JP2009204375A - Electrode base for biomolecule detection and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrode base for biomolecule detection not causing an electrochemiluminescence amount to lower even in case of a carbon electrode, and its manufacturing method. <P>SOLUTION: This electrode base for biomolecule detection and its manufacturing method are characterized in that acidic groups comprising either carboxyl groups or sulfonic acid groups bonding to only the surfaces of a plate substrate using any of glass, plastic resin, ceramics, and those of a carbon electrode using any carbon material out of carbon paste, glassy carbon, and DLC, on the plate substrate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a detection electrode substrate for electrochemically detecting a specific biomolecule present in a biochemical sample and a method for producing the same. More specifically, the present invention relates to a detection electrode substrate for detecting an electrochemical signal from a labeling agent with high sensitivity and a method for manufacturing the same.

  As a technique for detecting a specific biomolecule to be detected, such as a specific nucleic acid, antigen, or antibody, by using a specific binding ability such as a nucleic acid hybridization reaction or an antigen-antibody reaction, a labeling agent is bound to the biomolecule, Many methods for detecting by the presence or absence of a labeling agent have been proposed.

  Examples of the labeling agent used in these include radioisotopes, fluorescent dyes, and electrochemiluminescent materials. In particular, electrochemiluminescent materials emit light during an electrically reversible oxidation-reduction reaction. It is useful as a labeling agent that can be easily detected without the danger of a radioisotope and without the need for excitation light such as a fluorescent dye.

  As a method using such an electrochemiluminescent substance as a labeling agent, there is a method using magnetic fine particles. In this method, a sample containing a biomolecule to be detected, a magnetic fine particle to which a substance that specifically binds to a biomolecule is fixed, and an electrochemiluminescent substance to which a substance that specifically binds to a biomolecule is bound are mixed. Then, a complex in which the electrochemiluminescent substance is bonded to the magnetic fine particles through the biomolecule is formed. The composite is collected by magnetic force and washed to perform B / F (Bound / Free) separation that removes the unreacted sample and the electrochemiluminescent material. Thereafter, the composite is dropped on the electrode, and the composite is collected on the electrode surface by magnetic force. Then, by applying a voltage to the electrode and measuring an electrochemical signal from the labeling agent bound to the complex, the presence of a biomolecule to be detected is detected (for example, Patent Document 1). And Patent Document 2).

  A feature of the method using the magnetic fine particles is that the composite can be collected on the electrode surface by magnetic force. The electrochemical reaction is performed by exchanging electrons between the electrochemiluminescent substance and the electrode, and the reaction region is a very narrow region called several nanometers from the electrode surface called an electric double layer. Therefore, highly sensitive detection requires collecting more complex on the electrode surface so that more electrochemiluminescent material is present on the electrode surface. Body collection is an effective technique for increasing sensitivity.

  However, the use of magnetic fine particles also has problems. That is, the proportion of the electrochemiluminescent material contributing to the electrochemical reaction is very low. It is true that collecting magnetic particles on the electrode surface by magnetic force as described above is an effective technique, but not all of the electrochemiluminescent materials bound to the collected magnetic particles contribute to the electrochemical reaction. It is. This is because the size of the magnetic fine particles is on the order of sub-micron to micron, which is much larger than the electrochemical reaction region. Among the electrochemiluminescent materials bonded to the magnetic fine particles, the electrochemiluminescent materials that contribute to the electrochemical reaction are This is because it is only a small part in contact with the electrode surface.

  Furthermore, it is a problem that the magnetic fine particles collected on the electrode surface become a factor inhibiting light emission. The magnetic fine particles collected on the electrode surface using a magnet are present on the electrode surface in an aggregated and laminated state, and in order for the luminescence generated on the electrode surface to reach the photodetector above the electrode, It must pass between these agglomerated and laminated magnetic fine particles. Since the magnetic fine particles are opaque, light emission generated on the electrode surface is greatly attenuated while passing between the aggregated and laminated magnetic fine particles. Therefore, even if the luminescence generated on the electrode surface increases, the luminescence that can be detected is very small, and the luminescence generated on the electrode surface cannot be detected efficiently.

