JPS61231419A - Method for measuring minute amount of organic substance - Google Patents

Abstract

PURPOSE:To calculate the wt. of an org. substance corresponding to the quantity of the change in the inherent frequency of a quartz vibrator, by coating the quartz vibrator with a material adhering an org. substance in a film form to adhere the org. substance to the surface of said material and measuring the change in the inherent frequency of the quartz vibrator caused thereby. CONSTITUTION:For example, a silver electrode is mounted to the center of a quartz surface having a surface area of about 0.64cm<2> and lead wires 3, 3' are respectively led out from said quartz surface 1 and the silver electrode. A material for adhering an org. substance is applied to thus constituted quartz vibrator 4 in a film form. This coating is performed by an arbitrary method, such as a drop method or a dip method. Next, the org. substance is adhered to the surface of this coated film material and the change in the inherent frequency of the quartz vibrator caused by the adhesion of the org. substance is measured and the wt. of the org. substance corresponding to said change quantity is calculated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for measuring trace amounts of organic substances. More specifically, the present invention relates to a method for measuring the weight of a trace amount of an organic substance such as an immobilized enzyme.

[Prior Art] and [Problems to be Solved by the Invention] A balance has conventionally been used to measure trace amounts of organic substances attached to the surface of a specific material. In this case, weigh the sample weight of the specific material in advance.

Thereafter, an organic substance is attached to the surface of the sample and weighed again, and the weight of the attached organic substance is determined from the difference in weight. However, this method has low sensitivity and does not meet today's requirements in terms of operability.

In view of these circumstances, the present inventors have conducted various studies in search of a method that can extremely easily measure trace amounts of organic substances.
It has been found that this problem can be effectively solved by making effective use of the phenomenon that the natural frequency of a crystal resonator changes depending on the weight of an organic substance attached directly or indirectly to its surface.

SUMMARY OF THE INVENTION Accordingly, the present invention relates to a method for measuring trace amounts of organic substances.

The measurement of trace amounts of organic substances involves coating a crystal resonator with a material to which the organic substance is attached, and then attaching the organic substance to the surface of the coated film material. This is done by measuring the change in frequency and calculating the weight of the organic substance corresponding to the amount of change.

The method of the invention carried out in this way can be used in many applications, for example in the field of biotechnology, such as biosensors, bioreactors, antithrombotic materials, or in the field of the production of organic films, such as monolayers, cumulative films, vapor-deposited films, etc. It can be used as a basic weighing technology in the industrial field. Below, as a typical example, a case where a physiologically active substance is used as an organic substance will be mainly explained.

Physiologically active substances such as enzymes and microorganisms can be used as biosensors,
It is used in bioreactors and the like, and when these are used, it is often attached to the surface of a specific material and used in an immobilized state.

For example, in the case of an enzyme, a compound having a functional group capable of reacting with an amino group in the enzyme molecule, such as a dialdehyde compound or a diisocyanate compound, is used as the material to be adhered. Further, in the case of an antithrombotic material in which adhesion of proteins and the like is the most important issue, the same material as the antithrombotic material is used.

In addition, in the field of manufacturing organic films such as vapor deposited films, monomolecular films, and cumulative films, silicon nitrogen (
513N4), silicon oxide (Sin2), etc. are used.

In the method of the present invention, first, these materials are coated onto the crystal resonator in the form of a film. This coating may be performed by any method such as drop method, dip method, cast method, spinner method, vapor deposition method, sputtering method, plasma polymerization method, or the like.

One embodiment of the crystal resonator used is shown in a plan view in FIG. , 3' are drawn out. An example of the frequency measuring circuit of the crystal resonator 4 configured in this way is the second
As shown in the figure, the measured frequency is measured by a frequency counter 5.

【Effect of the invention〕

According to the method of the present invention, the weight of a minute amount of physiologically active substance attached to the surface of the coating material can be easily measured.

〔Example〕

Next, the present invention will be explained with reference to examples.

Example 1 A predetermined amount of an aqueous urease enzyme solution with a concentration of 5■/++FI was added to the surface of the crystal resonator (frequency: 6 MHz) shown in FIG. 1, and the frequency change after drying was measured. As shown in the graph, the frequency change per mg of urease enzyme was 53,750 Hz.

From this, by measuring the frequency change, in this case, the amount of urease enzyme attached can be calculated.

Separately, a similar crystal oscillator was fixed in a pell jar of a vapor deposition apparatus, and 100 μa of γ-aminopropyltriethoxysilane and 50% glutaraldehyde aqueous solution were applied at 80'C15 Torr for 30 minutes. It was deposited sequentially. The frequency change for both membrane materials after washing with water and drying is 2787, and from the calibration curve shown in Figure 3, which does not change depending on the type of substance, the weight of the deposited membrane is approximately 5.2 μg. it is conceivable that.

Next, 20μQ of an aqueous urease enzyme solution with a concentration of 5μ/mQ was added to the surface of the crystal resonator on which this vapor-deposited film had been formed, and when it was left in a cold room overnight, washed with water, and dried, the frequency change was The frequency is 479 Hz, and when converted to the weight of urease enzyme, it is approximately 8.9 μg.

That is, the amount of urease enzyme immobilized on the vapor deposited film on the crystal resonator is approximately 8.9 μg.

Example 2 A silicon oxide sputtering film was formed on the surface of the crystal resonator shown in FIG.
When a predetermined amount of mQ of urease enzyme aqueous solution is added and the frequency change after drying is measured, as shown in the graph of Figure 4, the frequency change per 1 inch of urease enzyme is 450.
It was 00Hz.

Next, on the surface of the crystal resonator on which this sputtered film was formed, a vapor deposited film was formed and an aqueous urease enzyme solution was added in the same manner as in Example 1, and the frequency changes were measured for each of 8077 and 3407. The obtained values correspond to approximately 18 μg as the weight of the deposited film and approximately 7.6 μg as the amount of urease enzyme immobilized.

Example 3 242 μQ of a benzene solution of arachidic acid having a concentration of 0.02 g/Ω was added to a monomolecular film production apparatus to produce a monomolecular film. This monomolecular film is compressed, and the interfacial pressure is 10.1 dynes/a.
A crystal resonator whose surface had been treated with silicon oxide was moved up and down in the immersion liquid 10 times under conditions of No. 11. When the obtained immersed product was measured with a frequency counter, a decrease in frequency was confirmed compared to before immersion, and it can be considered that a cumulative film was formed.

[Brief explanation of drawings]

FIG. 1 is a plan view of one embodiment of a crystal resonator used in the method of the present invention. FIG. 2 is an example of a frequency measuring circuit for a crystal resonator. Moreover, FIGS. 3 and 4 are graphs showing the relationship between the amount of urease enzyme and frequency change. (Explanation of symbols) 1...Crystal surface 2...Silver electrode 4...Crystal oscillator 5...Frequency counter

Claims (1)

[Claims] 1. After coating a crystal oscillator with a material to which an organic substance is attached in the form of a film, an organic substance is attached to the surface of the coated film material, and the resultant crystal oscillator is A method for measuring trace amounts of organic substances, which is characterized by measuring changes in natural frequencies and calculating the weight of organic substances corresponding to the amount of change. 2. Claim 1 in which the organic substance is a physiologically active substance
Method for measuring trace amounts of organic substances as described in Section 1. 3. The method for measuring trace amounts of organic substances according to claim 2, wherein the physiologically active substance is an immobilized enzyme.