JP4255323B2 - Sensor chip precursor, sensor chip and poison detection system - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a sensor chip precursor, a sensor chip, and a toxic substance detection system, and more particularly, to a sensor chip precursor, a sensor chip, and a toxic substance detection system used 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 frequently seen in newspapers. .
[0003]
For example, in the inspection at a water purification plant, these poisons are kept in a tank filled with raw water, and the system analyzes the movement of the fish and detects the contamination of the poison when taking abnormal behavior. Has been detected.
A poison sensor using nitrifying bacteria is also known. This is because, after adding a certain amount of ammonia, which is a bait for nitrifying bacteria, to the raw 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 poisoning contamination is determined based on the decrease in activity (see, for example, Non-Patent Document 1).
[0004]
[Non-Patent Document 1]
ISAJ NewsLetter No. 9, 1999 "Biosensor for detection of toxic substances in raw water"
[0005]
[Problems to be solved by the invention]
However, these methods can detect poisons almost in real time, but require maintenance such as feeding, and have a problem that it is difficult to determine the type and amount of the poisons. In addition, there is a problem that the detection apparatus and system become large and it is difficult for the general public to use.
[0006]
The present invention has been made in view of such problems of the prior art, and its object is to be maintenance-free (chips can be discarded or replaced) and to simplify the type and amount of sample toxicity. It is an object of the present invention to provide a sensor chip precursor, a sensor chip, and a toxic substance detection system that can be measured.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor arranges a lipid membrane in the gap between the reference solution and the object to be detected, and measures changes in membrane potential and the like through this lipid membrane. The present inventors have found that the above problems can be solved and have completed the present invention.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the sensor chip precursor of the present invention will be described in detail. In the present specification, “%” indicates a mass percentage unless otherwise specified.
[0009]
The sensor chip precursor of the present invention is formed by filling a lipid film forming part provided on a substrate with a reference solution and a lipid film forming component. When this sensor chip precursor is used as a sensor chip, the lipid film forming component is heated to form a lipid film.
Another sensor chip precursor of the present invention is formed by providing a lipid film forming part on a substrate, filling the lipid film forming part with a reference solution and a lipid film forming component in this order, and then drying or cooling. In order to use this sensor chip precursor as a sensor chip, the precursor may be immersed in a reference solution.
Making such a precursor is effective because it can be stored for a long time until the time of toxic measurement, and a sensor chip can be easily prepared at any time according to the type, concentration, amount, etc. of the object to be detected. is there.
[0010]
Here, the substrate is, for example, glass, silicon, polyvinyl alcohol, polyvinyl chloride, polyethylene, silicone rubber, polypropylene, polytetrafluoroethylene, metal coated with an insulator (for example, a nickel plate having a polytetrafluoroethylene film) ) Etc. can be used as materials.
The substrate has a lipid film forming part. That is, an arbitrary number of recesses (wells) having openings are provided on the substrate, and a lipid membrane is formed through the reference liquid surface and well inner walls filled therein.
For example, a thin silicon substrate can be etched to form a large number of holes, and a substrate having a lipid film forming portion can be formed by bonding to a glass or silicon substrate on which a gold electrode has been previously arranged. Utilizing such silicon technology is desirable because it facilitates mass production of sensor chips.
[0011]
Further, the substrate may be formed from a single material, but when a material having a contact angle with water of 60 ° or more is used for the lipid film forming portion, it is preferable because a lipid film is easily formed. Examples of such a material include polydimethylsiloxane (PDMS).
In addition, although the magnitude | size of a lipid membrane formation part changes with the kind of lipid membrane to form, reference | standard liquid, etc., an opening diameter can be about 0.1-100 micrometers and a depth can be about 0.1-100 micrometers. Moreover, a lipid film formation part is not limited to the single side | surface of a board | substrate, You may form on both surfaces.
[0012]
Furthermore, as the standard solution, typically, saline, potassium chloride water and the like can be used, and these can also contain toxic reactants such as glycolipids.
