JP3665720B2 - Sensor device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sensor device for measuring a toxic substance present in a sample.
[0002]
[Prior art]
In today's environment, there are many substances (broadly toxic substances) that are harmful to living organisms. For example, trihalomethanes in tap water and dioxins in the atmosphere come to mind, but there are only a few other examples, but pollution of raw water and groundwater by so-called poisons such as cyan and cadmium is also sometimes popular in newspapers. Yes. By the way, how are these poisons detected? Many inspections at water purification plants use a system in which fish are kept in an aquarium filled with raw tap water, the movement of the fish is analyzed, and poisoning is detected when abnormal behavior is taken. Alternatively, there is a poison sensor developed by Fuji Electric that uses nitrifying bacteria. This is because, after adding a certain amount of ammonia, which is a feed for nitrifying bacteria, to the tap water, the nitrifying bacteria are brought into contact with an oxygen sensor immobilized on the electrode surface, and the activity of the nitrifying bacteria is estimated from the decrease in dissolved oxygen concentration. In addition, poisoning contamination is determined based on a decrease in activity. However, these methods can detect poisons almost in real time, but require maintenance such as feeding, and it is difficult to determine the type and amount of the poison.
[0003]
Conventionally, a poison sensor having a lipid bilayer membrane proposed by the present inventors is also known. In explaining this poison sensor, let's first consider the action of the poison. A poisonous substance is a substance that harms a living body, but it can be rephrased as a substance that harms a living body “cell” when considered closely. Therefore, the action of the toxic substance is considered to have some influence on the “cell membrane”. The “some effect” mentioned here means adhesion or adsorption to the cell membrane or incorporation into the cell membrane. Therefore, by attaching an artificially produced pseudo cell membrane (artificial membrane) to the surface of an electrode or the like, it becomes possible to measure the action of a poison on the pseudo cell membrane in real time. That is, the physical information (membrane potential, electric capacity, ion permeability, luminescence, heat generation, endotherm, etc.) of the pseudo cell membrane that changes due to the reaction with the poison is measured, and the contamination of the poison is determined from the difference in the output values before and after the poison reaction. It can be done.
[0004]
Next, the type and amount of the poison are determined as follows. In general, a cell membrane is constituted by incorporating a molecule such as protein or sugar into a lipid bilayer membrane containing phospholipid as a main component or adhering it to the surface. Therefore, the pseudo cell membrane is artificially produced by blending various molecules such as proteins and sugars that act with poisons based on lipid membranes or polymer membranes instead of them. In some cases, it is not an essential condition in the present invention to add a substance that acts as a poison. Therefore, it is possible to make various toxic sensors by changing the type and amount of lipid, protein, sugar, etc. to be used, as well as the method for producing the pseudo cell membrane. Then, the poison response of each individual poison sensor (how the output value changes according to the kind and amount of the poison) is obtained in advance, and based on these response patterns, the actual measurement results (multiple sensors) The type and amount of the poison are determined from the output value of
[0005]
A method for producing such a lipid bilayer membrane is as follows.
[0006]
Prepare an appropriate lipid solution (eg, n-decane as a solvent, may contain an appropriate toxic reactant), and apply a certain amount of Teflon membrane or Teflon coating in a buffer solution using a micro syringe. Into the nickel substrate (having a hole with a diameter of 1 mm or less in the center). If left as it is, the lipid bilayer membrane is naturally reconstituted in the pores, and at the same time, toxic reactants (such as glycolipids) are taken into the membrane.
[0007]
A poison sensor using such a lipid bilayer membrane is configured as follows.
[0008]
(1) Reagents Triolein (also known as glyceryl trioleate, molecular weight: 885, melting point: 4-5 ° C.) and trichlorethylene (common name: trichlene) were manufactured by Wako Pure Chemical Industries, Ltd. Monoolein (also known as glyceryl monooleate, molecular weight: 357, melting point: 33-34 ° C.), n-decane, and acetonitrile were manufactured by Kanto Chemical Co., Inc. Moreover, the cholesterol (molecular weight: 387, melting | fusing point: 149 degreeC) used the product made from Sigma. In addition, all the reagents used in the experiment were used as they were without any purification of commercially available special grade products. As the water, ion-exchanged water was used through an ultrapure water production apparatus (Millipore, Milli-Q). In addition, the nickel substrate (pore diameter: 0.2 to 0.6 mm, thickness: 10 mm) having a single hole used for forming the lipid bilayer membrane is a custom-made product manufactured by Optonix Precision, After the surface was covered with a Teflon film, the hole was broken with a needle and used.
