JP2001091494A - Sensor device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a maintenance-free poison sensor capable of determining the type and the quantity of poison at real time. SOLUTION: This poison biosensor using a lipid bilayer 2 is so formed that lipid solution fills a hole detecting the destruction of the bilayer and automatically forming a lipid bilayer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a sensor device for measuring a poison present in a sample.

[0002]

2. Description of the Related Art In today's environment, there are many substances (toxic substances in a broad sense) that are harmful to living organisms. For example, trihalomethane in tap water and dioxin in the air come to mind, but there are few other cases,
Contamination of raw water and groundwater by so-called poisons such as cyanide and cadmium is also frequently appearing in newspapers. By the way, how are these poisons detected? Inspection at the water purification plant keeps fish in a water tank into which raw water from tap water is poured, analyzes the movement of this fish, and analyzes the image.
Many use a system that detects the incorporation of poison when taking abnormal behavior. Alternatively, there is a toxicant sensor utilizing nitrifying bacteria developed by Fuji Electric. This means that after adding a certain amount of ammonia, which is the food of nitrifying bacteria, to 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. The determination of toxic contamination is based on the decrease in activity. However, these methods can detect a poison in almost real time, but require maintenance such as feeding, and it is difficult to determine the type and amount of the poison.

[0003]

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and makes it possible to determine the type and amount of a toxic substance in a maintenance-free manner with high sensitivity in real time. Things.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a sensor device which enables maintenance-free and real-time determination of the type and amount of poisons.

First, let us consider the action of the poison. Toxic substances are substances that cause harm to living organisms,
When considered in depth, it can be rephrased as a substance that harms living "cells". So the effect of the poison is
It is thought to have some effect on the "cell membrane". The term “some influence” as used herein means, for example, adhesion or adsorption to a cell membrane, or incorporation into a cell membrane. Therefore, by attaching an artificially produced pseudo cell membrane (artificial membrane) to the surface of an electrode or the like, the effect of a toxic substance on the pseudo cell membrane can be measured in real time. That is, physical information (membrane potential, electric capacity, ion permeability, luminescence, heat generation / endotherm, etc.) of the simulated cell membrane that changes due to the reaction with the poison is measured,
Poisoning can be determined from the difference between the output values before and after the poisoning reaction. So how is the type and amount of poison determined? In general, cell membranes are constituted by incorporating molecules such as proteins and sugars into lipid bilayer membranes containing phospholipids as a main component, and adhering to the surface. Therefore, a simulated cell membrane is artificially prepared by blending various proteins such as proteins and sugars that act with a toxic substance on the basis of a lipid membrane or a polymer membrane that substitutes the lipid membrane. In some cases, the addition of a substance that interacts with a toxicant is not a prerequisite in the present invention, since a response may be exhibited only by a substance. Therefore, various types of toxic sensors can be produced by changing the types and amounts of lipids, proteins, sugars, and the like used, as well as the method of producing the pseudo cell membrane. Then, the poison response (how the output value changes according to the type and amount of poison) of each poison sensor is obtained in advance, and based on these response patterns, actual measurement results (a plurality of sensors) are obtained. The type and amount of the poison are determined from the output value of the poison.

In the toxicant sensor configured as described above, a response can be obtained almost in real time, and no maintenance such as feeding is required at all because the component is not an organism or a microorganism. Further, by using a deviation from the steady state as an output value, it is possible to use the sensor under a wide range of environmental conditions. Furthermore, since the output value from each toxicant sensor can be wirelessly transferred to the signal processing facility, it can be used freely in rivers and oceans.
In consideration of measurement sensitivity, the present inventors have found that a lipid bilayer membrane is optimal as a pseudo cell membrane. However, lipid bilayer membranes are unstable against external forces such as physical vibration and static electricity, and their lifespan is at most several days. Therefore, in putting a toxicant sensor using a lipid bilayer into practical use, the breakage of the lipid bilayer is automatically detected, and the lipid bilayer is automatically formed immediately and with good reproducibility at the film formation site. I came up with the device.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (Examples) The present invention will be described below in detail with reference to examples. (Example 1) Production of lipid bilayer membrane A method (example) of producing a lipid bilayer membrane is outlined below.

