JP2006126179A - Glass substrate for analysis tip, and analysis tip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a glass substrate for analysis tip, such as a biotip or a microchemical tip capable of improving the S/N ratio of the fluorescence intensity of each spot, when using the analysis tip and measuring accurately and with high sensitivity. <P>SOLUTION: The glass substrate is soda-lime based silica glass and is characterized by Fe content lower than 0.1 mass%, when converted to Fe<SB>2</SB>O<SB>3</SB>. Preferably, the surface used as the analysis tip of the aforementioned glass substrate for analysis tip is processed by finishing agents. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a glass base plate suitable for a biochip that aligns and immobilizes minute amounts of biopolymer oligomers such as DNA, RNA, sugar chains and protein fragments corresponding to hundreds to tens of thousands of genes or more. Further, the present invention is mainly used for chemical analysis and chemical reaction, and a fine channel having a width of about several hundred nm to several hundred μm is formed on or inside the substrate, and a liquid sample or The present invention also relates to a glass base plate suitable for a microchemical chip in which liquid reaction products are transported, mixed, reacted, separated and purified, and the like, from analysis pretreatment to detection are completed in the same substrate.

  First, a biochip, which is one of analysis chips, will be described. As a typical biochip, there is a DNA chip in which various types of DNA fragments (hereinafter referred to as detection DNA) are fixed on a substrate as hundreds to tens of thousands of minute spots. By applying (hybridization) what is to be evaluated with human or animal DNA (hereinafter referred to as evaluation DNA) to the DNA chip, multiple DNA sequences can be detected and evaluated at one time, and differences in sequence between individuals, cell status, etc. Differences in gene expression due to differences can be analyzed. The same applies to RNA, protein, sugar chain, and the like. In the following description, DNA will be described as a representative example.

  For biochips, solid-phase synthesis using photolithography and the Stanford method (a stamping method or a spotting method) in which many types of detection DNA prepared in advance are arranged on a substrate by immobilizing DNA on the substrate. It is roughly divided into As a means for detecting a mixed DNA fragment (hereinafter referred to as sample DNA) that acts on DNA immobilized on a substrate (hereinafter referred to as probe DNA) by any method, sample DNA is used in advance. In general, a fluorescent molecule is modified, applied to a fluorescence reader, and the relative intensity of the fluorescence intensity of each spot is examined when excitation light is applied.

  The fluorescence intensity is weak with respect to the excitation light intensity, and the concentration of the sample DNA varies from the thinnest DNA to that of the darkest DNA by 1000 to 10,000 times. In particular, for sample DNA having a low concentration, auto-fluorescence and reflection from the surface of the substrate itself and attached matter (dust, organic dirt) and the like become noise, and accurate detection is difficult.

  In order to improve the ratio of fluorescence intensity to noise (S / N ratio), a film having a high affinity with probe DNA is formed on a substrate having irregularities, thereby increasing the accuracy of spot formation and increasing the fluorescence density. There is a method (see Patent Document 2) in which fine scratches are randomly formed on the substrate surface by a method (see Patent Document 1), the surface area in the spot is increased to increase the accuracy of spot formation, and the fluorescence density is increased. Although all have been proposed, it is necessary to form irregularities on the surface of the substrate, which is disadvantageous in terms of cost, such as using a special material and requiring a processing step and a cleaning step. As in the present invention, there has not yet been proposed an invention for improving the S / N ratio by the substrate itself.

  In addition, quartz glass (sometimes referred to as synthetic quartz or silica glass) or borosilicate glass plates are known as biochip substrates with low fluorescence noise (fluorescence background) from the substrate surface. (See Patent Documents 1 and 2), which is manufactured by batch processing and has a high manufacturing cost because the surface is precisely polished, which is one of the obstacles to the spread of biochips. On the other hand, as a glass plate with excellent surface flatness and smoothness and low manufacturing cost, there is soda lime-based silica glass used for window glass, but it is fluorescent compared to quartz glass and borosilicate glass. There was a problem of high background.

  That is, the fluorescence background is as low as that of quartz glass or the like, and it does not require precision polishing when used as a substrate, and sufficient S / N is not required even if a very fine unevenness is not formed on the substrate surface. A cost-effective, low-cost biochip substrate has not yet been proposed.

