JPH05196578A - Evaluation of organic thin film for biosensor - Google Patents

Abstract

PURPOSE:To facilitate quick and firm inspection of enzyme activity and film surface by forming an organic film containing a pH indicating reagent and thereby optically measuring inside pH of the film. CONSTITUTION:The desired pattern is formed by painting a photoresist on a substrate 2, optically exposing from on a mask and then developing it. Onto the substrate 2, a mixture of a cattle serum albumin, a glutaric aldehyde solution and a phenol red as a pH reagent is painted. When this substrate 2 is soaked into an organic solvent such as an acetone to dissolve the photoresist, an organic thin film 1 of crosslinkage film of albumin-glutaric aldehyde which contains a pH indicating reagent, is patterned. Then, the substrate 2 is soaked into a buffer liquid 3 and measured using a microscopic system. That is to say, for pH measurement inside the organic thin film 1, a focus point of a microscope 4 is adjusted to an inside of the organic thin film 1 and color development of the pH indicating reagent is observed by a monitor 7, and therewith the pH inside the organic thin film 1 can be decided.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for evaluating an organic thin film for a biosensor.

[0002]

2. Description of the Related Art There have been many reports on biosensors in which an enzyme-immobilized film is provided on the surface of an electrochemical conversion element for detecting a change in interfacial potential or a change in current value between electrodes. For example, the following specific examples can be given as a glucose sensor in which the enzyme to be immobilized is glucose oxidase. That is, Biophysica Biochimica Acta, No. 320
Volume, 529-534, 1973; Analytical
Letters (Analytical Letters), Volume 19, 1973
~ 1986, 1986. The former is glucose-based by measuring the gluconic acid produced by glucose due to the catalytic action of glucose oxidase from the pH change.
The principle is to find the concentration On the other hand, the latter is also based on the principle that the glucose concentration is determined by measuring the hydrogen peroxide generated when glucose is oxidized by the catalytic action of glucose oxidase.

The enzyme activity of the enzyme-immobilized membrane is greatly affected by the pH in the membrane. Therefore, in order to realize a highly sensitive biosensor, it is necessary to design the enzyme-immobilized membrane so that the pH in the membrane matches the optimum pH of the enzyme. For that purpose, it is first necessary to accurately measure the pH in the enzyme-immobilized membrane. There has been an attempt to insert a capillary type pH electrode into the enzyme-immobilized membrane to measure the pH in the membrane. In addition, a planar type has been developed in place of the glass electrode type as a method for mass-producing a biosensor at low cost.
That is, an electrochemical conversion element is formed on a substrate such as silicon by using a semiconductor microfabrication technique, and an organic thin film such as an enzyme immobilization film is further patterned on the surface using a photoresist.
Have been proposed. In this way, hundreds or more biosensors can be manufactured from one wafer. The pattern of the enzyme-immobilized membrane formed by this method is 50 μm × 100 μm, and the thickness is 1 μm. When the response of the biosensor manufactured in this manner was evaluated in the wafer state, the probe was brought into contact with the wiring part one by one, and the buffer solution was dropped on the enzyme-immobilized film. Alternatively, the characteristics of the biosensor could not be revealed until they were cut out from the wafer one by one and mounted on a connector or the like.

[0004]

Since it is impossible to insert a conventional glass-type pH electrode into a fine film such as an organic thin film for biosensor, there is no suitable method for measuring the pH in the film. It was Further, in order to measure the enzyme activity held by the enzyme-immobilized film in a wafer state, it is necessary to measure the sensors one by one with a prober and evaluate all the sensors on one wafer. It took enormous amount of time and effort. Further, it is known that it is possible to control the measurement concentration range of the glucose sensor by forming a restricted permeation membrane outside the immobilized enzyme membrane. This limited permeation film needs to be free of defects such as pinholes, but in order to inspect it, it is still necessary to measure the sensors one by one with a prober. It took a great deal of time and effort to evaluate the sensor.

FIG. 3 is a schematic block diagram of a conventional method for measuring the enzyme activity of a biosensor in a wafer state. The conventional method will be described by taking the ISFET type biosensor as an example. ISFET
An organic thin film 24 is formed on the ion sensitive portion 21 and a source and drain electrode 26 is formed. In the buffer solution 23 dropped on the organic thin film 24,
The bar 22 is inserted, and the prober 27 is also brought into contact with the source and drain electrodes 26. The prober is connected to the measuring device 25 and the sensor output is measured. When all the sensors on the wafer are evaluated, an enormous amount of time is required because the operation of dropping the buffer solution on each sensor and bringing the prober into contact is repeated for each sensor. As described above, in the conventional method, the evaluation of the sensor in the wafer state requires time and cannot be automated. An object of the present invention is to solve the above problems and provide a method for surely evaluating the characteristics of a biosensor in a short time.