[0008] An appropriate lipid solution (for example, n-decane is used as a solvent, and may contain an appropriate toxic reactant) is prepared, and a certain amount of a Teflon membrane is used in a buffer solution using a small syringe. Or into a Teflon-coated nickel substrate (having a hole with a diameter of 1 mm or less in the center). If left as it is, the lipid bilayer is spontaneously reconstituted in the pores, and at the same time, toxic reactants (such as glycolipids) are also incorporated into this membrane. (Example 2) Toxicological sensor using lipid bilayer membrane (manual preliminary test) (1) Reagents Triolein (alias: glyceryl trioleate, molecular weight: 885, melting point: 4-5 ° C) and trichloroethylene (commonly known as trichlene) Manufactured by Wako Pure Chemical Industries, Ltd. was used. Monoolein (alias: glyceryl monooleate, molecular weight: 357, melting point: 33 to 34 ° C), n-decane and acetonitrile were manufactured by Kanto Chemical Co., Ltd. Cholesterol (molecular weight: 387, melting point: 149)
℃) was used from Sigma. In addition, the reagents used in the experiments were all commercial grade products without any particular purification. As the water, ion-exchanged water was used through an ultrapure water production device (Millipore, Milli-Q). A nickel substrate having a single hole (pore diameter: 0.2 to 0.6 mm, thickness: 10 m) used for forming a lipid bilayer membrane
m) is a custom-made product manufactured by Optonics Seimitsu Co., Ltd., whose surface was covered with a Teflon (registered trademark) film, and the hole was broken with a needle before use. (2) Electrode and Measurement Cell FIG. 1 shows a schematic diagram of the measurement device used. A nickel substrate is placed in a quartz glass measurement cell (each part has a capacity of about 25 mL, a custom-made product made by the company) 10 using a silicon packing, and a lipid bilayer membrane is formed in a hole formed in the substrate by brush coating. Was prepared. The brush coating method is a method in which lipid is put into a hole using a paintbrush containing a lipid solution, and left naturally to form a bilayer membrane (also called a natural thinning method). The production status of the lipid bilayer membrane was observed using a digital microscope (VH-6300) manufactured by Keyence Corporation. Through this film, the measurement electrode Ag / A
A gCl electrode (Toa Denki Co., Ltd., special order product) 11 was inserted into the solution, and the potential difference between the membranes (membrane potential) was measured with an electrometer (Advantest Corp., TR8411) 12. The data is a Yokogawa Hewlett-Packard recorder (Type
e: 3047) 13. The measurement cell and the electrode were placed on a vibration isolator to prevent physical vibration, and the whole was placed in an electrically insulating box for measurement. (3) Experimental method A nickel substrate whose surface was covered with a Teflon film was set in a measuring cell via a silicon packing, and 20 mL of a 10 mM saline solution 14 was added to each part, and then the membrane potential (in this state, lipid No film is formed)
Let stand for about 15 minutes until is stabilized. 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 is generated, so that the membrane potential changes rapidly. After that, when the mixture is left quietly, the membrane potential settles to a constant value, and a lipid bilayer is formed. Thereafter, 10 μL of trichloroethylene 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 there was a concern about the influence of electrical noise and physical vibration from the stirrer. (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 a 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 trichloroethylene solution was added, the membrane potential immediately decreased and reached a minimum value, and then gradually increased to a constant membrane potential (about 1 mV change compared to the initial membrane potential). It turns out that it becomes. In this case, it was also clarified that when trichlorethylene was repeatedly added, the equilibrium potential gradually changed with the number of additions (increase in trichlorethylene concentration). However, in the fourth addition, almost no change in the membrane potential was observed (the membrane was broken 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. From the above, it was clarified that a single lipid bilayer called monoolein shows a membrane potential response to trichlorethylene,
It can be said that the basic principle of the toxicant biosensor using the lipid bilayer was confirmed. However, the instability of the lipid bilayer was also confirmed. (Example 3) Toxicological sensor using lipid bilayer membrane (investigation of automatic lipid bilayer preparation device 1) A schematic diagram (example) of an automatic bilayer preparation device is shown in FIG. In this example, it is considered that the brush coating method of Example 2 is automated. That is, a certain amount of lipid solution is automatically injected into a hole for preparing a lipid membrane (a few micro L level). It is assumed that a micro pump is applied to the discharge unit 1. The timing of injecting the lipid solution is when the membrane potential, which is constantly monitored, disappears. This is because when the lipid bilayer 2 is broken, the membrane potential disappears. It is very simple compared to the optical detection method. When the lipid solution is injected into the hole, a membrane potential of ± 10 mV or more is generated, and gradually settles to a constant potential as a stable bilayer membrane is formed. The actual poison measurement is performed after the membrane potential becomes constant. The storage tank 3 stores an appropriate amount of lipid solution. 4 is a controller, 5 is an Ag / AgCl electrode,
6 is a Teflon-coated nickel substrate, and 7 is a liquid tank. (Example 4) A toxicant sensor using a lipid bilayer membrane (investigation of an automatic lipid bilayer preparation device 2) In the embodiment of the third embodiment, a discharge unit adopts an ink jet type (using a piezoelectric element) pump. is there. It is characterized in that it is smaller and less expensive than a micropump. (Example 5) Toxic sensor using lipid bilayer membrane (actual toxicant measurement result) The result of measuring cis-dichloroethylene (DCE) using the toxicant sensor device of Example 3 is shown in FIG.
Using this device, DCE even at 50 ppb order
It has been found that can be measured.

