JP2006090784A - Substrate for immobilizing biosubstance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate for immobilizing a biosubstance capable of shielding fluorescence of a background emitted from a base body, and capable of holding densely and firmly an inexpensive and secure biosubstance used genegally, onto a metal film. <P>SOLUTION: This substrate for immobilizing the biosubstance is layered with the metal film 12, a dielectric multilayer film 13 for increasing reflected light, a glass film 14 and a biosubstance immobilizing film 15 in order on an upper face of the base body 11. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a biological material-immobilized substrate for measuring the arrangement of biological materials such as DNA and RNA. More specifically, the present invention blocks and measures background fluorescence from a biological material detection substrate by a fluorescence detection method. An object of the present invention is to improve and stabilize detection sensitivity with respect to a method for further increasing the gain of fluorescence from a phosphor modified with a biological substance.

  Biological substance-immobilized substrates such as DNA microarrays have bioimmobilized membranes such as aminosilane, aminoalkylsilane, or poly-L-lysine formed on a glass substrate, and oligonucleotides and cDNA are deposited on it. A nucleic acid sequence is analyzed by reacting a sample DNA that has been fluorescently modified in advance and detecting a fluorescence level that varies depending on the reaction level. For this reason, background fluorescence emitted from the glass substrate causes a reduction in resolution. In particular, in the DNA microarray, the Stanford University method is inexpensive, but the detection accuracy is inferior to that of the Affimetrix type, so that an improvement in sensitivity is required to improve the detection accuracy.

  As a countermeasure against this, it is possible to form a biological immobilization film such as aminosilane, aminoalkylsilane or poly-L-lysine and then apply a blocking material or a quenching material for attenuating the fluorescence emitted from the glass substrate. It is being considered.

For example, as shown in FIG. 4, the following Patent Document 1 proposes that a quencher 54 is coated on the upper surface of the glass substrate 51 in order to improve the detection accuracy. As shown in FIG. 5, a biological material-immobilized substrate in which a metal film 52 is formed on the upper surface of a glass substrate 51 and a biological material-immobilized film 53 having a functional group having a good bondability with a metal is formed on the surface of the metal film 52. Has been proposed.
JP 2003-84002 A JP 2002-323498 A

  However, the method of coating a quencher or blocking agent 54 as shown in Patent Document 1 is a problem that the background fluorescence cannot be completely removed because the quencher or blocking agent 54 is a translucent organic substance. Had a point. In addition, as shown in Patent Document 2, the method of forming the biological material fixed film 53 having a functional group having a good binding property with a metal on the surface of the metal film 52 can block the fluorescence of the glass substrate 51 by the metal film 52. However, it is necessary to use an expensive noble metal such as Au or Pt, and it is necessary to use a special material having a functional group having a good binding property to the metal as the biological material immobilization film 53, which is relatively expensive. In addition, since a toxic substance is used, there is a problem that care is required in handling.

  On the other hand, it is conceivable to try blocking the background fluorescence and reflecting the measured fluorescence using a dielectric multilayer film. However, since a very large number of layers from 40 layers to 60 layers is required, the light absorption also increases. The reflectance does not increase as much as that of metal, and the occurrence of defects due to contamination increases.

  Therefore, the present invention has been completed in view of the above problems, and its purpose is to block background fluorescence emitted from the substrate and to enhance measurement fluorescence by the reflective action of a metal film or dielectric multilayer film. An object of the present invention is to provide a biological material-immobilized substrate that is strong and can hold a generally inexpensive and safe biological material-immobilized film on a reflective film densely and firmly.

  The biological material-immobilized substrate of the present invention is characterized in that a metal film, a dielectric multilayer film for increasing reflected light, a glass film, and a biological material-immobilized film are sequentially laminated on the upper surface of a base.

  In the biological material-immobilized substrate of the present invention, it is preferable that the metal film is at least one of Ti, Cr, Ni, Au, Ag, Pt, Rh, Al, Ni—Cr alloy, and Fe—Cr—Ni alloy. It is characterized by having a seed as a main component.