  Therefore, as a technique for removing the influence of the magnetic fine particles, a technique for separating and detecting the labeling agent from the magnetic fine particles has been proposed. In this method, a labeling agent is separated from the complex after B / F separation, and a solution containing the separated labeling agent is detected (see, for example, Patent Document 3 and Patent Document 4).

Here, in the method using the electrochemiluminescent substance as described above as a labeling agent, the electrode used at the time of detection also discloses a carbon electrode in addition to an electrode made of a noble metal material such as a gold electrode or a platinum electrode. Has been
Japanese National Patent Publication No. 6-509212 JP-A-11-125601 JP 2000-105236 A JP 2004-121231 A

  However, there is no disclosure or suggestion of a change in the amount of electrochemiluminescence when the carbon electrode is used in the conventional technique. In reality, when a carbon electrode is used, the amount of electrochemiluminescence is reduced to 10% or less as compared with the case where a gold electrode is used.

  An object of the present invention is to solve the above-mentioned conventional problems, and to provide an electrode substrate for biomolecule detection that does not cause a decrease in the amount of electrochemiluminescence even in a carbon electrode, and a method for producing the same.

  In order to solve the conventional problems, the biomolecule detection electrode substrate of the present invention comprises a plate base material and a carbon electrode made of a carbon material having an acidic group bonded to the plate base material. It is what.

  The method for producing the electrode substrate for biomolecule detection according to the invention includes an electrode forming step of forming a carbon electrode on a plate substrate, and an acidic group binding step of binding an acidic group to the surface of the carbon electrode formed on the plate. It is characterized by comprising.

  According to the gene detection substrate and the method for producing the same of the present invention, a decrease in electrochemiluminescence is prevented by electrostatically adsorbing the labeling agent to the electrode surface by binding an acidic group to the surface of the carbon electrode. be able to.

Embodiments of a biomolecule detection electrode substrate and a method for producing the same according to the present invention will be described below in detail with reference to the drawings.
(Embodiment 1)
Hereinafter, specific embodiments of the electrode substrate for biomolecule detection of the present invention will be described.

  FIG. 1 is a top view of a biomolecule detection electrode substrate according to Embodiment 1 of the present invention, and FIG. 2 is a cross-sectional view taken along line A-A ′ in FIG. 1. Three carbon electrodes of a working electrode 1, a counter electrode 2, and a reference electrode 3 are formed on the plate substrate 4. These three electrodes are electrodes for performing electrochemiluminescence by a three-electrode method. A working electrode 1, which is an electrode for dropping the labeling agent, is formed in a circular shape at the center of the electrode plate substrate 4, and a counter electrode 2 is disposed so as to surround the working electrode 1, and in the gap between the working electrode 1 and the counter electrode 2. A reference pole 3 is arranged. An insulating film 5 is disposed so as to surround these electrodes. The three electrodes are extended toward the end of the plate substrate 4, which is a contact portion for connecting to the voltage application portion.

  As the plate base material 4, an insulating substrate material can be used, and examples thereof include glass, plastic resin, and ceramic.

  The carbon electrode formed on the plate substrate 4 is a carbon electrode with a carbon material exposed on the surface, and is formed of, for example, carbon paste, glassy carbon, or DLC. The carbon electrode may be formed by coating the carbon material layer on the metal material layer in addition to the carbon material layer described above, as long as the carbon material is exposed on the surface. But you can. Although it does not specifically limit as a metal material, Low resistance metal materials, such as silver, copper, and aluminum, can be used. With such a two-layer structure, the conductivity can be improved, which is more preferable.

The insulating film 5 is provided to define the area of the electrode, and may be any insulating material, and examples thereof include SiO ₂ , SiN, polyimide, resist paste, and the like.

  An acidic group 6 is bonded to the surface of the carbon electrode formed on the plate substrate 4. The acidic group 6 does not need to be bonded to all three electrodes, but may be at least applied to the working electrode 1 to which the labeling agent is dropped. Examples of the acidic group 6 to be bonded include a carboxyl group and a sulfonic acid group.

  Here, the function of the acidic group 6 bonded to the carbon electrode surface will be described. The above-mentioned acidic group 6 has a negative polarity due to the large electronegativity of oxygen, and the surface of the carbon electrode has a negative charge by binding the acidic group 6. On the other hand, a labeling agent that exhibits electrochemiluminescence is a substance having a positive charge, as represented by a ruthenium complex. Therefore, a positively charged labeling agent can be electrostatically adsorbed on the negatively charged carbon electrode surface by the binding of the acidic group 6, and the labeling agent is also present in the light emitting region near the electrode surface even with the carbon electrode. Many come to exist.