  Similarly, in a microchemical chip, which is one of analysis chips, accurate and highly sensitive fluorescence measurement can be performed on a small amount of liquid analyte flowing in a channel formed on or inside the chip. A low-cost microchemical chip substrate that can be produced has not been proposed yet. In the present specification, the biochip and the microchemical chip are hereinafter collectively referred to as an analysis chip.

JP 2003-14744 A (Embodiment of the Invention) JP 2003-107086 A (Embodiment of the Invention)

  An object of the present invention is to provide a low-cost glass substrate for an analysis chip that has a low fluorescence background during use, improves the S / N ratio of each spot, and enables accurate and highly sensitive measurement.

The present invention provides a glass substrate for an analysis chip, wherein the glass substrate is soda lime-based silica glass and the Fe content is 0.1% by mass or less in terms of Fe ₂ O ₃ .

  When the glass substrate for an analysis chip of the present invention is used as a glass substrate for a biochip, the fluorescence background from the sample DNA is relatively high and an excellent S / N ratio is obtained because the fluorescence background is low. In addition, since the glass of the glass substrate is soda-lime-type silica glass, the raw material price is low compared to quartz glass and the like, and it is easy to manufacture, which is advantageous in terms of cost. Since it can be manufactured by the float method that can stably obtain a flat and smooth surface, and can be used as it is as a glass substrate for biochips without performing precise polishing, there is no need for a polishing process or a cleaning / drying process after the polishing process. It is also advantageous in terms of productivity. Therefore, it can greatly contribute to the spread of biochip diagnosis.

  Moreover, when the glass surface used as the biochip of the glass substrate for biochip of the present invention is treated with a surface treatment agent having high affinity with the probe DNA, an excellent S / N ratio can be obtained. As a result, the reproducibility of the data is improved.

  In this way, the substrate itself has an excellent S / N ratio over the entire surface, so that it can be used as it is without any special modification of the conventional biochip utilization method, measurement equipment, etc., and to obtain a fluorescent signal Thus, it is possible to measure with high accuracy over a wide range from high-concentration sample DNA in which fluorescence intensity is easily saturated to low-concentration sample DNA in which fluorescence intensity is weak.

  Thereby, the spot diameter of each sample on the biochip can be reduced to achieve higher density and higher integration, the amount of information per biochip can be improved, and the number of biochips used can be reduced. If the number of biochips used in one measurement is only one, it is not necessary to correct the data by calculating variations in the data between the substrates, so the quality and reliability of the measurement data is improved.

  Similarly, when the glass substrate for an analysis chip of the present invention is used for a microchemical chip, a minute amount of liquid analysis object flowing in a channel formed on the chip or inside the chip can be accurately measured with high sensitivity. As a result, the flow path can be designed more finely, the analytical concentration limit can be lowered, and the quality and reliability of the measurement data can be improved.

The glass substrate for an analysis chip of the present invention (hereinafter referred to as the present substrate) is a glass substrate used for immobilizing a biopolymer oligomer such as DNA or for forming a flow path for flowing a small amount of a liquid analyte. a is characterized by Fe content is soda lime silica glass is less than 0.1 wt% in terms of Fe ₂ O _3.

In the case of soda lime-based silica glass, the inventor of the present invention uses fluorescent light for excitation light of 532 nm and 635 nm mainly used for biochips when the Fe content is 0.1% by mass or less in terms of Fe ₂ O _3. It was found that the background strength was lowered. Similarly, the fluorescence background intensity used in the microchemical chip is also low. In the present substrate, preferably the Fe content of the soda-lime-silica glass is at most 0.07 wt% calculated as _Fe 2 _{O 3,} Fe content is 0.05 mass% or less in terms _{of Fe} 2 _{O 3} Is particularly preferred.

In this substrate, the composition other than the Fe content of the soda lime-based silica glass is not particularly limited as long as it can be produced by the float process, but the SiO ₂ content is 65 to 75 mass% based on the oxide, Al _2. When O ₃ is contained in an amount of 0 to 5% by mass, Na ₂ O is contained in an amount of 10 to 16% by mass, K ₂ O is contained in an amount of 0 to 5% by mass, CaO is contained in an amount of 5 to 15% by mass, and MgO is contained in an amount of 0 to 7% by mass. This is preferable. More preferably, K ₂ O is 0.1% by mass, Cl is less than 0.1% by mass, and Al ₂ O ₃ is 1.5% by mass or more.