  In the biological material-immobilized substrate of the present invention, preferably, the metal film has an arithmetic average roughness Ra on the upper surface of 0.05 μm or less.

  In the biological material-immobilized substrate of the present invention, preferably, the dielectric multilayer film is mainly composed of at least one of a tantalum oxide film, a titanium oxide film, a calcium fluoride film, a zirconium oxide film, and a niobium oxide film. It is characterized by doing.

  In the biological material-immobilized substrate of the present invention, preferably, the dielectric film constituting the dielectric multilayer film has a wavelength of fluorescence emitted from the biological material whose thickness is modified by the biological material-immobilized film and the dielectric. It is characterized by being (2n-1) / 4 times (n: natural number) of the product of the refractive index of the body film.

  In the biological material-immobilized substrate of the present invention, preferably, the glass film is composed of a number of glass columnar bodies grown on the upper surface of the metal film.

  In the biological material-immobilized substrate of the present invention, preferably, the glass film has a silicon oxide content of 70% by mass or more.

  In the biological material-immobilized substrate of the present invention, preferably, the biological material-immobilized film has at least one of an amino group, an aldehyde group, a mercapto group, a silane group, and a carboxyl group. To do.

  In the biological material-immobilized substrate of the present invention, a metal film, a dielectric multilayer film for increasing reflected light, a glass film, and a biological material-immobilized film are sequentially laminated on the upper surface of the substrate. Thus, the fluorescence emitted from the phosphor modified with the biological material bound to the biological material-immobilized membrane can be reflected with light efficiency.

  Furthermore, the reflected light can be enhanced by the interference action of the reflected light reflected by the surface of each dielectric film and the surface of the metal film constituting the dielectric multilayer film. The detection accuracy can be made extremely high.

  In the biological material-immobilized substrate of the present invention, preferably, the metal film is at least one of Ti, Cr, Ni, Au, Ag, Pt, Rh, Al, Ni—Cr alloy, and Fe—Cr—Ni alloy. As a main component, it is chemically stable due to its chemical stability, and also reflects fluorescence emitted from a phosphor modified with a biological material bound to a biological material-immobilized membrane with high reflectance. And high detection sensitivity can be stably maintained.

  In the biological material-immobilized substrate of the present invention, preferably, since the arithmetic average roughness Ra of the upper surface of the metal film is 0.05 μm or less, from the phosphor modified with the biological material bound to the biological material-immobilized film. Scattering and reflection of the emitted fluorescence can be prevented, and the fluorescence gain can be further increased.

  In the biological material-immobilized substrate of the present invention, preferably, the dielectric multilayer film is mainly composed of at least one of a tantalum oxide film, a titanium oxide film, a calcium fluoride film, a zirconium oxide film, and a niobium oxide film. Therefore, it is possible to form a dielectric multilayer film for increasing reflected light that can cope with various fluorescence wavelengths, and it is possible to easily control the surface physical properties so that the adhesion to the metal layer or the glass layer is good.

  In the biological material-immobilized substrate of the present invention, preferably, the wavelength of the fluorescence emitted from the biological material whose thickness of the dielectric film constituting the dielectric multilayer film is modified to the biological material-immobilized film and the refractive index of the dielectric film (2n-1) / 4 times (n: natural number) of the product of, and interference between reflected light reflected by the surface of each dielectric film or the surface of the metal film by the fluorescence emitted from the biological material The fluorescence intensity can be enhanced very well by the action, and high gain measurement is possible.

  In the biological material-immobilized substrate of the present invention, preferably, the glass film is composed of a large number of glass pillars grown on the upper surface of the metal film. For this reason, the amount of fluorescence emitted from the biological material is increased. In addition, the entire surface of the biological material-immobilized substrate is not covered with glass, and the fluorescence reflected by the dielectric multilayer film or the metal film is well taken out from the gap between the glass columnar bodies. The occurrence of loss can be effectively suppressed, and the detection accuracy can be increased.