[0009]

By using the sensor device of the present invention, it is possible to supply a maintenance-free and real-time sensor for toxic substance detection and quantification in a wide range.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a measuring device.

FIG. 2 shows an example of a membrane potential response when 5 ppm (final) of trichloroethylene (TCE) is repeatedly added (application of a brush coating method, use of a monoolein bilayer membrane).

FIG. 3 is a schematic diagram (example) of an automatic bilayer membrane production device.

FIG. 4 shows an example of membrane potential response when 50 ppb (final concentration) cis-1,2-dichloroethylene (DCE) is added (using a monoolein / cholesterol bilayer membrane) [pore size: 0.2 mm, automatic lipid bilayer membrane] Device usage].

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Discharge part 2 Lipid bilayer membrane 3 Storage tank 4 Controller 5 Ag / AgCl electrode 6 Teflon-coated nickel substrate 7 Liquid tank

Claims (6)

[Claims] 1. A toxicant sensor having a lipid bilayer capable of acting on a toxicant, wherein a lipid bilayer is automatically detected when a lipid bilayer is destroyed, and a lipid membrane is automatically formed inside a hole in a substrate. Sensor device. 2. The method according to claim 1, wherein the toxicant sensor comprises a container for storing the lipid solution, a mechanism for discharging the lipid solution, a mechanism for introducing the lipid solution into a hole in the substrate, and a mechanism for detecting breakage of the lipid bilayer. The sensor device according to claim 1, wherein: 3. The sensor device according to claim 2, wherein a mechanism that detects a signal of lipid bilayer destruction and automatically operates is linked to the mechanism for discharging the lipid solution. 4. The sensor device according to claim 3, wherein a pump or an ink jet system is applied as the discharge mechanism. 5. The sensor device according to claim 3, wherein a change in membrane potential is used as the detection signal. 6. The sensor device according to claim 1, wherein a plurality of types of single sensors composed of the lipid bilayers having different compositions are used.