As components other than those described above, SrO, BaO, ZnO, ZrO _{2 and the} like may be contained in the range of, for example, 1% by mass or less for the purpose of adjusting the mechanical properties or thermal properties of the glass substrate or as impurities. Sb ₂ O _3, F, respectively Cl or the like as a fining agent, or impurities such as may be contained in an amount of 0.5 mass% or less. For example, SnO ₂ may be contained in the range of 0.5% by mass or less for the purpose of adjusting the reduction degree of the glass or as an impurity, respectively.

  The production method of the substrate is not particularly limited, but production by the float method is preferable because it is advantageous in terms of cost because a substrate having excellent smoothness and flatness can be produced in large quantities. When manufacturing by a method other than the float process, it is desirable to perform precision polishing or the like as necessary. The surface roughness Ra of the substrate is preferably 100 nm or less because a surface treatment agent such as a silane coupling agent can be easily applied, the hybridization can be made uniform, and the focal point becomes uniform when reading the fluorescence signal of the specimen. . The surface roughness Ra of the substrate is more preferably 10 nm or less, and particularly preferably 1 nm or less.

  In this substrate, when the analysis chip is used as a biochip, the surface of the glass to be used is subjected to a surface treatment agent so that the glass surface has a functional group highly reactive with the probe DNA. Can be bonded to the surface of the glass substrate more firmly, an excellent S / N ratio can be obtained, and the reproducibility of the biochip data is increased.

  Similarly, in the present substrate, when the analysis chip is used as a microchemical chip, the surface of the portion used as the flow path has affinity or reactivity with the liquid analysis object flowing in the flow path. The treatment with a non-surface treatment agent is preferable because the liquid analyte can be prevented from being clogged in the flow path, and an excellent S / N ratio can be obtained or the reproducibility of data can be improved.

  Such a surface treatment agent is not particularly limited as long as it has the above function, and a silane coupling agent is preferable. Although the detailed cause is unknown, the bond between the glass substrate and the probe DNA is strengthened by the mediation of the silane coupling agent, and the reaction point with the silane coupling agent is because the glass is soda-lime-type silica glass. It seems to be related to the fact that there are many on the surface and uniform surface treatment is easy.

The silane coupling agent typically has an RSiX ₃ chemical structure, where R is an organic functional group and X is a hydrolyzable group that reacts with an inorganic material. As the organic functional group R, vinyl, Preferred are those having groups such as glycidoxy, methacryl, amino, mercapto, aldehyde, epoxy, carboxyl and hydroxyl group. On the other hand, preferred examples of X include chlorine and an alkoxy group. Examples of the alkoxy group include methoxy group, ethoxy group, propoxy group, butoxy group, pentyloxy group, hexyloxy group, and the like.

  An example of the silane coupling agent includes γ-aminopropyltriethoxysilane. As the silane coupling solution, the above silane coupling agent was diluted with an alcohol having the same structure as the hydrolyzable group, and an appropriate amount of water was added to activate the hydrolyzable group. Are preferably used.

  In the present substrate, the method of using the surface treating agent is not particularly limited, but a preferable method is coating or dipping. For example, a method such as wiping the surface of the substrate with a cloth or cleaning and drying with an alkali or organic solvent, immersing the substrate in a silane coupling solution for a predetermined time, drying, and heat-dehydrating bonding Is done. The thickness of the surface treatment agent on the glass substrate is preferably 1 nm to 100 nm, more preferably 2 nm to 50 nm, and particularly preferably 3 nm to 30 nm.

  Examples of the present invention are shown below.

[Glass substrate]
First, this substrate used in the experiment was manufactured by the float process, soda-lime-type silica glass (manufactured by Asahi Glass Co., Ltd.) with a thickness of 1 mm, was cut into a rectangle of about 25 mm × 76 mm, and the surface deposits were removed. After immersing in a 10% by mass aqueous sodium hydroxide solution for 30 minutes, the sample was rinsed thoroughly with distilled water to obtain a specimen. The surface roughness Ra measured by a surface roughness measuring machine (TALYSURF manufactured by TAYLOR HOBSON) was about 0.6 nm.

  On the other hand, a commercially available soda-lime-type silica glass (manufactured by Telechem, trade name: SuperClean, about 25 mm × 76 mm, thickness of about 1 mm) was used as a comparative substrate. The cleaning method was the same as this substrate. The surface roughness Ra, measured in the same manner as this substrate, was about 5 nm.