[0006]

The first aspect of the present invention is to evaluate an organic thin film for a biosensor, which comprises forming an organic thin film containing a pH indicator and optically measuring the pH in the film. Is. A second aspect of the present invention is to form an organic thin film containing a pH indicator, and optically measure the pH in the film to measure the activity of the enzyme immobilized in the film. It is a method of evaluating an organic thin film. A third aspect of the present invention is to form an organic thin film containing a pH indicator, and optically detect the pH in the film to detect defects on the film surface. Is. In the above three inventions, p
It is preferable that the H indicator is a pH-responsive fluorescent indicator.

The fourth aspect of the present invention is to evaluate an organic thin film for a biosensor, which comprises forming an organic thin film containing a redox indicator and optically measuring the activity of an enzyme immobilized in the film. Is the way. A fifth aspect of the present invention is a method for evaluating an organic thin film for a biosensor, which comprises forming an organic thin film containing a redox indicator and optically detecting defects on the film surface.

[0008]

In the present invention, the pH in the film can be optically measured by forming an organic thin film containing a pH indicator. In addition, it is possible to evaluate the enzyme activity and defects of the organic thin film for biosensor in a wafer state. Also, by forming an organic thin film containing a redox indicator, the enzyme activity and defects of the thin film can be evaluated in a wafer state.

[0009]

Embodiments of the present invention will be described below with reference to FIGS. FIG. 2 is a schematic configuration diagram showing the configuration when the in-film pH of the organic thin film containing the pH indicator described in claim 1 of the present invention is measured using a microscope system. The organic thin film 1 is formed on the substrate 2. The method of forming the organic thin film 1 by the lift-off method will be described below by taking an albumin-glutaraldehyde crosslinked film as an example. A desired pattern is formed by applying a photoresist on the substrate, exposing it from above the mask, and developing it. A 15% bovine serum albumin solution, a 1% glutaraldehyde solution and 0.1% phenol red as a pH indicator are mixed on this substrate and applied using a spinner. When this substrate is immersed in an organic solvent such as acetone to dissolve the photoresist, the albumin-glutaraldehyde crosslinked film containing the pH indicator is patterned. In the case of a biosensor film, the size is 50 microns × 100 microns and the film thickness is 1 micron. The substrate is immersed in the buffer solution 3 and the measurement is performed with the microscope system. The microscope system includes a microscope 4, an XY stage 5, a measuring device 6, and a monitor 7.
It is composed of The pH in the organic thin film can be determined by focusing the microscope inside the film and observing the color of the pH indicator on the monitor 7 to determine the pH in the organic thin film. When measuring pH accurately, a microspectrophotometer is used instead of the microscope. Since the absorbance of the pH indicator is proportional to pH, it is possible to determine the pH value by measuring the absorbance at an appropriate wavelength.

When pH is to be measured with higher accuracy, a pH-responsive fluorescent reagent such as fluorescein isocyanate is used as a pH indicator as described in claim 4 of the present invention, and a microscopic fluorometer is used as a microscope. To use. p
Since the fluorescence intensity of the H-responsive fluorescent reagent fluorescein isocyanate is proportional to pH, it is possible to determine the pH by irradiating the excitation light of wavelength 495 nm and measuring the fluorescence intensity of wavelength 520 nm. Strictly speaking, it is necessary to find the relationship between the fluorescence intensity and pH, and the method is shown below. For example, the fluorescence intensity is measured using a pH 7 buffer containing 1 mol of sodium chloride. At this time, a charged film such as an albumin-glutaraldehyde crosslinked film swells and the pH inside the film and the pH outside the film (in the solution) are the same. That is, the fluorescence intensity at this time corresponds to pH 7. Repeating this measurement with buffer solutions of different pH values, fluorescence intensity and p
It is possible to obtain the relationship of H (calibration curve).

FIG. 1 (a) is a schematic diagram showing the method for measuring the enzyme activity described in claim 2 of the present invention, and FIG. 1 (b) is an S diagram.
A biosensor formed on an i-wafer, as shown in FIG.
It is a schematic plan view of one biosensor. Here, a specific example of the biosensor will be described using an ion sensitive field effect transistor (ISFET). ISFET is a semiconductor sensor that detects pH in a solution. In case of glucose sensor for measuring glucose concentration, Fig. 1 (c)
As shown in, the organic thin film 12 having glucose oxidase immobilized thereon is formed on the ion sensitive part of the ISFET 11. Glucose is converted to gluconic acid by glucose oxidase, and the concentration of glucose can be determined by detecting the change in pH caused by this by ISFET. FIG. 1B shows the wafer 13.
The biosensor 14 formed above is shown. About 200 sensors are formed on a 2-inch wafer. FIG. 1 (a) shows a method for measuring enzyme activity using a microscope system. Since the pH changes due to the enzyme reaction and the color of the indicator changes, the presence or absence of the enzyme activity can be determined by observing this. The microscope system includes a microscope 15, an XY stage 16, an image processing device 17, and a monitor 18. For example, the wafer 13 is dipped in a buffer solution 19 containing 100 mg / dl glucose and the monitor 1
Wafer 1 by observing the color change of the indicator at 8.
It is possible to test the enzyme activity of 200 sensors on 3 at the same time. Further, in order to perform measurement with high sensitivity, a pH-responsive fluorescent reagent is used as a pH indicator and a fluorescence microscope is used as a microscope, as described in claim 4 of the present invention.
According to the method shown in FIG. 1 (a), the time required for the evaluation can be significantly shortened and automation can be performed, as compared with the method for measuring the enzyme activity of the conventional biosensor shown in FIG.