  In the biological material-immobilized substrate of the present invention, preferably, since the glass film has a silicon oxide content of 70% by mass or more, a glass film having excellent transparency can be formed at low cost. The biological material-immobilized membrane used on the conventional glass can be used as it is, and it is not necessary to use a biological material having a special functional group, and the material cost can be reduced.

  In the biological material-immobilized substrate of the present invention, preferably, the biological material-immobilized film has at least one of an amino group, an aldehyde group, a mercapto group, a silane group, and a carboxyl group. The adsorptivity of biological materials such as cells can be increased, and more biological materials can be fixed to increase detection accuracy.

  Next, the biological material fixed substrate of the present invention will be described in detail with reference to the accompanying drawings.

  FIG. 1 is a cross-sectional view of an example of an embodiment of a biological material fixed substrate of the present invention. As shown in FIG. 1, 11 is a substrate, 12 is a metal film, 13 is a dielectric multilayer film for increasing reflected light, 14 is a glass film, and 15 is a biological material immobilization film.

  The substrate 11 is a plate made of glass, metal, ceramics or the like, but is not limited thereto, and the surface can be flattened into a mirror surface, and the chemical resistance and heat and humidity resistance related to the treatment of biological substances are satisfied. Anything is fine.

  The metal film 12 is a metal that can reflect fluorescence and has good adhesion to the glass film 14 and has chemical resistance. Preferably, Ti, Cr, Ni, Au, Ag, Pt, Rh, It is preferable that at least one of Al, Ni—Cr alloy and Fe—Cr—Ni alloy is a main component. As a result, the reflectance of the fluorescence can be made very high, the adhesion between the glass film 14 and the metal film 12 can be made very high, and the metal film 12 is a chemically stable metal, so that the metal The chemical resistance of the membrane 12 can be made very high. In order to improve the film adhesion with the glass film 14, for example, the glass film 14 may be adhered to the metal film 12 such as Au or Ag via an adhesion layer such as Ti or Cr.

  The thickness of the metal film 12 is preferably 0.05 μm or more in the process of growing from an island structure that transmits light to a film structure that does not transmit light. In particular, when the thickness is about 0.2 μm, the film adhesion is relatively high, and corrosion of the base glass 11 due to the penetration of the chemical solution does not occur. If it exceeds 1 μm, although it depends on the film forming method, film peeling due to breakage of the glass 11 of the substrate tends to occur due to internal stress of the film, so that the thickness is preferably 1 μm or less.

  Further, in selecting the film forming conditions, it is necessary to limit the film forming conditions in consideration of smoothness. As for the smoothness of the metal film 12, Ra is preferably 0.05 μm or less including the smoothness of the glass 11 of the substrate. Thereby, diffused reflection of fluorescence can be suppressed and the gain of fluorescence can be increased.

  The dielectric multilayer film 13 for increasing reflected light is a so-called optical thin film, and is formed by laminating a plurality of dielectric films. The thickness of these dielectric films is controlled so that the fluorescence is enhanced by the interference of the reflected lights reflected by the respective surfaces of the dielectric film and the surface of the metal film 12.

  Such a dielectric multilayer film 13 preferably contains at least one of tantalum oxide, titanium oxide, calcium fluoride, zirconium oxide and niobium oxide as a main component. Thereby, it is possible to form the dielectric multilayer film 13 that can correspond to the fluorescence wavelengths of various wavelengths, and the film adhesion to the underlying metal film 12 and the surface glass film 14 is also improved. Further, since the film formation speed of the dielectric film can be slowed, film formation with an accurate film thickness is possible.