Furthermore, the lipid film forming component can be appropriately selected depending on the reference solution, the material of the lipid film forming part, and the like. Typically, monoolein, triolein + cholesterol, phosphatidylcholine, phosphatidylethanolamine and the like can be mentioned.
[0013]
Next, the sensor chip of the present invention will be described in detail.
Such a sensor chip is formed using the above-described sensor chip precursor, and has a plurality of lipid film forming portions. Further, each of these lipid film forming portions is filled with a reference solution and a lipid film is formed thereon. The reference liquid and the constituent material of the lipid film can be changed for each lipid film forming unit, and a plurality of types of objects to be detected can be measured with one sensor chip. In addition, a specific object to be detected can be measured more accurately by using a reference solution made of the same type of constituent material or by providing a plurality of the same type of lipid membrane.
In addition, the lipid membrane included in the sensor chip is preferably a lipid bilayer membrane. As a result, various toxic substances that are harmful to the living body can be measured, and the toxic substance can be measured as a pseudo cell membrane by blending protein and sugar. For example, a lipid bilayer such as monoolein can be employed.
[0014]
Next, the poison detection system of the present invention will be described in detail.
Such a poisonous substance detection system is configured by disposing the above-described sensor chip in a solution containing an object to be detected. The sensor chip includes a lipid film composed of a plurality of types of lipids, and further includes an electrode in contact with the reference solution and an electrode in contact with the solution.
Here, the “poisonous substance” that can be detected by the present system means a substance that is harmful to a living body, and includes, for example, environmental pollutants such as trihalomethane, dioxin, cyanide, and cadmium. In addition, if this “poisonous substance” is investigated, it can be rephrased as a substance that harms the living body “cell”. Therefore, it is conceivable that the toxic substance has some influence on the “cell membrane”, that is, it is adhered or adsorbed to the cell membrane or taken into the cell membrane.
[0015]
Therefore, in the toxin detection system of the present invention, for example, by mounting an artificially produced pseudo cell membrane (artificial membrane) on the surface of an electrode or the like, it is possible to measure the action of the poison on the pseudo cell membrane in real time. Become. That is, physical information (membrane potential, electric capacity, ion permeation amount, luminescence amount, calorific value, endothermic amount, etc.) of the lipid membrane that changes due to the reaction with the toxicant is measured, and the difference in the output value before and after the toxicant reaction is measured. Mixing can be determined. In addition, it is preferable that a plurality of the sensor chips are provided. At this time, it can be disposable or replaced at any time according to the lifetime of each sensor chip.
[0016]
In the poison detection system of the present invention, 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.
Thus, a pseudo cell membrane can be artificially prepared by blending various proteins such as proteins and sugars that act with poisons on the basis of a lipid membrane or a polymer membrane instead thereof. Depending on the type of poison, there may be a response with only the lipid membrane. In addition, the sensor chip can be made compatible with various poisons by appropriately selecting the type and amount of lipid, protein, and sugar to be used, and the method for producing a pseudo cell membrane. Then, the poison response of each sensor chip (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 poison can be determined from the output value from the chip).
[0017]
Specifically, a sensor chip or the like can be provided as measurement means, and further data storage means and analysis means can be provided. In the data storage means, when the solution contains a toxic substance, the membrane potential through the lipid membrane, the electric capacity, the ion permeation amount, the luminescence amount, the calorific value / the endothermic value, and values relating to any combination thereof The changes are stored as a database. Further, the data obtained from each well (electrode) of the sensor chip is transmitted to the analysis means through a telephone line, a wireless LAN, etc., and the type and amount of the detected object are specified by collating with the database. Is done.
When a disposable sensor chip including a sample pretreatment unit such as a filter or a reference electrode is used as the measurement means, the problem of contamination between samples can be overcome. FIG. 3 shows a usage example of the disposable sensor chip.
