JPH0232238A - Heat measuring biosensor - Google Patents

Abstract

PURPOSE:To output an exact signal with simple constitution and to extend the life of immobilized active enzyme by admitting a sample fed from a liquid feed tube at an equal proportion together with a buffer soln. to the two chambers of a column. CONSTITUTION:The sample fed from the liquid feed tube 3 is admitted together with the buffer soln. at the equal proportion to the two chambers 25a, 25b of the column 25. Heat generation is caused by the reaction by the immobilized enzyme in the chamber 25a in which a carrier 22 immobilized with the enzyme, etc., having activity is packed. The specific reaction does not take place in the other chamber 25b in which a carrier 23 immobilized with the deactivated enzyme, etc., is packed. The temps. in these two chambers 25a, 25b are measured by thermistors 21a, 21b and the exacter determination of the sample in which the non-specific detection component is removed by the differential signal thereof is executed. The activity of the active enzyme immobilized to the carrier 22 in the column 25 is lost by magnetic force and, therefore, magnetic force is applied to the column 25 when the sensor is not used. The immobilized enzyme is reactivated by eliminating the magnetic force at the time of use.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a hot needle fI11 biosensor that is applied to fermentation industrial process monitoring, environmental measurement, clinical chemical analysis, and the like.

[Conventional technology]

The above thermal measurement biosensor includes a specific selective sensing element such as an enzyme immobilized on a carrier such as porous glass or Eupergit, a column packed with this carrier, and a thermistor that detects thermal changes in the specific selective sensing element. The conventional configuration is shown in FIGS. 5 and 6.

The conventional example shown in Fig. 5 is a flow-type measurement system, in which 1 is a buffer tank containing a buffer solution 2, and 3 is a buffer tank 1 with a filter 4 attached to the tip. Tubes 5 and 6 are a Verisf pump and an injection valve that are connected in series to this liquid feeding tube 3, 7 is a sample injector that injects the sample into the injection valve 6, and 8 is a constant temperature bath. , a heat exchange section configured in a coil shape on the downstream side of the injection valve 6 of the liquid feeding tube 3; a column 11 connected to the downstream end of the liquid feeding tube 3 and filled with a carrier 10 on which an enzyme is immobilized; A thermistor 12 for detecting the calorific value of the immobilized enzyme in the column 11 and a thermistor probe 13 surrounding the thermistor 12 are arranged.

14 is a bridge amplifier connected to the thermistor 12 by a lead wire 15, and 16 is a recorder.

In the above configuration, the sample injected into the injector valve 6 by the sample injector 7 is sent in the buffer solution 2, and after being adjusted to a predetermined temperature in the heat exchange section 9 in the thermostatic chamber 8, it flows into the column 11. Here, it comes into contact with the immobilized enzyme on the carrier 10, and is then discharged. The immobilized enzyme in the column 11 performs a reaction according to the sample, and generates heat in response to the reaction. Thermistor 1 detects the temperature change due to this heat generation.
The sample is quantified by detection at step 2.

On the other hand, the one shown in Figure 6 is of the split flow type.
In the thermostatic chamber, the liquid feeding tube 3 is connected to the branch pipe 17.
Two branch liquid feeding tubes 18a.

Each branch liquid feeding tube 18a is branched into 18b.

18b with peristaltic pumps 19a, 19b and column 20a
, 20b are connected, and each column 20a, 2
Thermistor 21a is on 0b.

21b is the attachment. And the double force rams 20a, 2
A carrier 22 on which an active enzyme is immobilized is on one column 0b (active immobilized enzyme), and a carrier 23 on which an inactivated enzyme is immobilized on the other column 20b (inactive immobilized enzyme).
are each filled.

In this configuration, the buffer solution and sample sent from the liquid sending tube 3 are divided into two equal parts by the branch pipe 17, and are passed through the branch liquid sending tubes 18a and 18b, respectively, to the columns 20a and 20b. Inflow.

In the column 20a packed with the carrier 22 on which an active enzyme is immobilized, heat is generated due to the reaction of the active immobilized enzyme therein, and in the other column 20b, heat is generated due to the enzyme reaction because the enzyme therein is inactive. No specific fever.

Both thermistors 21
a.

21b, and the sample is quantified by obtaining the difference signal.