  In addition, Table 1 shows values measured with a fluorescent X-ray analyzer (trade name: ZSX100e, manufactured by Rigaku Corporation) for the composition of the present substrate and the comparative substrate. In addition, the glass composition of this board | substrate is not limited to the composition of a present Example.

[Evaluation methods]
With respect to the test substrate, the fluorescence reflection device (Axon GenePix 4000B) was used to set the excitation light energy to the maximum value (output 100%, energy 1000), and the fluorescence reflection of the substrate with respect to the excitation wavelengths 532 nm and 635 nm was observed. The fluorescence reflection of the substrate was observed in a central portion of about 25 mm × 25 mm out of a rectangle of about 25 mm × 76 mm. The observation results for the excitation wavelengths of 532 nm and 635 nm were captured in a TIFF format image of a single color 65536 tone (16 bits), respectively, on an attached computer. The resolution of the image was about 1000 pixels square.

  The obtained image was subjected to histogram analysis using GIMP, which is standard image processing software, while maintaining 16-bit gradation information. The histogram here refers to the ratio of pixels in the image where the pixels of each brightness from black (brightness 0) to white (brightness 65535) occupy the image, the horizontal axis represents the brightness, and the vertical axis represents the brightness. The number of existence is shown in the graph. The lower the brightness shown by each pixel (0, that is, close to black), and the sharper distribution in the lower brightness, the lower the fluorescence background of the substrate, and the better the substrate for an analysis chip (biochip). Show.

  The analysis result for the excitation wavelength of 532 nm is shown in FIG. 1, and the analysis result for the excitation wavelength of 635 nm is shown in FIG. From FIGS. 1 and 2, the present substrates (11, 21) have lower brightness than the comparative substrates (12, 22), have sharp peaks, and have a low fluorescence background, so they are excellent as glass substrates for biochips. You can see that Table 2 shows the results of obtaining the average brightness, standard deviation, and central brightness for FIGS.

  Since this substrate has a low fluorescence background from the substrate itself, the S / N ratio of the fluorescence intensity from the sample DNA or the like is high and accurate information can be obtained. In particular, high-precision analysis can be expected even for low-expressed sample DNA, which has been conventionally difficult to analyze with high precision. Further, since the S / N ratio is high, the spot can be miniaturized and a highly integrated biochip can be provided. This substrate is a glass substrate that has the above-mentioned excellent characteristics and can be manufactured by a float process, so that it is low cost, and greatly contributes to the spread of biochip gene-related research and gene analysis.

  Similarly, this substrate can provide a microchemical chip capable of accurate detection and analysis.

Fluorescence background measurement results for an excitation wavelength of 532 nm. Fluorescence background measurement results for an excitation wavelength of 635 nm.

Explanation of symbols

11: Fluorescent background characteristics from the substrate for an excitation wavelength of 532 nm.
12: Fluorescent background characteristics from the comparative substrate for the excitation wavelength of 532 nm.
21: Fluorescent background characteristic from this substrate with respect to excitation wavelength of 635 nm.
22: Fluorescent background characteristics from the comparative substrate for the excitation wavelength of 635 nm.

Claims (6)

A glass substrate for analysis chip, wherein the glass substrate is soda-lime-based silica glass and the Fe content is 0.1% by mass or less in terms of Fe ₂ O ₃ . The composition other than the Fe content of the soda-lime-based silica glass is 65 to 75% by mass of SiO ₂ , 0 to 5% by mass of Al ₂ O ₃ , 10 to 16% by mass of Na ₂ O, based on oxides, K _{2. The} glass substrate for an analysis chip according to claim 1, wherein 0 to 5% by mass of ₂ O, 5 to 15% by mass of CaO, and 0 to 7% by mass of MgO.   The glass substrate for analysis chips according to claim 1 or 2, wherein a surface used as an analysis chip of the glass substrate for analysis chips is treated with a surface treatment agent.   The glass substrate for an analytical chip according to claim 1, wherein the surface treatment agent is a silane coupling agent.   The glass substrate for an analytical chip according to any one of claims 1 to 4, wherein the soda-lime-based silica glass is a glass plate produced by a float process. An analysis chip comprising the glass substrate for an analysis chip according to claim 1.

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