[0018]
As described above, the toxic substance detection system of the present invention can obtain a response in almost real time, and does not use organisms or microorganisms as constituents, and therefore does not require any maintenance such as feeding. Further, by using the deviation from the steady state as the output value, the sensor chip can be used in a wide range of environmental conditions. Furthermore, since the output value from each sensor chip can be wirelessly transferred to the signal processing facility, it can be freely used in rivers and oceans. Furthermore, a compact device (system) using a sensor chip that is inexpensive, disposable, and replaceable can be provided, particularly when used in a general home.
In consideration of measurement sensitivity, it is more preferable to employ a lipid bilayer membrane that can be a pseudo cell membrane as the lipid membrane of the sensor chip. On the other hand, lipid bilayer membranes are unstable to external forces such as physical vibrations and static electricity, and their lifetime is at most several days. In the present invention, the number and size of sensor chips to be used and the number of lipid films provided on the sensor chip can be adjusted according to the lifetime of the lipid film, and the sensor chip can be replaced at any time as a disposable type. The period of use is not limited by the lifetime.
[0019]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to these Examples.
[0020]
Example 1
<1> Mold production method (1) A silicon wafer cut into 4 cm × 4 cm was subjected to ultrasonic cleaning in pure water for 2 minutes. Subsequently, it wash | cleaned, heating with acetone. Further, the silicon wafer was immersed in a solution of HF: NH4F = 1: 6 for 3 minutes. Thereafter, it was washed with running water, and water on the silicon wafer was blown off.
(2) A photoresist (SU-8, manufactured by MicroChem) was dropped onto a silicon wafer, and a SU-8 thin film (30 to 40 μm) was formed using a spin coater.
(3) Using a hot plate, it was heated to 90 ° C. in 30 minutes, and further heated and maintained at 90 ° C. for 30 minutes.
(4) A photomask was aligned with the silicon wafer and exposed.
(5) Using a hot plate again, it was heated to 90 ° C. in 30 minutes, and further heated and held at 90 ° C. for 30 minutes.
(6) Using a SU-8 developer, the unexposed portion of SU-8 was peeled off.
[0021]
<2> PDMS (polydimethylsiloxane) sheet production method (1) The PDMS solution was degassed for 15 minutes.
(2) One drop of PDMS was dropped on the mold and a sheet was placed. This was sandwiched between the silicon sheet and metal plate from both sides and firmly fixed with clamps.
(3) (2) was placed in an oven and heated at 80 ° C. for 1.5 hours.
(4) Before cooling, the clamp was removed, the metal plate / sheet was peeled off, the solidified PDMS sheet was peeled off from the mold, and stored in ethanol.
[0022]
<3> Attachment of PDMS sheet (1) The slide glass was washed with dilute hydrochloric acid and then with acetone.
(2) The PDMS sheet stored in ethanol was taken out and washed with acetone.
(3) The PDMS sheet of (2) was placed on the slide glass of (1) and dried.
(4) Oxygen plasma treatment was performed for 30 seconds with a RIE apparatus (Reactive Ion Etching System) to obtain a sensor chip substrate provided with a lipid film forming part.
The mold preparation and PDMS sheet affixing work were all performed in a clean room. Moreover, after vapor-depositing chromium and gold on the slide glass, a PDMS sheet was pasted to prepare an electrode. FIG. 1 shows an image of a lipid membrane chip, and FIG. 2 shows an enlarged photograph thereof.
[0023]
<4> Formation of lipid membrane (1) First, each well of the sensor chip substrate was filled with 10 mM saline as a reference solution (the substrate was immersed in saline). At this time, a gold (Au) electrode was formed on the bottom of each well, and lead wires were connected from the back surface.
(2) Next, 5% monoolein (n-decane solution) was layered on the surface of each well with a brush and allowed to stand at room temperature.
(3) Waiting for the solvent decane to evaporate (about 1 hour), the entire sensor chip substrate was rapidly cooled to 10 ° C. or lower.
Through the above operation, a sensor chip precursor in which the surface of each well of the sensor chip substrate was coated with a monoolein thin film was obtained. Note that the sensor chip can be stably supplied by refrigerated storage in this state.