[0009]
(2) Electrode and Measuring Cell FIG. 1 is a schematic diagram of a measuring device. A nickel substrate is placed in a quartz glass measurement cell (capacity of each par is about 25 mL, a custom-made product made in-house) 10 using silicon packing, and a lipid bilayer membrane is formed by brush coating in a hole formed in the substrate. Was made. The brush coating method is a method of creating a bilayer film by placing a lipid in a hole using a paintbrush containing a lipid solution and leaving it naturally (also called a natural thinning method). The production status of the lipid bilayer was observed using a digital microscope (VH-6300) manufactured by Keyence Corporation. Through this membrane, an Ag / AgCl electrode (manufactured by Toa Denki Co., Ltd., special order) 11 as a measuring electrode is inserted into the solution, and the potential difference (membrane potential) between the membranes is measured with an electrometer (Advantest, TR8411) 12. Measured.
[0010]
Data was recorded with a recorder (Type: 3047) 13 manufactured by Yokogawa Hewlett-Packard Company. The measurement cell and electrode were placed on a vibration isolation table to prevent physical vibration and the whole was placed in an electrical insulation box for measurement.
[0011]
(3) Experimental method A nickel substrate whose surface was coated with a Teflon membrane was set in a measurement cell via silicon packing, and 20 mL of 10 mM saline solution 14 was added to each part. Leave for about 15 minutes until the film is stable. Next, an appropriate amount of lipid is put into the hole of the nickel substrate by using a paintbrush containing a suitable lipid solution (the tip is slightly bent for easy operation). When lipid enters the hole, membrane resistance occurs, and the membrane potential changes rapidly. Then, when left gently, the membrane potential settles to a constant value and a lipid bilayer membrane is formed.
[0012]
Thereafter, 10 μL of trichlorethylene or cis-1,2-dichloroethylene (each acetonitrile solution) was added to the right part, and the same amount of acetonitrile was added to the left part. The measurement liquid was not stirred. This is because of concern about the influence of electrical noise and physical vibrations coming out of the stirring device.
[0013]
(4) Experimental results FIG. 2 shows an example of the measurement results of trichlorethylene (final concentration: 5 ppm). In this experiment, only monoolein was used as the lipid, and all measurements were performed at room temperature. The hole diameter of the nickel substrate was 0.6 mm. As is clear from this result, when the trichlorethylene solution is added, the membrane potential immediately decreases and reaches a minimum value, and then gradually increases to a constant membrane potential (change of about 1 mV compared to the initial membrane potential). I understand that In this case, it was also clarified that when trichlorethylene is repeatedly added, the equilibrium potential gradually changes with the number of times of addition (increase in trichlorethylene concentration). However, almost no change in membrane potential was observed after the fourth addition (the membrane was destroyed after the fourth addition). The change in the absolute value of the membrane potential is important, and the change in the polarity (+ side or-side) depends on the characteristics of the electrode to be used, so it is considered that it is not so important. From the above, it has been clarified that a single lipid bilayer membrane called monoolein exhibits a membrane potential response to trichlorethylene, and it can be said that the basic principle of a toxic biosensor using a lipid bilayer membrane has been confirmed.
[0014]
The poison sensor configured as described above does not require any maintenance to feed since the component is not a living organism or a microorganism. Further, by using the deviation from the steady state as an output value, the sensor can be used in a wide range of environmental conditions. Furthermore, since the output value from each toxic sensor can be transferred wirelessly to the signal processing facility, it can be used freely in rivers and oceans. In consideration of measurement sensitivity, the inventors have clarified that a lipid bilayer membrane is optimal as a pseudo cell membrane.
[0015]
[Problems to be solved by the invention]
However, lipid bilayer membranes are unstable with respect to external forces such as physical vibrations and static electricity, and their lifetime is at most several days. In addition, the work to form the lipid bilayer in the hole of the nickel substrate is done manually using a paintbrush containing a lipid solution, so it takes time and is difficult to measure in real time. It was insufficient for the practical application of poison sensors.
[0016]
Therefore, the present invention automatically detects the breakage of the lipid bilayer membrane and puts the lipid bilayer membrane automatically in the reproducible place in the reproducibility when putting the poison sensor using the lipid bilayer membrane into practical use. It is an object of the present invention to provide a device that can be formed automatically.