  Preferably, the thickness of the dielectric film constituting the dielectric multilayer film 13 is the product of the product of the wavelength of fluorescence emitted from the biological material modified by the biological material fixed film 15 and the refractive index of the dielectric film (2n -1) / 4 times (n: natural number). Thereby, the fluorescence emitted from the biological material (fluorescence emitted from the phosphor modified with the biological material) is enhanced by the interference action of the reflected lights reflected on the surface of each dielectric film and the surface of the metal film 12. It can be performed very well and high gain measurement is possible.

  Then, (2n-1) / 4 times (n: natural number) of the product of the wavelength of the fluorescence emitted from the biological material modified by the biological material-immobilized film 15 and the refractive index of the dielectric film. The number of layers of the dielectric films 13-1 to 13-N (refer to the enlarged sectional view of FIG. 2) is increased by the interference action of light of about 2n power rule as the number of layers increases with a relatively small number of layers. However, when the number of layers is several tens, the thickness of the dielectric multilayer film 13 is increased and the fluorescence is easily absorbed, resulting in inefficiency. Therefore, the thickness of the dielectric multilayer film 13 is 1 μm or less. There should be.

  The glass film 14 may be any material as long as it is transparent and does not absorb the fluorescence wavelength, and the biological material-immobilized film 15 is well fixed. In particular, if the thickness is half the product of the emission wavelength of the phosphor modified with the biological material and the refractive index of the glass film, absorption due to interference occurs, and the product of the wavelength of the phosphor and the refractive index of the glass film. If the thickness is ¼ of the thickness, there is an increase due to light interference, so a slight variation in the thickness of the film increases the variation in the measurement of the fluorescence intensity. Therefore, it is a quarter of the product of the fluorescence wavelength and the refractive index of the glass film 14. The thickness should be smaller than 1.

Further, the glass film 13 preferably contains 70% by mass or more of SiO ₂ as a component composition. As a result, almost all general biological material-immobilized membranes 15 such as aminoalkylsilane and poly-L-lysine can be used.

  Further, FIG. 3 is a partial cross-sectional view showing another example of the embodiment of the biological material-immobilized substrate of the present invention. In this way, the film forming conditions of the glass film 24 are controlled, and the dielectric multilayer film 23 is controlled. It is preferable that the glass film 24 is formed by growing a large number of glass columnar bodies having many gaps between adjacent particles. Thereby, since the surface area of the glass film 24 becomes very large, the amount of the biological material to be immobilized is greatly increased, and the gain of fluorescence is further increased. In addition, the entire surface of the biological material-immobilized substrate is not covered with glass, and the fluorescence reflected by the dielectric multilayer film 23 and the metal film 22 is favorably extracted from the gap between the glass columnar bodies. It is possible to effectively suppress the occurrence of light loss and to improve the detection accuracy.

  The glass film 24 composed of a large number of glass pillars grown on the upper surface of the dielectric multilayer film 23 is produced by, for example, low temperature high gas pressure sputtering or oblique deposition.

  The biological material immobilization film 5 is selected from aminoalkylsilane, poly-L-lysine and the like. In general, any biological material-immobilized film 5 used on the glass substrate 1 may be used, and more preferably has at least one of an amino group, an aldehyde group, a mercapto group, a silane group, and a carboxyl group. preferable. Thereby, the adsorptivity of biological substances such as proteins and cells can be enhanced, and more biological substances can be fixed to increase detection accuracy.

  Examples of the biological material-immobilized substrate of the present invention will be described below.

  The glass of the substrate 1 was prepared as borosilicate glass having a size of 26 mm × 76 mm × 1.1 mm.

  The prepared borosilicate glass is alkali degreased and thoroughly washed with pure water, then neutralized with dilute hydrochloric acid, sufficiently washed with pure water, and after light etching the glass surface with dilute hydrofluoric acid, The pure water was washed. The arithmetic average roughness Ra was controlled by the immersion time of dilute hydrofluoric acid. Drying was carried out with alcohol vapor after alcohol substitution.

