-PAKI and Q-PAK■). The diagram shows the results of glucose t-measurement using an immobilized COD column.

In 24 cases of human serum, the correlation coefficient 7 = 0.983, which is large, and Figure 1111 is for the identified uricase column.
The results of uric acid determination were shown, and the correlation coefficient was 7=(L973) for human serum concentration S%, and both glucose and uric acid showed a good correlation.

Example 2 A biological sample analyzer similar to that shown in Fig. 6 was prepared except that an immobilized COD column and an immobilized uricase column were used as the immobilized oxidase column, and the simultaneous preparation of glucose and uric acid was carried out. Do it.

The optimum pH of each enzyme is different, but since it is technically difficult to change the pH for each item, we investigated buffer solutions and found that by using phosphate buffer, pH & 0, both It was found that it can be used without significantly inhibiting oxidase activity. In addition, it was found that luminescence was inhibited when the concentration of the buffer solution was made of gold, so it was decided to take the stability of the buffer solution into account and perform the measurement using a 10mM phosphoric acid buffer OYt. Other conditions such as the concentration of the light reagent, pre-mixing, flow rate, etc. were kept under the optimum conditions described above.

Guk: f-su (Final concentration WL2s, 50% too.

150.200% 250 da/dl) and uric acid (final concentration 1.0% λO% 10%&O% 110,140 da/dl)
The results of quantitative analysis using a mixed solution of
Shown in Figure 2.

mKu Showing the relationship between glucose and uric acid concentrations and their luminescence amount, each sample amount is measured in order of uric acid Zpl (U in the figure)
, glucose has two voices! (Gl in the figure) and 4-I (Gl in the figure)
G.) introduced. In addition, the counter feel f is Shuhata/l
I chose Goose 8. As a result, linearity could not be obtained at high concentrations of glucose and uric acid #1 degree of 1 sov, /az, and 11 da/dj, respectively. Next, 10 samples of the same
The sample amount was diluted twice and the amount was adjusted to uric acid Spl, glucose t.
A similar experiment was conducted with spl and 10pl introducedvr
As shown in FIG. 13, the counter feeling f is L 6 /
Samples diluted 10 times with t211 showed good linearity. Linearity was also obtained in experiments using samples diluted with Mara sO.

[Brief explanation of the drawing]

Figure 1g1 shows Ikko 113 of the biological sample analyzer of this invention.
The figure is alla! mixing means in the apparatus shown in ll;
Jk! showing the connection state IIN of the flow cell and photoreceiver. It is a u sectional view. 144 Figure U% Collection 70 of the device shown in Figure 1
- A plan view showing the cell. FIG. 5 is a block diagram corresponding to FIG. 1 showing another specific example of the biological sample analyzer Q of the present invention; FIG. be. Figure m7 and Figure 8 are graphs showing -Nt- of the measurement results of a glucose solution using the biological sample analyzer equivalent to Figure 1. Figure m9 is a graph showing an example of the measurement results of a uric acid solution using a biological sample analyzer equivalent to Figure 1. Figure 910 and Figure 15 are graphs showing the correlation between the measured values of glucose concentration and uric acid concentration in good serum using a biological sample analyzer equivalent to Figure 1, and the values observed by the respective autoanalyzers. @ tM, 12 figures and i! FIG. 13 is a graph showing the measurement results of a glucose solution and a uric acid solution, respectively, using the multi-item biological sample analyzer of this invention similar to W45-. (1) ...Test component supply channel, (2), (!A),
(2B), (ZC)...immobilized oxidase column, (5
) - Luminescent agent supply channel, 161 - Luminol solution supply channel, (7) - Red blood salt solution supply channel, (8) - Spiral tubular premixing section, 191 - Peristaltic pump, scream ...4 jet mixer, ■--spiral flow cell in the same plane, υ... light receiver, (to)-- photon counter, (2)-- light receiving surface, 171... light transmitting surface , mackerel, concave, ■, ■−・electromagnetic no(rupu, (branch)−・
Feed pump. Fig. 1 4 Fig. 2 Fig. 7 Fig. 8 Fig. 9

Claims (1)

[Claims] The test component is passed through the immobilized oxidase column.
a luminescent agent supply channel that supplies a luminol solution and a red blood salt source at a predetermined flow rate through a spiral tubular pre-construction part; a test component-luminescent agent mixing means that connects the
A flow cell in the form of a 1,000 m rounded flow channel connected to the wrinkle mixing means, a light receiver whose light receiving surface is surface-coupled with a certain area on the light transmitting surface of the skeleton flow cell, and the amount of light received by the light receiver is integrated for a predetermined period of time. Biological sample analyzer using chemiluminescence, which uses 1111g from a photon counter. 1 A test sample having a plurality of different immobilized oxidase column tubes connected in parallel through pulp, and supplying the test component together with a buffer at a predetermined flow rate through one of the columns in the valve 4M11. A component supply flow path, a luminescent agent supply flow path that supplies a luminol solution and a red blood salt solution at a predetermined flow rate through a spiral premixing tube, and a test component-luminescent agent mixing means that connects both of the flow paths. a spiral flow cell in the same plane connected to the mixing means; a light receiver having a light-receiving surface coupled to the light transmitting surface of the flow cell with a certain area; and a photon counter that integrates the amount of light received by the light receiver for a predetermined period of time. A multi-item biological sample analyzer using chemiluminescence.