[Problem to be solved by the invention]

The flow type measurement system shown in Fig. 5 above is called a single type, and although it is easy to manufacture and configure, it has a large baseline drift and the accuracy of the measurement results is limited to 8.
There was a problem with the accuracy of the measurement results, as the measurement depended heavily on the temperature control ability of the meter.

On the other hand, with the split flow measurement system shown in Figure 6, more accurate measurement results can be obtained by eliminating baseline drift, non-specific heat, etc., but this system requires an increased number of parts. This results in a complicated configuration and high cost. Furthermore, the speed of the buffer solution passing through the columns 20a and 20b must be controlled approximately equally, which results in a lack of operability.

Furthermore, in the thermal measurement biosensor mentioned above, the lifespan of the active immobilized enzyme may be short, and for this reason, conventionally, the column filled with the carrier on which the active enzyme is immobilized must be removed and refrigerated when not in use. In addition, there was the problem that it was complicated to attach and detach the column each time the measuring instrument was used, and that the enzyme deactivated early in places where refrigerated storage was not possible.

The present invention has been made in consideration of the above, and has a single flow type measurement system and a simple configuration between roads, etc.
It has the same performance as the sprint flow type, that is, it can remove baseline drift and output accurate signals.
Furthermore, it is an object of the present invention to provide a thermal measurement biosensor that can extend the life of the active immobilized enzyme without refrigeration.

[Means to solve the problem]

In order to achieve the above object, the thermometer all+ according to the present invention
A biosensor consists of a specific selective detection element such as an enzyme immobilized on a carrier, a column that is filled with the carrier and connected to a liquid delivery tube that delivers the sample together with a buffer solution, and a temperature sensor that generates heat within the column. In the thermal needle Δ11 biosensor consisting of a thermistor that detects changes, the column is separated into two chambers parallel to the liquid feeding direction by a partition,
One chamber is filled with a carrier on which an active enzyme, etc. is immobilized, the other chamber is filled with a carrier on which an inactivated enzyme, etc. is immobilized, and a thermistor is attached to the outside of each chamber of the column. It becomes.

Furthermore, a magnetism generating element capable of turning on and off the effect of magnetic force is disposed on the outside of the column filled with a carrier on which enzymes and the like having the above-mentioned activity are immobilized.

[For production]

The sample sent from the liquid sending tube flows equally into both chambers of the column along with the buffer solution. Heat is generated during the reaction, and in the other chamber filled with a carrier immobilized with inactivated enzymes, there is no specific heat generation, and the temperature in both chambers is measured with a thermistor, and the difference signal is used to detect non-specific heat. A more accurate quantification of the sample excluding target bone is performed.

Furthermore, since the immobilized enzyme immobilized on the carrier within the column loses its activity due to magnetic force, when the column is not in use, a magnetic force is applied to the column, and when in use, the magnetic force is removed to restore the immobilized enzyme.

〔Example〕

Embodiments of the present invention will be described based on FIGS. 1 to 4. In the description of this embodiment, the same components as those of the conventional example shown in FIGS. 5 and 6 are given the same reference numerals, and the description thereof will be omitted.

FIG. 1 shows an embodiment of the first invention. In the figure, 25 is a column connected to the tip of the liquid feeding tube 3, and this column 25 is divided into two by a partition wall 26 that crosses the cross section of the column. There are two chambers 25 parallel to the direction of buffer solution flow.
a and 25b are configured. And these rooms 25a
.

The upstream side of 25b is connected to the liquid feeding tube 3, and the downstream side is connected to the thermistor probe 27 with equal cross-sectional areas. Also both parts above! 25a.

A thermistor 21a is installed on the outside of each downstream side of 25b.
, 21b is the attachment. Both rooms 25 in column 25 above
One of the chambers 25a and 25b is filled with a carrier 22 on which an active enzyme is immobilized, and the other chamber 25b is filled with a carrier 23 on which an inactivated enzyme is immobilized.

Note that the above-mentioned column 25 and thermistor 21a.

21b is placed in a constant temperature bath (not shown) as in the conventional example, and the upstream side of the liquid feeding tube 3 is connected to a heat exchange section placed in the constant temperature bath.