A metal film 2 was formed on the upper surface of the dried substrate 11 using a sputtering apparatus. Next, two types of dielectric films to be the dielectric film multilayer film 13 were alternately formed on the upper surface of the metal film 12 by an optical thin film deposition apparatus. Further, a SiO ₂ film having a predetermined thickness was formed as a glass film 14 on the upper surface of the dielectric multilayer film 13. At this time, in forming the SiO ₂ film, consideration was given to the heating temperature, the film forming speed, and the degree of vacuum so that the film does not become dense.

  The thickness of the dielectric film constituting the dielectric layer 13 was set to ¼ times the product of the fluorescence wavelength of the phosphor modified with the biological material to be used and the refractive index of the dielectric film.

  After the film formation, the substrate 1 was washed with RCA and sufficiently washed with pure water, followed by substitution with alcohol, and drying was performed by alcohol vapor drying.

  Next, a coating solution of 3-aminopropyltriethoxysilane (hereinafter abbreviated as APS) was prepared as the biological material immobilization film 15. The coating solution was mixed with 94% by volume of isopropyl alcohol (hereinafter abbreviated as IPA) and 4% by volume of APS as a solvent, and then 2% by volume of pure water was added and immediately stirred well, followed by washing after film formation. The glass was immersed for 30 seconds. Thereafter, the glass was rinsed with IPA for 5 seconds and then dried with a rinser dryer. Next, in order to advance the fixation of APS, curing was performed at 150 ° C. for 10 minutes to obtain the biological material fixed substrate of the present invention. Similarly, other biological material-immobilized substrates of the present invention were also prepared.

  As a comparative example, borosilicate glass was subjected to the same cleaning and then subjected to the same APS coating. For evaluation, a 15-base oligo DNA whose 5 'end was modified with a Cy3 phosphor was used. The oligo DNA was dissolved in pure water to a concentration of 0.5 μM to obtain a spot solution. After spotting on the prepared substrate, it was allowed to stand at 25 ° C. for 12 hours in a wet box and fixed to the substrate on which the oligo DNA was prepared. Next, it was washed with a citrate buffer, rinsed with pure water, and blow-dried.

  The substrate prepared at an excitation wavelength of 532 nm was scanned, and the fluorescence intensity was digitized. The DNA can be evaluated if the fluorescence intensity is 50 or more. Further, the film adhesion was evaluated by performing a tape peeling test, and a film having no film peeling was marked with ◯, and a film with little peeling was marked with x.

  Hereinafter, a description will be given based on Table 1.

  Table 1 shows the evaluation of the material and thickness of the metal film 12, the material and thickness of the dielectric film constituting the dielectric multilayer film 13, the material and thickness of the glass film 14, and the obtained fluorescence intensity and film adhesion. Results are shown.

  The influence of the thickness of the metal film 12 was evaluated by Cr as a representative example. The fluorescence intensity is low at a thickness of 0.03 μm, but favorable results were obtained in the range of 0.05 to 0.5 μm. At 1.0 μm, the glass of the substrate 11 was easily damaged due to the internal stress of the film.

  Next, the fluorescence intensity was evaluated by changing the number of layers of the dielectric multilayer film 13. As the number of layers increased, the fluorescence intensity improved dramatically.

  Next, the material of the metal film 12 was changed and investigated. Although there was a slight difference depending on each metal, there was no significant deterioration in fluorescence intensity.

  Next, the influence by the thickness of the glass film 14 was investigated. When the glass film thickness is 0.2 μm, the fluorescence intensity is low and no obvious effect is obtained, but when the glass film thickness is 0.03 μm or more, a large effect is obtained. Further, when the thickness is about 130 nm or more, which corresponds to ¼ of the product of the wavelength of the phosphor and the refractive index of the glass film 14 with respect to the fluorescence wavelength of around 540 nm, it is considered that the thickness of the glass film 14 varies. Variation in fluorescence intensity occurred. For this reason, it turned out that the thickness of the glass film 14 has more preferable 1/4 or less of a measurement wavelength.