  This electrostatic adsorption of the labeling agent is the same as that which occurs for an electrode using a noble metal material such as gold. An electrode using a noble metal material such as gold has a property of having a negative charge on the electrode surface, and electrostatic adsorption of the labeling agent occurs. Therefore, by attaching an acidic group to the surface of the carbon electrode, as in the case of an electrode using a noble metal material such as gold, a carbon electrode can have a large amount of labeling agent in the light emitting region near the electrode surface, and the amount of emitted light It is possible to prevent a decrease in the above.

  Hereinafter, specific embodiments of the method for producing a biomolecule detection electrode substrate of the present invention will be described. FIG. 3 shows a schematic view of a method for producing the biomolecule detection electrode substrate according to Embodiment 1 of the present invention.

  First, the plate base material 4 shown in FIG. Next, as shown in FIG. 3B, the working electrode 1 and the counter electrode 2 which are carbon electrodes are formed. These carbon electrodes may be formed by a known method. For example, a method of performing screen printing using a carbon paste, a method of adhering a molded glassy carbon film, or a method of DLC film formation by CVD can be mentioned.

  In addition, when a metal material layer such as silver, copper, or aluminum is formed below the carbon electrode, for example, the metal electrode layer is formed on the plate base material by screen printing using a paste, sputtering, or vacuum deposition. Then, the working electrode 1 and the counter electrode 2 which are electrodes covered with a carbon material can be formed by performing the above-described carbon electrode forming method.

Next, the insulating film 5 is formed as shown in FIG. The method for forming the insulating film 5 is not particularly limited. For example, screen printing using a resist paste, photolithography using a photosensitive polyimide, film formation of SiO ₂ or SiN by CVD, plastic resin And silicon rubber adhesion.

  Next, as shown in FIG. 3D, the acidic group 6 is bonded to the surface of the working electrode 1 which is a carbon electrode. The bonding of the acidic group 6 may be performed by oxidizing the surface of the working electrode 1. Examples of the oxidation treatment include known methods such as gas phase oxidation treatment using oxygen plasma and UV ozone.

  Oxidation treatment with oxygen plasma generates plasma by applying high-frequency power in a vacuum chamber into which oxygen gas has been introduced, and oxygen radicals generated at that time react with carbon on the electrode surface to bond carboxyl groups. For example, it can be performed using a reactive ion etching apparatus or the like. In addition, since this process should just be able to process only the carbon electrode surface, it is not necessary to perform for a long time.

  In the oxidation treatment with UV ozone, oxygen gas is irradiated with UV light to generate ozone, and this ozone reacts with carbon on the electrode surface to bond carboxyl groups. Note that this treatment need not be performed for a long time because only the surface of the working electrode 1 needs to be treated, as in the case of oxygen plasma.

  Further, as the oxidation treatment, liquid phase oxidation treatment can be performed in addition to the above gas phase oxidation treatment. The liquid phase oxidation treatment is performed by immersing the carbon electrode in a solution containing an oxidizing agent. As the oxidizing agent, when a carboxyl group is bonded, for example, nitric acid, hydrogen peroxide, ozone water, iodine water, hypochlorite, chlorite, permanganate, dichromate, peroxonitrate. Examples thereof include sulfate and bisulfite. In the case of bonding a sulfonic acid group, a sulfonated pyridine salt, amidosulfuric acid, fluorosulfuric acid, chlorosulfuric acid, sulfur trioxide, and fuming sulfuric acid are exemplified. As described above, the biomolecule detection electrode substrate of the present invention can be manufactured.

  Next, a specific embodiment will be described in detail for an example of detection of a gene sample using the above-described biomolecule detection electrode substrate of the present invention.

(1) Gene sample In the gene sample, AATTGAATGA AAACATCAGG ATTGTAAGCA CCCCCTGGAT CCAGATATGC AATATTTGTC CCACTATGA sequence of the cytoplasm P-450 derived from human cytochrome P-450 sequence A 100 base oligodeoxynucleotide was used.

