JP2794763B2 - Quartz crystal protein sensor - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a crystal oscillator protein sensor. More specifically, the present invention relates to a protein sensor that uses a quartz oscillator to achieve simple detection and quantification of protein.

[Prior art] and [Problems to be solved by the invention] For the quantification of protein, the conventional buret method, Lowry
Method, a fluorescence method, a dye binding method, a method of measuring absorption at a wavelength of 280 nm, and the like are used. However, these methods have problems such as a relatively long measurement time, a high level of skill in biochemical experiment techniques, and the need to use an expensive spectrophotometer. .

An object of the present invention is to provide a crystal oscillator protein sensor that avoids such problems and simplifies the detection and quantification of protein.

[Means for solving the problem]

The quartz oscillator protein sensor of the present invention that achieves the above object has a protein binding dye-immobilized film formed on the surface of the quartz oscillator.

The formation of the protein-binding dye-immobilized film on the surface of the quartz oscillator is performed by applying an approximately 1 to 10% aqueous solution of γ-aminopropyltriethoxysilane to the surface of the quartz oscillator, and heating at approximately 45 to 80 ° C. for approximately 1 hour. After drying by heating for about 30 minutes, immersing in about 0.1 to 5% glutaraldehyde aqueous solution for about 1 to 24 hours, and further immersing in about 1 to 5% dye dye solution for about 1 to 24 hours Done. Examples of this dye include Coomassie Brilliant Blue G250, Ponceau 3R, Nigrosine,
Synthetic or natural protein binding dyes such as Amido Black 10B, Xylene Brilliant Cyanine G, and Naphthalene Blue Black are used.

As the crystal unit, those having an AT cut and a vibration frequency of 5 MHz or more are preferably used.
It is shown as a plan view in the figure, for example, with a surface area of about 0.64
A silver electrode 22 is mounted in the middle of the crystal surface 21 of cm ² , and lead wires 23 and 2 are provided from each of the silver electrodes also mounted on the back side.
3 'has been pulled out.

[Function] and [Effect of the Invention] As shown in the following formula, the frequency of a crystal oscillator changes in response to the weight change of a substance attached to a surface. The white mass can be measured with high sensitivity.

ΔF = K · Δm ΔF: Frequency change Δm: Adhesion weight change K: Constant The crystal oscillator protein sensor according to the present invention is obtained by simply forming a protein-binding dye-immobilized film on the surface of the crystal oscillator. Therefore, it is inexpensive in terms of cost, and the measuring operation using it is also simple.

Examples of the protein measured by the crystal oscillator protein sensor include bovine serum albumin, bovine gamma globulin, and other biologically derived protein components.

〔Example〕

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

Example 1 A 5% aqueous solution of γ-aminopropyltriethoxysilane was applied to the surface of a quartz oscillator having the shape shown in FIG.
After heating and drying at 60 ° C. for 10 minutes, immersed in a 1% glutaraldehyde aqueous solution for 12 hours, and then at a constant temperature of 4 ° C., a 2% staining solution of Coomassie Brilliant Blue G250 dye (methanol 45 ml, glacial acetic acid 5 ml, water 50 ml) ) Dipped in the solution for 12 hours to immobilize the dye.

The vibrator surface of the crystal resonator protein sensor thus prepared is brought into contact with an aqueous solution of bovine serum albumin having a concentration of 12.5, 25, 50 or 100 ng / ml, and the frequency is measured using a vibration frequency measuring device. did. The result obtained is the third
As shown in the graph of the figure, it can be seen from the results that there is a correlation between the concentration of the protein solution and the amount of change in frequency.

Comparative Example In place of the bovine serum albumin solution, the same measurement was performed for an aqueous solution in which sucrose was dissolved in distilled water at the same concentration, but no change in frequency was observed.

Example 2 As a result of comparing healthy urine with protein-containing urine using the above-mentioned sensor, the protein mass of healthy urine was 5 mg /
dl, whereas the mass of protein in urine in patients with nephritis was 30
mg / dl, and a clear difference was observed, confirming that this sensor can be effectively used as a urine protein detector.

[Brief description of the drawings]

FIG. 1 is a plan view of one embodiment of a crystal resonator used in the present invention. FIG. 2 is an example of a frequency measuring circuit of the crystal resonator protein sensor according to the present invention. FIG. 3 is a graph showing the relationship between the protein concentration and the frequency change in Example 1. (Explanation of reference numerals) 21: quartz surface 22: silver electrode 24: quartz oscillator 25: quartz oscillator protein sensor 26: frequency counter

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 5/02 G01N 33 / 483,33 / 493,33 / 68

Claims (1)

(57) [Claims] 1. A crystal oscillator protein sensor comprising a crystal-binding surface on which a protein-binding dye-immobilized film is formed.