[0024]
<Evaluation measurement>
As shown in FIG. 3, a test tank provided with a reference electrode was prepared, and the sensor chip precursor was installed in this test tank. Thereafter, 10 mM saline as a sample solution was injected into the test tank at a low temperature, and the sensor chip precursor was immersed therein. Then, the salt solution was heated to 30 ° C. or higher. As a result, the monoolein thin film was dissolved, and a bilayer structure was naturally formed to function as a sensor chip. The toxic substance detection system was configured by connecting the independent lead wires (covered with an insulator) included in the sensor chip and a reference electrode to an external potentiometer.
As a result of measuring the membrane potential response for each well using the above system, a membrane potential of about 15 mV was observed for each well. Subsequently, cis dichloroethylene was added to the test tank so that it might become 1 ppm, and it measured again. As a result, a membrane potential increase of about 10 mV was observed for each.
[0025]
In the toxic substance detection system of the present invention, in order to analyze the type and concentration of toxic substances in detail, it can be estimated by providing different lipid membranes for each well and comparing their membrane potential responses. Further, by providing the same lipid membrane in a plurality of wells, it is possible to improve the analysis accuracy. Furthermore, even if the lipid membrane is in a thick film state, environmental pollutants can be measured by the same measurement method. Further, the sample solution may be the same type of solution as the reference solution, or the concentration of the electrolyte may be appropriately changed according to the measuring method (quantitative analysis or qualitative analysis) of the toxic substance.
[0026]
【The invention's effect】
As described above, according to the present invention, a lipid membrane is disposed in the gap between the reference solution and the object to be detected, and changes in membrane potential and the like through this lipid membrane are measured. It is possible to provide a sensor chip precursor, a sensor chip, and a toxic substance detection system that can easily measure the type and amount of toxicity of a sample.
[Brief description of the drawings]
FIG. 1 is a schematic view showing an example of a sensor chip.
FIG. 2 is an enlarged photograph showing another example of a sensor chip.
FIG. 3 is a schematic view showing an example of a poison detection system using a disposable sensor chip.

Claims (9)

A substrate, at least one concave lipid film forming part provided on the substrate, a reference liquid filled in the lipid film forming part, and a lipid film forming component that forms a lipid film by heat treatment, A sensor chip precursor comprising: A sensor chip precursor comprising a substrate, at least one concave lipid film forming portion provided on the substrate, and a reference liquid and a lipid film forming component filled in the lipid film forming portion. ,
A sensor chip precursor , wherein a membrane film formed by drying or cooling of the lipid film-forming component covers the reference solution . A sensor chip using the sensor chip precursor according to claim 1 or 2,
The sensor chip precursor has a plurality of the lipid film forming parts,
The plurality of lipid film forming units correspond to different objects to be detected,
A sensor chip comprising a lipid film formed from the lipid film-forming component formed on the reference solution .   The sensor chip according to claim 3, wherein the lipid membrane is a lipid bilayer membrane.   5. The sensor chip according to claim 3, wherein a material having a contact angle with water of 60 ° or more is used for at least a portion of the substrate where the lipid film is formed. A poison detection system comprising the sensor chip according to any one of claims 3 to 5 disposed in a solution containing an object to be detected,
The toxin detection system, wherein the sensor chip has a lipid film composed of a plurality of types of lipids, and includes an electrode in contact with the reference solution and an electrode in contact with the solution.   7. The toxic substance detection system according to claim 6, wherein said electrode comprises means capable of detecting a change in membrane potential through a lipid membrane.   The poison detection system according to claim 6 or 7, wherein a plurality of sensor chips are arranged, and each sensor chip can be disposable at any time and / or can be replaced at any time. A measuring means, a data storing means and an analyzing means, wherein the measuring means is at least one value selected from the group consisting of membrane potential through the lipid membrane, electric capacity, ion permeation amount, luminescence amount and calorific value / endothermic amount. The data storage means stores the change in the value when the solution contains the object to be detected, and the analysis means collates the measurement result and the storage data to identify the object to be detected. The poison detection system according to claim 6 .

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