WO2013179342A1

WO2013179342A1 - Biochip

Info

Publication number: WO2013179342A1
Application number: PCT/JP2012/003561
Authority: WO
Inventors: 民谷　栄一; 大木　誠; 真人斉藤; 瑞穂村橋; 準向井; 守中西
Original assignee: 株式会社バイオマーカーサイエンス; 国立大学法人大阪大学
Priority date: 2012-05-30
Filing date: 2012-05-30
Publication date: 2013-12-05
Also published as: JPWO2013179342A1

Abstract

A biochip provided with: a substrate (1) in which a metal layer has been deposited on the upper surface; a plurality of partitioning parts (2) provided on the substrate (1), each partitioning part (2) extending in parallel fashion; and a cover member (3) provided on the plurality of partitioning parts (2). A plurality of flow channels (4), each extending in parallel fashion, is formed on the substrate (1) by the substrate (1), the partitioning parts (2), and the cover member (3). A plurality of solid-phase-forming probes (5) is formed in the flow channels (4) in respective fashion on the substrate (1). The gap in the flow channels (4) between the substrate (1) and the cover member (3) has a size that allows a liquid to deploy by capillary phenomenon from one end of the flow channels (4) to the other end.

Description

Biochip

The present invention relates to a biochip, and more particularly to a biochip for easily detecting or quantifying biomolecules in a biological sample.

In recent years, biochips have been used for analysis of biomolecules such as DNA and proteins. In a conventional biochip, a predetermined probe is immobilized on the substrate according to a substance to be detected among DNA, protein, sugar chain, etc., and the probe and a biological sample such as blood are reacted, A desired biomolecule is bound to the probe. This enables detection and quantification of biomolecules in the biological sample. Various tests can be performed simultaneously by fixing various probes on the substrate.

Conventionally, protein detection and quantification are performed by labeling a target protein with a fluorescent substance or the like and measuring the fluorescence intensity. Since such a measuring method requires a labeling step, it takes a long time to perform a series of steps, and inhibits the high recognition ability of biomolecules, leading to a decrease in sensitivity and complication of work. It will be.

Therefore, in recent years, a non-labeling method using localized plasmon resonance (LSPR) has attracted attention as a method for detecting and quantifying biomolecules without performing such labeling (for example, , See Patent Document 1). LSPR is a nonlinear optical phenomenon that appears in noble metal fine particles such as gold, silver, and platinum. In the measurement method using LSPR, a biomolecule is bound to a noble metal particle portion deposited on a substrate of a biochip through a probe, the measurement light is irradiated to the noble metal particle portion, and the intensity of transmitted or reflected light and The magnitude of the shift of the absorption peak wavelength is measured. A desired biomolecule can be quantified using the size as an index. When such a non-labeling method is used, it is not necessary to perform a labeling step, and a test can be easily performed.

Biochips are used for the purpose of disease prevention and treatment. For example, the patient's condition can be examined by detecting and quantifying a biomarker such as a protein related to a disease in the patient's blood with a biochip using the patient's blood as a sample. In addition, when a biochip is used, a plurality of biomarkers can be detected at the same time, and a plurality of patient biological samples can be simultaneously measured.

JP 2002-365210 A

However, in the conventional biochip, a plurality of probes are fixed in a matrix on the substrate, and it is necessary to sequentially drop a small amount of biological sample on each probe, which requires a great deal of labor and time. In addition, even if a device that can automatically drop a sample is used, such a device can continuously drop a single sample, but when dropping a plurality of samples, The process becomes complicated, and there is a risk of contamination between samples in the apparatus. For example, when human blood is used as a sample and dropped onto a biochip, and a standard solution for creating a calibration curve is dropped onto the same biochip, there is a risk of contamination and the like. Similarly, there is a risk of contamination or the like when blood from a plurality of patients is dropped on the same biochip.

The present invention has been made in view of the above problems, and an object of the present invention is to obtain a biochip capable of easily measuring both various substances in a sample and creating a calibration curve without using an expensive apparatus. There is to do.

In order to achieve the above-described object, the present invention has a configuration in which a biochip has a plurality of flow paths, and the flow paths have gaps that allow the liquid to develop by capillary action.