Next, the case of using the redox indicator described in claim 5 of the present invention will be described. Hydrogen peroxide is produced during the reaction between glucose oxidase and glucose. O-dianisidine, which is a redox dye, changes from colorless to red when it reacts with hydrogen peroxide. Therefore, by forming an organic thin film containing o-dianisidine and observing the color change with a microscope system, it is possible to test the enzyme activity as in the case of using a pH indicator. When quantifying the enzyme activity, a microspectrophotometer is used instead of the microscope, and the absorbance at a wavelength of 400 nm is measured.

Also when inspecting for defects on the surface of the permeable membrane described in claim 3 of the present invention, as in the case of measuring the enzyme activity, the wafer is immersed in a buffer solution containing glucose to adjust the pH.
Observe the change. When the limiting permeation membrane has a defect such as pinhole, glucose is locally permeated in a short time and
Cause a change in H. By finding such a local pH change on the monitor, it becomes possible to inspect defects in the permeation limiting membrane. Similarly, when the redox indicator described in claim 6 of the present invention is used, the defect on the film surface can be inspected by detecting the color change of the indicator due to the enzymatic reaction.

[0014]

In the past, it was impossible to insert a capillary type pH electrode with a micro membrane such as an organic thin film for biosensors, so there was no suitable method for measuring the pH in the membrane. In the present invention, by adding a pH indicator to form an organic thin film and optically measuring with a microscope system,
It is possible to measure the pH in the membrane. Further, in order to measure the enzyme activity retained by the enzyme-immobilized film in the conventional wafer state, it is necessary to measure the output of each sensor one by one using a prober, and It took a great deal of time and effort to evaluate the sensor. Further, in order to inspect for defects such as pinholes in the limited permeation film, it is necessary to measure each sensor with a prober in the same manner, which also requires a huge amount of time and labor. In the present invention, by adding a pH indicator or a redox indicator to form an organic thin film such as an enzyme-immobilized film and observing it with a microscope system, it is possible to reliably inspect the enzyme activity and the film surface in a short time. It is possible.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of an example of a measurement system that uses an evaluation method of the present invention.

FIG. 2 is a schematic configuration diagram of an example of a measurement system using the evaluation method of the present invention.

FIG. 3 is a schematic configuration diagram showing a conventional method.

[Explanation of symbols]

1,12,24 Organic thin film 2 Substrate 3,19,23 Buffer solution 4,15 Microscope 5,16 XY stage 6,25 Measuring device 7,18 Monitor 11,21 ISF
ET 13 Wafer 14 Biosensor 17 Image processing device 22, 27 Pro
26 electrodes

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[Procedure amendment]

[Submission date] March 9, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction content]

When pH is to be measured with higher accuracy, a pH-responsive fluorescent reagent such as fluorescein isocyanate is used as a pH indicator as described in claim 4 of the present invention, and a laser fluorescence microscope or a laser microscope is used as a microscope.
Use a device that combines a fluorescence microscope and a spectrophotometer . pH-responsive fluorescent reagent fluorescein isocyanate
Is the fluorescence intensity when irradiated with excitation light of wavelength 450 nm
Not affected by pH, but illuminated with excitation light of wavelength 550 nm
When irradiated, the fluorescence intensity is proportional to pH. Therefore, the excitation light
At wavelengths of 530 nm when is 450 nm and 550 nm.
The relationship between the fluorescence intensity ratio and the pH (calibration curve) is calculated in advance.
The pH of the organic thin film can be measured by
Wear.

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Claims (6)

[Claims] 1. A method for evaluating an organic thin film for a biosensor, which comprises forming an organic thin film containing a pH indicator and optically measuring the pH in the film. 2. An organic thin film for a biosensor, which comprises forming an organic thin film containing a pH indicator, and optically measuring the pH in the film to measure the activity of the enzyme immobilized in the film. Thin film evaluation method. 3. A method for evaluating an organic thin film for a biosensor, which comprises forming an organic thin film containing a pH indicator and optically detecting the pH in the film to detect defects on the film surface. 4. The method for evaluating an organic thin film for a biosensor according to claim 1, wherein the pH indicator is a pH-responsive fluorescent indicator. 5. A method for evaluating an organic thin film for a biosensor, which comprises forming an organic thin film containing a redox indicator and measuring the activity of an enzyme optically immobilized in the film. 6. A method for evaluating an organic thin film for a biosensor, which comprises forming an organic thin film containing a redox indicator and optically detecting defects on the film surface.