(2) Immobilization of nucleic acid probe on the surface of magnetic fine particles Streptavidin magnetic beads CM01N / 5896 (manufactured by Bangs Laboratories) having a particle size of 0.35 μm were used as magnetic fine particles. A nucleic acid probe having a sequence complementary to the gene sample of AATTTGTTAT GGGTTCCCGG GAAATAATCA (SEQ ID NO: 2) from the 5 ′ end and modified with biotin via a phosphate group at the 5 ′ end was used.

  First, 1 mg of magnetic fine particles were sampled and prepared so as to have a volume ratio of TTL buffer (500 mmol / L Tris-HCl (pH 8.0): Tween 20: 2 mol / L lithium chloride: ultrapure water = 2: 10: 5: 3). ) And then replaced with 20 μL of TTL buffer. Thereafter, 5 μL of 100 nmol / L nucleic acid probe was added and shaken at room temperature for 15 minutes.

  This solution was decanted, and the remaining magnetic fine particles were washed with a 0.15 mol / L sodium hydroxide aqueous solution. Then, TT buffer (500 mmol / L Tris-HCl (pH 8.0): Tween 20: ultrapure water = 1: 2: 1).

  After washing, the solution was replaced with TTE buffer (500 mM Tris-HCl (pH 8.0): Tween 20: 200 mM Na 2 EDTA (pH 8.0): ultrapure water prepared to have a volume ratio of 5: 10: 1: 4)). Instable binding was removed by incubation at 80 ° C. for 10 minutes. As a result, magnetic fine particles having a nucleic acid probe immobilized thereon were obtained.

(3) Labeling agent The labeling agent shown in the following (Chemical Formula 1) in which a nucleic acid probe having a sequence complementary to a gene sample was bound to a ruthenium complex that is an electrochemiluminescent substance was used.

  The nucleic acid probe used here is a 30-base oligodeoxynucleotide having a sequence of TGCTTACAAT CCTGATGTTT TCATTCAATT (SEQ ID NO: 3) from the 5 'end. By using this labeling agent, it is possible to obtain electrochemiluminescence having a peak wavelength of 620 nm from the ruthenium complex.

(4) Hybridization 14 μL of 2XSSC was added to the magnetic microparticles on which the nucleic acid probe had been immobilized, and 4 μL of the gene sample and labeling agent prepared at 5 μmol / L were added thereto, and a hybridization reaction was performed at 70 ° C. for 1 hour. .

(5) B / F separation After the hybridization reaction, magnetic fine particles were collected with a magnet, the solution was decanted, washed with 2XSSC heated to 40 ° C, B / F separation was performed, and the labeling agent was a gene sample. As a result, a composite bonded to the magnetic fine particles was obtained.

(6) Separation of labeling agent Next, nuclease DNase I (manufactured by Takara Bio Inc.) was used to separate the labeling agent from the magnetic fine particles. DNase I treatment solution (10 × DNase I Buffer 5 μL, DNase I 2 μL, DPEC treated water 40 μL) is added to the complex after B / F separation, and the labeling agent is separated from the magnetic fine particles by shaking at 37 ° C. for 30 minutes. I let you. Thereafter, the supernatant solution was collected while collecting magnetic fine particles with a magnet to obtain a labeling agent separation solution.

(7) Production of carbon electrode substrate Next, a carbon electrode substrate was produced. 4 is a top view of the produced carbon electrode substrate, and FIG. 5 is a cross-sectional view taken along the line BB ′ in FIG.

  First, a 20 mm square Lumirror E20 # 250 (manufactured by Toray Industries, Inc.) is prepared as a PET substrate 7, and a silver paste Electrodag 6022SS (manufactured by Japan Atchison Co., Ltd.) is screen-printed on the PET substrate 7 at 130 ° C. for 1 minute. It was made to dry and the silver electrode 8 with a film thickness of 10 micrometers was formed. On the silver electrode 8, carbon paste JEF-120 (manufactured by Nippon Atson Co., Ltd.) is screen-printed and dried at 130 ° C. for 1 minute to form a carbon electrode 9 having a film thickness of 15 μm. Three electrode patterns of a counter electrode 2 and a reference electrode 3 were formed. The pattern shape of the carbon electrode 9 is almost the same as that of the silver electrode, but is 200 μm larger than the pattern of the silver electrode, and the electrode surface is a surface where the carbon material is exposed. Thereafter, resist paste CCR-232CFV (manufactured by Asahi Chemical Laboratory Co., Ltd.) was screen-printed and dried at 130 ° C. for 1 minute to form an insulating film 5 having a thickness of 20 μm. Note that a screen printer LS-34TV (manufactured by Neurong Seimitsu Kogyo Co., Ltd.) was used for the screen printing, and an oven DOV-450 (manufactured by As One Co., Ltd.) was used for drying after paste printing.