Specifically, the biochip according to the present invention is provided on a substrate having a metal layer formed on the upper surface, a plurality of partition portions extending in parallel to each other, and a plurality of partition portions. A plurality of flow paths extending in parallel with each other by the substrate, the partition portion, and the cover member, and each flow path on the substrate has a plurality of solid phase probes. The gap between the substrate and the cover member in the flow channel is a size that allows the liquid to develop from one end of the flow channel to the other end by capillary action.

According to the biochip of the present invention, a plurality of flow paths are formed on the substrate, and the gap between the substrate and the cover member in the flow paths can be developed from one end of the flow path to the other end by capillary action. Because of the size, a large number of samples can be easily measured simultaneously without using an expensive apparatus. It is also possible to create a calibration curve along with sample measurement. Furthermore, since a metal layer is formed on the upper surface of the substrate, LSPR can be used for measurement, and it is not necessary to perform a labeling step, and highly sensitive measurement can be performed more easily. .

The biochip according to the present invention preferably further includes a liquid discharge means that is detachably provided at one end or the other end of each flow path and discharges the liquid in the flow path.

In this way, the liquid in each flow path can be quickly and easily discharged.

In this case, the liquid discharging means may be a water-absorbing material having water absorption.

Furthermore, the liquid discharging means may be a suction pump that can suck liquid.

In the biochip according to the present invention, the metal layer may be formed via an anodized alumina film having a porous layer on the upper surface of the substrate.

In the biochip according to the present invention, the substrate may have a concavo-convex structure on its upper surface, and the metal layer may be formed on the concavo-convex structure.

In this case, the concavo-convex structure is preferably formed by a nanoimprint method.

In the biochip according to the present invention, the metal layer may be formed on a plurality of silica beads arranged two-dimensionally on the upper surface of the substrate.

In the biochip according to the present invention, the metal layer may be formed by arranging metal nanoparticles directly on the substrate.

In the biochip according to the present invention, the metal layer may include gold.

In the biochip according to the present invention, the partition portion and the cover member may be integrated.

In this way, the partition portion and the cover member can be provided in the same process, and the production of the biochip becomes easier.

The biochip according to the present invention can easily measure a large number of samples and create a calibration curve without using an expensive apparatus. For this reason, it is possible to easily measure a plurality of biomolecules of one patient, or to measure a single or a plurality of biomolecules of a plurality of patients.

FIG. 1 is a perspective view showing a biochip according to an embodiment of the present invention. FIG. 2 is a plan view showing an example of a biochip partition section according to an embodiment of the present invention. FIG. 3 is a perspective view showing a mode in which liquid is discharged from the flow path in the biochip according to the embodiment of the present invention. FIG. 4 is a graph showing the results of measuring transthyretin using the biochip of the present invention and a conventional biochip. FIG. 5 is a graph showing the results of measuring C-reactive protein using the biochip of the present invention and a conventional biochip. FIG. 6 is a graph showing the results of measuring cystatin C using the biochip of the present invention and a conventional biochip.

A biochip according to an embodiment of the present invention will be described with reference to FIGS.

As shown in FIG. 1, the biochip 10 according to the present embodiment is formed with a plurality of partition portions 2 extending in parallel on the upper surface of a substrate 1 made of, for example, glass. A cover glass 3 as a cover member is provided on the substrate 1 and the partition 2. A plurality of flow paths 4 extending in parallel are formed by the substrate 1, the partition portion 2, and the cover glass 3, and a plurality of solid-phase probes 5 are formed in the flow paths 4 on the upper surface of the substrate 1. Here, the gap between the substrate 1 and the cover glass 3 in the flow path 4 is such a size that the liquid can be developed from one end of the flow path 4 to the other end by capillary action. Specifically, the size of the gap is about 100 μm to 1000 μm. If it is less than 100 μm, it becomes difficult to discharge the liquid from the flow path 4, and if it is greater than 1000 μm, the light is not focused when irradiating the solid-phased probe 5, making measurement difficult. In FIG. 1, one end of the flow path 4 is formed inside the end of the substrate 1, but it may extend to the end of the substrate 1 like the other end. Conversely, the other end of the flow path 4 may be formed inside the end of the substrate 1.

