JPH02309247A - Vasopressin detection sensor - Google Patents

Abstract

PURPOSE:To make the title sensor inexpensive and to enhance the capacity thereof by using a measuring pH-ISFET covered with a vasopressin monoclonal antibody immobilized film. CONSTITUTION:A drain 2 and a source 2' are formed on a P<->-type silicon substrate and drain and source electrodes 5, 6 are formed thereto by vapor deposition. An SiO2 film 7 is formed to the surface of an element by thermal oxidation and an Si3N4 film is formed by a CVD method. A vasopressin monoclonal antibody immobilized film 10 reacting only with vasopressin is provided and the part other than a gate part is molded by a resin mixture 9. This electrode is used as a detection electrode and an electrode having the same structure as said electrode but no vasopressin monoclonal antibody immobilized film 10 is used as a comparing electrode.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is applicable to acetylcholine, dopamine, serotonin,
Even when other substances such as γ-aminobutyric acid and glutamic acid are present, only pasopressin is felt, and it has attracted attention as one of the substances related to senile dementia, and is present in the paraventricular nucleus and supraoptic nucleus of the hypothalamus of the cerebrum. The present invention relates to a vasopressin detection sensor that detects the concentration of vasopressin.

Conventional technology The detection of vasobrecin, which has attracted attention as a substance related to senile dementia, has conventionally been performed using a combination of liquid chromatography and radioimmunoassay as separation and quantitative methods, or radioimmunoassay (radioimmunoassay). Immunoassay RIA)
It was done well.

When combined with a liquid chromatography column, a specific substance is adsorbed using an antigen/antibody reaction that reacts only when a specific substance arrives, and the substance is marked with an isotope to determine its radiation amount. Quantification is performed based on the strength of The isotope to mark is 35S, 125
A lot of tools are used.

Problems to be Solved by the Invention However, the above-mentioned method involves extracting, separating and quantifying body fluid from a living body, and does not directly measure local information within the body. In addition, the equipment used to detect the radiation amount (liquid syntellinometer, r-ray hectometry, scintillation capacunter, etc.) is large, and the half-life of S is 87.9 days, and that of 125I is stable at 60 days. Problems include: time-consuming detection, low accuracy of detectors, need for facilities to handle irradiated materials and radioactive materials, strict protection against radiation, limited number of people who can handle them, and high total cost. was there.

In order to eliminate the drawbacks of the conventional method, the present invention uses the action of a vasopressin monoclonal antibody to react only with vasopressin to form a vasopressin monoclonal antibody-immobilized membrane proportional to its concentration, thereby changing the membrane potential. Potential)! I-i provides a compact and inexpensive I8FET sensor for detecting pasopressin that can be easily measured. Note that this detection sensor is also capable of measuring in vivo.

Means for Solving the Problems In order to achieve the above object, the technical solution of the present invention is as follows:
A measurement ISFET (ion-sensitive field effect transistor) covered with a membrane immobilized with a Pazoprene monoclonal antibody whose membrane potential changes depending on the antibody/antigen reaction on one substrate, and a reference ISFET without an immobilized membrane are installed on one substrate. By preparing it and compensating for the environmental temperature, it senses only pasopressin and indicates its concentration.

Effects According to the present invention, even if various substances coexist, only pasopressin undergoes an antibody-antigen reaction with the vasopressin monoclonal antibody, and a pasopressin monoclonal antibody-immobilized membrane is formed. Inside the membrane, the membrane potential changes depending on the concentration of pasopressin present outside, which changes the gate voltage and induces electrons in the channel of the FET, causing a current to flow between the source and drain. It is subject to changes even if the membrane potential or temperature of the external solution changes.
By installing ISFETs in parallel that sense only the membrane potential and temperature of the external solution, this is compensated for by a push-pull differential circuit and the concentration of only pasopressin in the solution is detected.

Example Embodiments of the present invention will be described in detail below with reference to the drawings.

Figure 1 (a) is a top view of the measured ISFET (ion-sensitive field effect transistor), and Figure 1 (b) is (
It is a sectional view taken along 1-1' of a). 1 is a P-type silicon substrate. 2.2 is a drain and source n+ region made by doping it with phosphorus by diffusion. 3 is P
-It is the channel part of the mold.

4 is a P+ channel stopper in which boron is diffused and formed on the surface other than the gate portion at the tip. 5 is a drain electrode on which Au-Cr is deposited. 6 is a source electrode on which Au--Cr is deposited. 5.6 are the drain and source electrodes AI! It is a terminal. 7 is a 5iOz film formed on the element surface by thermal oxidation. 9 is a mixed resin of epoxy and silicon which is molded except for the gate portion. 10 is a pasopressin monoclonal antibody that reacts only with pasopressin.

FIG. 2 shows the configuration of a reference pH-ISFET (ion-sensitive field effect transistor), and FIG. 2(a) is a plan view,
FIG. 2(b) is a sectional view taken along line 1-1' of FIG. 2(a). The reference pH-ISFET is at the measured pH shown in Figure 1.
- It has almost the same configuration as the ISFET, but differs in that no immobilization film is provided in the gate part.