  The working electrode 1 was formed at the center of the PET substrate 7, the counter electrode 2 was disposed so as to surround the working electrode 1, and the reference electrode 3 was disposed in the gap between the working electrode 1 and the counter electrode 2. In this embodiment, the diameter of the circular part of the working electrode 1 is 3 mm, the gap to the counter electrode 2 is 1 mm, the diameter of the outer periphery of the circular part of the counter electrode 2 is 7 mm, and the line width of the reference electrode 3 in the gap is 0.2 mm. did. The insulating film 5 is arranged so as to surround these electrodes in a circular shape, and the inner diameter is 7.5 mm.

  Next, the surface of the carbon electrode 9 was oxidized. For the oxidation treatment, an oxidation treatment using oxygen plasma was performed using a reactive ion etching apparatus RIE-200L (manufactured by Samco Corporation). The oxidation treatment is performed in a state in which a PET substrate having an opening so as to expose a portion of the working electrode 1 of the carbon electrode 9 is overlaid on the carbon electrode substrate, and the working electrode 1 of the carbon electrode substrate is formed. Only was exposed to oxygen plasma. Table 1 shows the oxygen plasma conditions.

  In Table 1, the oxygen gas flow rate, pressure, and power conditions were all the same, and the treatment time was set to four types of 0, 15, 30, and 60 seconds. Here, 0 seconds means that no oxidation treatment is performed, and is a condition set for clarifying the difference between the prior art and the present invention.

  In order to confirm the surface state of the carbon electrode substrate obtained as described above, X-ray photoelectron spectroscopy (XPS) measurement was performed.

  FIG. 5 shows the XPS measurement result of the surface of the carbon electrode treated with oxygen plasma under the conditions shown in Table 1. In FIG. 5, COOH that did not exist in 0 seconds when the treatment was performed for 15, 30, and 60 seconds was detected, and a carboxyl group that is an acidic group on the surface of the carbon electrode by oxygen plasma treatment. It can be seen that are combined.

  For comparison with the carbon electrode substrate of the present invention, a conventional gold electrode substrate was prepared. The gold electrode substrate is manufactured by forming 200 nm of gold on a glass substrate with a sputtering apparatus SH-350 (manufactured by ULVAC, Inc.) with a titanium of 10 nm as a base, and forming an electrode pattern similar to the above carbon electrode by a photolithography process. After that, a gold electrode substrate was obtained by forming an insulating film by screen printing in the same manner as the carbon electrode described above.

(8) Comparison with Carbon Electrode Substrate and Gold Electrode Substrate by Electrochemical Measurement 1 μL of a labeling agent separation solution is dropped on each working electrode of the carbon electrode substrate and the gold electrode substrate obtained as described above, It was dried in the atmosphere for 5 minutes. Thereafter, silicon rubber having a thickness of 1 mm for liquid reservoir was attached to the electrode substrate. The silicon rubber was provided with an opening having a diameter of 8 mm, and was pasted so that the center of the opening and the center of the working electrode were the same. Thereafter, 80 μL of a solution prepared by mixing 500 μL of 0.2 mol / L PBS (sodium phosphate buffer at pH 7.4) and 500 μL of 0.2 mol / L triethylamine as an electrolyte solution was dropped into the reservoir. The amount of electrochemiluminescence generated by applying a voltage to was measured. The voltage was applied by scanning from 0 V to 1.3 V in 1 second and then holding 1.3 V for 4 seconds. The electrochemiluminescence amount was measured by using a photomultiplier tube (H7360-01 manufactured by Hamamatsu Photonics) and measuring the maximum luminescence amount during voltage application.