On the upper surface of the substrate 1, for example, a metal layer (not shown) formed by depositing gold is formed. The solid-phase probe 5 formed on the substrate 1 includes, for example, an antibody, and specifically, the antibody or the like is fixed to a metal layer deposited on the substrate 1. The kind of the immobilized probe 5 can be selected depending on the biomolecule to be detected or quantified. In addition to the antibody, when detecting a predetermined DNA, a nucleic acid such as a DNA fragment capable of binding to the DNA can be used. The metal layer may be a metal layer that can perform detection or measurement using localized plasmon resonance (LSPR), and may use silver or platinum in addition to gold.

The metal layer is preferably formed on the substrate 1 via an anodized alumina film having a porous layer. Further, the metal layer may be formed on a plurality of silica beads arranged two-dimensionally on the substrate 1. Further, the metal layer may be formed by arranging metal nanoparticles directly on the substrate 1.

In addition to these, an uneven structure having a desired shape may be formed on the substrate 1 and a metal layer may be formed thereon. In order to form a concavo-convex structure on the substrate 1, for example, a nanoimprint method can be used. In the case of using the nanoimprint method, for example, a polymer resin can be used for the substrate 1. After the upper surface of the substrate 1 is embossed using a mold having a specific shape, a metal layer may be deposited on the upper surface. The shape of the mold is preferably a shape suitable for depositing the metal layer on the upper surface of the substrate 1.

The partition portion 2 is provided so as to be interposed between the substrate 1 and the cover glass 3, thereby forming a gap between the substrate 1 and the cover glass 3. Moreover, the partition part 2 is fluid-adhered with the board | substrate 1 and the cover glass 3, and the flow path 4 is formed between the partition parts 2 by this, respectively. The partition portion 2 has a thickness capable of forming a gap between the substrate 1 and the cover glass 3 so that a liquid can be developed from one end to the other end of the flow path 4 by capillary action. Specifically, the thickness of the partition 2 is about 100 μm to 1000 μm. In this way, the sample is developed in the flow path 4 in which the plurality of immobilized probes 5 are formed simply by dropping a liquid sample onto one end of each flow path 4, so that an expensive apparatus is not used. Multiple samples can be easily measured simultaneously. In addition, it is possible to create a calibration curve together with the measurement of the sample, and it is also possible to prevent contamination between samples when the sample is dropped. Furthermore, as described above, the liquid can be discharged from the flow path 4, and when the solid-phased probe 5 is irradiated with light, the light is easily focused and measurement is possible.

For the partition part 2, for example, a double-sided tape having the above-mentioned thickness can be used. Moreover, you may form the partition part 2 by apply | coating an adhesive agent with the said thickness. In addition to these, the partition portion 2 can be formed by adhering a gasket made of a plurality of resins or the like to the substrate 1. Further, instead of a plurality of gaskets, a partition member 20 such as a comb-shaped gasket having a plurality of partition portions 2 as shown in FIG. 2 may be used. Moreover, you may form the partition part 2 in the both sides of a groove | channel by forming in the board | substrate 1 or the cover glass 3 the some groove | channel extended in parallel mutually. That is, the substrate 1 or the cover glass 3 and the partition portion 2 may be integrated. Moreover, in this embodiment, although the cover glass 3 is used as a cover member, you may use the cover member which consists of a transparent film.

A plurality of immobilized probes 5 are formed in each flow path 4 surrounded by the substrate 1, the partition 2 and the cover glass 3. For example, when 12 flow paths 4 are formed, if 8 immobilized probes 5 are formed in each flow path 4, 96 immobilized probes 5 are formed in a matrix on one biochip. it can. Here, the respective immobilized probes 5 may be the same type or a plurality of types. The numbers of the flow paths 4 and the immobilized probes 5 are not limited to the above numbers.

Moreover, as shown in FIG. 3, the water absorbing material 6 which has a water absorption which is a liquid discharge means for discharging the liquid expand | deployed in each flow path 4 is provided in one end part or the other end part of the flow path 4. It can be detachably provided. For the water absorbing material 6, for example, filter paper and resin having high water absorption can be used. By providing the water absorbing material 6 at the other end of the flow path 4, the water absorbing material 6 absorbs the sample in the flow path 4, so that the sample can be discharged from the flow path 4. In addition, a suction pump using a motor or the like can be used as the liquid discharging means. By such a liquid discharge means, the liquid in each flow path 4 can be discharged quickly and easily.