[0017]
[Means for Solving the Problems]
The sensor device of the present invention includes a liquid tank for storing an aqueous solution, a substrate in which a hole for forming a lipid film is inserted, and electrodes provided in the liquid tank on both sides of the substrate. A container for storing the lipid solution for forming the lipid film, a lipid solution discharge mechanism for discharging the lipid solution in the container into a hole for forming the lipid film formed on the substrate, and the hole for forming the film And a mechanism for detecting the breakage of the lipid film formed on the surface, and when the breakage of the lipid film is detected by this means, the lipid solution ejection mechanism is operated.
[0018]
According to the sensor device of the present invention having such a configuration, it is possible to obtain a sensor device that can determine the kind and amount of the poison in real time without maintenance.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below.
[0020]
FIG. 3 is a schematic diagram showing a configuration of a device for automatically producing a bilayer membrane in a poison sensor using a lipid bilayer membrane. In the present embodiment, the brush coating method used in the conventional measuring apparatus shown in FIG. 1 is automated. That is, an appropriate amount of lipid solution is stored in the storage tank 3. 4 is a controller, 5 is an Ag / AgCl electrode, 6 is a Teflon-coated nickel substrate, and 7 is a liquid bath. The discharge unit 1 automatically injects a predetermined amount (several micro-L level) of the lipid solution stored in the storage tank 3 into a hole in which the lipid film 2 is formed on the Teflon-coated nickel substrate 6. For example, a micro pump is applied to the discharge unit 1. The timing of the lipid solution injection is when the membrane potential constantly monitored by the controller 4 connected to the Ag / AgCl electrode 5 disappears. This is because the membrane potential disappears when the lipid bilayer membrane 2 is destroyed. Compared to the optical detection method, it becomes very simple. When the lipid solution is injected into the hole, a membrane potential of ± 10 mV or more is generated, and gradually settles at a constant potential as a stable bilayer membrane is formed. The actual toxic measurement is performed after the membrane potential becomes constant. As the lipid bilayer membrane 2, a monoolein / cholesterol bilayer membrane was used, and the hole diameter formed in the Teflon-coated nickel substrate 6 was 0.2 mm.
[0021]
FIG. 4 is a graph showing the results of measuring cis-dichloroethylene (DCE), which is an actual poison, using the poison sensor device configured for this purpose. It became clear that DCE can be measured even with the order of 50 ppb using this device.
[0022]
In the above-described embodiment, a micro pump is applied to the discharge unit 1, but an ink jet type (using piezoelectric element) pump may be used instead. It is characterized by being small and inexpensive compared to a micropump.
[0023]
It is also possible to use a plurality of types of single sensors composed of lipid bilayer membranes having different compositions.
[0024]
【The invention's effect】
By using the sensor device of the present invention, it is possible to supply a wide range of sensors for detecting and quantifying toxic substances with high sensitivity in real time without maintenance.
[Brief description of the drawings]
FIG. 1 is a schematic view of a conventional poisonous measuring apparatus.
FIG. 2 is a graph showing an example of membrane potential response when 5 ppm (final) trichlorethylene (TCE) is repeatedly added to the toxic substance measuring apparatus shown in FIG. 1 (application of a brush coating method, use of a monoolein bilayer).
FIG. 3 is a schematic diagram showing a configuration of a sensor device capable of automatically producing a bimolecular film according to an embodiment of the present invention.
4 shows an example of membrane potential response when 50 ppb (final concentration) cis-1,2-dichloroethylene (DCE) is added to the sensor device shown in FIG.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Discharge part 2 Lipid bilayer membrane 3 Reservoir tank 4 Controller 5 Ag / AgCl electrode 6 Teflon film nickel substrate 7 Liquid tank

Claims (5)

A liquid tank for storing an aqueous solution, a substrate in which a hole for forming a lipid film inserted in the liquid tank is formed, an electrode provided in the liquid tank on both sides of the substrate, and the lipid film forming A lipid solution storage mechanism for discharging the lipid solution in the container into a lipid film forming hole formed in the substrate, and a lipid film formed in the film forming hole. A sensor device comprising: a mechanism for detecting breakage; and a controller for operating the lipid solution discharge mechanism when breakage of the lipid membrane is detected by this means. The sensor device according to claim 1, wherein the mechanism for detecting breakage of the lipid membrane is configured by electrodes provided in the liquid tank, and these electrodes are connected to the controller. The sensor device according to claim 2, wherein the discharge mechanism is a micropump. The sensor device according to claim 2, wherein the discharge mechanism is an ink jet pump. The sensor device according to claim 2, wherein the controller detects the breakage of the lipid membrane using a change in membrane potential between electrodes provided in the liquid tank as a detection signal.

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