Further, the two thermistors 21a and 21b are each connected to a bridge amplifier so that both outputs are amplified to obtain a differential signal.

In the above configuration, the buffer solution and sample sent from the liquid sending tube 3 are transferred to both chambers 25a and 25 of the column 25.
It is divided into two parts and flows into b.

In the chamber 25a filled with the carrier 22 on which the active enzyme is immobilized, heat is generated due to the reaction of the active immobilized enzyme therein, and in the other chamber filled with the carrier 23 on which the inactivated enzyme is immobilized. In 25b, the enzyme within it is inactive and therefore does not generate a specific heat generation. And these rooms 25a
.

25b and both thermistors 21a.

21b, a difference signal is obtained, and the above-mentioned sample is quantified.

The above operation is substantially similar to the conventional example shown in FIG.

3 and 4 show an embodiment of the second invention, in which a magnet 27 having a shape surrounding the column 11 shown in FIG. 5 is placed on the outside and its magnetic force is turned on.

Arrange it so that it can be turned off. As shown in FIG. 3, this magnet 27 can be made by making semi-cylindrical magnets 28a and 28b using permanent magnets and attaching these semi-cylindrical magnets around the column 11 in a detachable manner, or
or coil 29 connected to the power supply around column 11.
The magnetic force acting on the column 11 by the coil 29 may be turned on or off by arranging the coil 29 and turning on or off the current of this coil 29. Further, in the case of this coil, it may be embedded outside the column 11.

In this configuration, when a magnetic force is applied to the column 11 by the magnet 27, the immobilized enzyme in the column 11 loses its activation, and when the magnet is removed, it becomes activated again.

Therefore, when the sensor is used, the magnetic force of the magnet 27 is removed to keep the immobilized enzyme in the column 11 in an activated state, and when the sensor is not used, the magnetic force is applied to keep the immobilized enzyme in an inactivated state.

The life of the immobilized enzyme in the column 11 is increased by the amount of time it is kept in an inactivated state, and therefore the life of the biosensor is increased by that amount.

Although the above embodiment shows an example in which the magnet 27 is arranged around the column 11 shown in FIG. 5, the magnet 27 may also be arranged around the columns 20a and 25 shown in FIGS. 1 and 6. Needless to say, it's a good thing.

〔Effect of the invention〕

According to the present invention, with a single flow type measurement system and a simple configuration such as between roads, it is possible to achieve the same performance as a split flow type, that is, to remove baseline drift and output accurate signals. I can do it.

In addition, the immobilized enzymes in columns 11, 20a and 25 can be easily controlled by turning on and off the magnetic force using magnetic generating elements, which eliminates the trouble of refrigerating the columns each time they are not in use. It is possible to extend its lifespan without spending too much time on it.

[Brief explanation of the drawing]

1 and 2 show essential parts of an embodiment of the first invention, FIG. 1 is a front view, FIG. 2 is a cutaway perspective view, and FIGS. 3 and 4 are views of the second invention. Figures 5 and 6 show conventional examples, Figure 5 is a circuit diagram showing a single flow type biosensor, and Figure 6 is a split diagram. FIG. 2 is an explanatory diagram showing the main parts of a flow type biosensor. 3 is a liquid feeding tube, 11, 20a, 20b. 25 is a column, 25a and 25b are rooms, and 27 is a magnet.

Claims (2)

[Claims] (1) A specific selective detection element such as oxygen immobilized on a carrier, a column filled with this carrier and connected to a liquid feeding tube 3 through which the sample is fed together with a buffer solution, and an exothermic temperature inside the column. In a thermometric biosensor comprising a thermistor for detection, the column 25 is separated into two chambers 25a and 25b parallel to the liquid feeding direction by a partition wall 26,
One chamber 25a is filled with a carrier 22 on which an active enzyme, etc. is immobilized, and the other chamber 25b is filled with a carrier 23 on which an inactivated enzyme, etc. is immobilized. Thermistor 21a, 21 on the outside
A thermal measurement biosensor characterized in that b is attached. (2) Turn on and off the action of a magnet on the outside of the column 11 filled with the carrier 22 on which enzymes etc. having the above activity are immobilized.
A heat measurement biosensor characterized in that a magnetism generating element such as a magnet 27 that enables F is disposed.