  FIG. 6 shows the electrochemiluminescence amounts of the carbon electrode substrate and the gold electrode substrate in the first embodiment of the present invention. In FIG. 6, the light emission amount is very low at 0 seconds when oxygen plasma treatment is not performed, which is 4% of the light emission amount of the gold electrode substrate. However, when the oxygen plasma treatment is performed, the amount of light emission is greatly increased. The amount of light emission is 10 times or more of 0 seconds when the treatment is not performed, and the light emission amount of the gold electrode substrate is 15 seconds at the highest light emission amount. It was possible to increase to 67%. This is a sufficient amount of light emission for practical use.

  From the above results, the surface of the carbon electrode is oxidized, and acidic groups are bonded to the surface of the carbon electrode, so that the labeling agent can be electrostatically adsorbed on the surface of the electrode, and the amount of electrochemiluminescence at the carbon electrode Can be greatly improved.

  The electrode substrate for biomolecule detection and the manufacturing method thereof according to the present invention can detect biomolecules to be detected in a biochemical sample with high sensitivity, and are used for genetic diagnosis, infectious disease diagnosis, food inspection and the like. Applicable.

Top view of biomolecule detection electrode substrate in accordance with the first exemplary embodiment of the present invention. Sectional drawing of the electrode substrate for biomolecule detection in Embodiment 1 of this invention Schematic of the manufacturing method of the electrode substrate for biomolecule detection in Embodiment 1 of this invention Top view of carbon electrode substrate according to Embodiment 1 of the present invention Sectional drawing in B-B 'of the carbon electrode substrate in Embodiment 1 of this invention The graph which shows the XPS measurement result of the carbon electrode surface which carried out the oxygen plasma processing in Embodiment 1 of this invention The graph which shows the electrochemiluminescence amount of the carbon electrode board | substrate and gold electrode board | substrate which carried out the oxygen plasma processing in Embodiment 1 of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Working electrode 2 Counter electrode 3 Reference electrode 4 Plate base material 5 Insulating film 6 Acid group 7 PET substrate 8 Silver electrode 9 Carbon electrode 10 Carboxyl group

Claims (12)

An electrode substrate for biomolecule detection used for electrochemical detection,
A plate substrate;
A biomolecule detection electrode substrate comprising: a carbon electrode made of a carbon material having an acidic group bonded to the plate base material. The electrode substrate for biomolecule detection according to claim 1, wherein the acidic group is bonded only to the surface of the carbon electrode. The electrode substrate for biomolecule detection according to claim 1 or 2, wherein the acidic group is any one of a carboxyl group and a sulfonic acid group. The biomolecule detection electrode substrate according to claim 1, wherein the plate base material is one of glass, plastic resin, and ceramic. The electrode substrate for biomolecule detection according to claim 1, wherein the carbon material is any one of carbon paste, glassy carbon, and DLC. The electrode substrate for biomolecule detection according to claim 1, wherein the carbon electrode is obtained by forming a carbon material layer on a metal material layer. An electrode forming step of forming a carbon electrode on the plate substrate;
An acidic group binding step of binding an acidic group to the surface of the carbon electrode formed on the plate;
The manufacturing method of the electrode substrate for biomolecule detection which consists of. The method for producing an electrode substrate for biomolecule detection according to claim 7, wherein the acidic group is any one of a carboxyl group and a sulfonic acid group. The method for producing an electrode substrate for biomolecule detection according to claim 7, wherein the acidic group binding step is performed by an oxidation treatment of a carbon electrode surface. The method for producing an electrode substrate for biomolecule detection according to claim 9, wherein the oxidation treatment is a gas phase oxidation treatment using either oxygen plasma or UV ozone. The oxidation treatment is nitric acid, hydrogen peroxide, ozone water, iodine water, hypochlorite, chlorite, permanganate, dichromate, peroxodisulfate, bisulfite, sulfonated pyridine salt The method for producing an electrode substrate for biomolecule detection according to claim 9, which is a liquid phase oxidation treatment using any one of amidesulfuric acid, fluorosulfuric acid, chlorosulfuric acid, sulfur trioxide, and fuming sulfuric acid. The method for producing an electrode substrate for biomolecule detection according to claim 7, wherein the electrode forming step is performed by forming a metal material layer on the plate base material and forming a carbon material layer on the metal material layer. .

Images