Next, the dielectric multilayer film 13 was investigated with niobium oxide, zirconium oxide, and calcium fluoride.

  Next, description will be made according to Table 2.

  In Table 2, when the arithmetic average roughness Ra of the metal film 12 was 0.05 μm or less, high fluorescence measurement intensity was shown, but when Ra was 0.1 μm, the fluorescence measurement intensity was greatly reduced. This is thought to be due to irregular reflection of fluorescence due to surface irregularities. For this reason, it was found that the arithmetic average roughness Ra is more preferably 0.05 μm or less.

  Next, about the glass film 14 which consists of many columnar bodies, the specific surface area was digitized by measuring the amount of adsorption | suction moisture. This is because when the amount of adsorbed moisture in the mirror-finished glass film is 1, when the glass film 14 becomes columnar and the specific surface area increases, the amount of adsorbed water increases in proportion to this. According to Table 2, the fluorescence measurement intensity increased dramatically as the specific surface area increased 5 times or 10 times. For this reason, it turned out that it is more preferable if the glass film 14 becomes columnar shape that the specific surface area becomes large.

Next, the material of the glass film 14 was investigated. Although a good result was shown when the mass of silicon oxide was 70% or more, the fluorescence intensity was weak when 63%, and it was found that the silicon oxide concentration of the glass film was more preferably 70 mass% or more.

Table 3 shows the results when DNA was fixed by coating aminoalkylsilane directly on a glass substrate as a comparative example.

  Note that the present invention is not limited to the above-described best modes and examples, and various modifications may be made without departing from the scope of the present invention.

It is sectional drawing which shows an example of embodiment of the biological material detection board | substrate of this invention. It is sectional drawing which shows the other example of embodiment of the biological material detection board | substrate of this invention. It is sectional drawing which shows the other example of embodiment of the biological material detection board | substrate of this invention. It is sectional drawing of the conventional biological material detection board | substrate. It is sectional drawing of the conventional biological material detection board | substrate.

Explanation of symbols

11, 21, 31: Substrate 12, 22, 32: Metal film 13, 23, 33: Dielectric multilayer film 14, 24, 34: Glass film
15, 25, 35: Biological material immobilization membrane

Claims (8)

A biological material-immobilized substrate, wherein a metal film, a dielectric multilayer film for increasing reflected light, a glass film, and a biological material-immobilized film are sequentially laminated on an upper surface of a substrate. The metal film is mainly composed of at least one of Ti, Cr, Ni, Au, Ag, Pt, Rh, Al, Ni-Cr alloy and Fe-Cr-Ni alloy. 1. The biological material-immobilized substrate according to 1. The biological material-immobilized substrate according to claim 1 or 2, wherein the metal film has an arithmetic average roughness Ra of 0.05 µm or less on an upper surface thereof. 4. The dielectric multilayer film as a main component comprising at least one of a tantalum oxide film, a titanium oxide film, a calcium fluoride film, a zirconium oxide film, and a niobium oxide film. A biological material-immobilized substrate according to any one of the above. The dielectric film constituting the dielectric multilayer film has a thickness (2n-1) of the product of the wavelength of fluorescence emitted from the biological material whose thickness is modified by the biological material-immobilized film and the refractive index of the dielectric film. The biological material-immobilized substrate according to any one of claims 1 to 4, wherein the biological material-immobilized substrate is multiplied by 4) (n: natural number). 6. The biological material-immobilized substrate according to claim 1, wherein the glass film is composed of a number of glass pillars grown on the upper surface of the metal film. The biological material fixed substrate according to any one of claims 1 to 6, wherein the glass film has a silicon oxide content of 70 mass% or more. The said biological material fixed film | membrane has at least 1 sort (s) of an amino group, an aldehyde group, a mercapto group, a silane group, and a carboxyl group, The Claim 1 thru | or 7 characterized by the above-mentioned. Biological material immobilization substrate.

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