Next, a method for immobilizing the Pazobrelen monoclonal antibody on a membrane will be described.

1 of (3-aminopropyl)triethquinsilane and water
Add IMHcJ to 0=1 solution and adjust to p)('y,
Place the gate electrode part in a water bath heated to 50℃ for 2 hours.
5iaN4 is chemically modified by r-soaking reaction. The chemically modified gate electrode is made of cellulose triacetate 250.
mg to dichloromethane 10mJ? and further 50
After immersing in a solution containing 100 μl of glutaraldehyde and 500 μl of 4-aminomethyl-1,8-octanediamine, the sample was stored in a desiccator for 1 day to allow the crosslinking reaction to proceed. Furthermore, aldehyde groups were introduced onto the surface of the organic film by reacting it with a 1% glutaraldehyde solution for 1 hour at room temperature, and after thorough washing with a pH 7 phosphate buffer, the sapphire substrate FET gate was coated with pasopressin monoclonal antibody at 4°C. The pasopressin monoclonal antibody is immobilized on the membrane by substituting the amino group with the aldehyde group by immersing it in a saturated dispersed solution.

FIG. 3 is a measurement circuit diagram of I S FET for detecting pasopressin.
A constant drain current Id always flows through another current source 18.
A constant voltage Vd=RIl is applied between the source and drain of the measured ISFET II.

Even if the interfacial potential changes due to the membrane potential in the solution, the measurement IS
Since Id and Vd of FETII cannot be changed, the source,
The potential between the drains changes by the amount of change in the interfacial potential, and v
The change is output to Outl. In order to improve response characteristics to changes in solution concentration and uniform membrane potential changes, the reference ISFET 12 outputs Vout 2 in the same manner. 24 is a differential amplifier for taking the difference.

To measure, first set up a tiny microsyringe,
A small amount of body fluid (volume: 0.5 μl) was taken out into this solution, and a measurement ISFET II on which vasopressosin monoclonal antibody was immobilized and a bare ISFET without immobilization (Reference I
SFET) 12 and Ag with chloride on the surface
/Ag The three electrodes of the C1 reference electrode 13 are brought into liquid junction. Both ISFETs by op amp 14.15.16.17
A drain current of 10 .mu.A is always passed through U and 12, and a constant DC voltage (1 to 2 V) is applied between the source and drain through the current source 18, 19, 20, 21 and resistor 22.23. Then, the temperature change and membrane potential change of the measurement solution are compensated for by the differential measurement circuit, and the differential amplifier 24 obtains an output that depends only on the concentration of bazobrene, resulting in the output result shown in FIG. 4.

Effects of the Invention As described above, the effects of the present invention are as follows: Measurement I 5FET covered with a membrane on which Pazobrene monoclonal antibody is immobilized on one substrate and reference JSF without the immobilized membrane.
Two types of ET have been prepared, and the measurement is easy and inexpensive, with a small size that could not be obtained conventionally, and it is not affected by the temperature of the solution or membrane potential, does not require temperature compensation, and can coexist with various types of substances. However, since this pasopressin monoclonal antibody has selectivity, it is possible to provide a detection sensor that only acts on pasopressin. Note that this detection sensor is also capable of measuring in vivo (local information within the living body can be measured by the current between the source and drain).

[Brief explanation of drawings]

FIG. 1(a) is a plan view of an ISFET for measuring vasobrenosine in one embodiment of the present invention, and FIG.
), FIG. 2(a) is a plan view of the Pasopressin reference 18FET, FIG. 2(b) is a sectional view along the 1-1' plane of FIG. The figure shows a measurement circuit diagram of the I 8 FET for detecting pasopressin of this example, and FIG. 4 shows the relationship between the concentration of vasobrenosine and the current between the source and drain. Engineering...P-type silicon, 2...Drain n region, 2'
...Source n region, 3...P-type channel, 4
... Channel stopper (P), 5... Drain electrode, 5'-Drain electrode Al terminal, 6... Source electrode, 6'8.・Source electrode AJ terminal, 7...SiO2
, 8... Si3N4.9... Mixed resin of epoxy and silicone, 10... Pasopressin monoclonal antibody solidified membrane, 11 Measurement pH-FET, 12... Reference p1 E-FET. 13 ・Ag/Ag (J reference pole, 24...differential amplifier. Name of agent: Patent attorney Shigetaka Awano and 1 other person No. 1
Figure (Q,) 10? i#C rise Figure 2 Figure 3 1δ ISF Hill T/2 Figure 4 PP” Conquest

Claims (1)

[Claims] Measurement ion-sensitive field-effect transistor (ISFET) whose gate was coated with a membrane immobilized with a pasopressin monoclonal antibody whose membrane potential changes due to antibody-antigen reaction with pasopressin, and a reference ion-sensitive field-effect transistor without coating. 1. A pasopressin detection sensor, which is characterized in that it includes a magnetic field effect transistor and determines the concentration of pasopressin based on the compensated outputs of both.