Next, a method for detecting and measuring a desired biomolecule in a sample using the biochip according to the present embodiment will be described.

For example, when detecting a biomarker related to a disease in blood using human blood as a sample, an antibody or a nucleic acid that binds to the desired biomarker is immobilized in advance as a solid phase probe on the biochip according to the present embodiment. To do. Here, a plurality of solid-phase probes may be formed in each flow path. As described above, for example, when 12 rows of flow paths are formed, when solid-phase probes are formed at 8 locations in each flow path, 96 immobilized probes can be formed in a matrix on one biochip.

Next, a sample such as human blood or serum is dropped on one end of the flow path of the biochip. The dropped sample develops in the channel by capillary action and reaches the other end of the channel. Here, the dropping of the sample may use an automatic dispensing device, or may be performed manually with a normal pipette or dispenser. However, when a plurality of types of samples are dropped onto one biochip, it is preferable to use a pipette that can easily replace the chip from the viewpoint of preventing contamination. In addition, for example, a protein standard solution can be dropped onto the same biochip together with the dropping of the sample in order to create a calibration curve. In this step, the liquid discharging means is not attached to the biochip.

Next, the sample and the immobilized probe are reacted at a predetermined temperature and time. As for the reaction time and the like, suitable conditions can be appropriately selected according to the type of sample and the type of probe.

Next, the sample is discharged from the other end of the flow path by attaching the liquid discharge means to the biochip. For example, when a water absorbing material is used for the liquid discharging means, the water absorbing material is placed on the other end of the channel of the substrate so as to be in contact with the sample. Thereby, since a water absorption material absorbs the sample in a flow path, a sample can be discharged | emitted from a flow path. When a suction pump is used as the liquid discharge means, the sample can be discharged from each channel by placing the suction port of the suction pump on the other end of the channel. Note that the sample may be discharged from one end portion by providing it at one end portion of the flow path without providing the liquid discharge means at the other end portion of the flow path. After discharging the sample, the liquid discharging means is removed from the biochip.

Next, a predetermined cleaning solution that is a buffer solution such as phosphate buffered saline (PBS) is dropped onto one end of the flow path. The dropped sample develops in the channel by capillary action and reaches the other end of the channel. Thereafter, like the sample, the cleaning liquid is discharged from the other end of the flow path by the liquid discharging means. This washing is performed about 2 to 3 times.

Next, after the biochip is dried, a desired biomolecule is detected and measured by measuring the absorbance. Here, the absorbance is measured in advance before reacting the sample and the probe, and compared with the absorbance after the reaction. It is measured by the shift amount of the absorption peak wavelength before the reaction and the absorption peak wavelength after the reaction.

According to the biochip according to an embodiment of the present invention, a plurality of immobilized probes that bind to the biomolecule to be measured are formed in a flow path having a gap that allows the liquid to be developed by capillary action. The reaction between each immobilized probe and a sample containing a biomolecule can be easily performed. Therefore, it is possible to easily measure a large number of biomolecules without using an expensive apparatus. In addition, a calibration curve can be created using the same biochip together with the measurement of biomolecules.

Hereinafter, specific examples of the biochip of the present invention will be shown. In this example, the amount of transthyretin (TTR), C-reactive protein (CRP) and cystatin C in human serum was measured using the biochip of the present invention, and the results were measured using a conventional method. And compared with the measured results.

(Production of biochip substrate)
First, an aluminum film having a thickness of about 3 μm was deposited on the upper surface of a commercially available ordinary slide glass. Subsequently, the anodized alumina film was formed on the formed aluminum film by using a 0.3 M oxalic acid solution at an applied voltage of 40 V for the first time of 60 minutes at 0 ° C. . Thereafter, the formed alumina film was subjected to ultrasonic treatment for 10 minutes and washed with ultrapure water.

Next, the anodized alumina film formed in the above process was removed. The removal of the anodized alumina film is performed by reacting at 60 ° C. to 80 ° C. for 3 minutes using a solution obtained by adding 50 g / L of chromic acid to a solution containing phosphoric acid and water at a ratio of 8: 2. It was. After the reaction, ultrasonic treatment was performed for 10 minutes, and washing was performed using ultrapure water.

Next, using a 0.3 M oxalic acid solution, an anodizing treatment is performed for the second time at an applied voltage of 40 V under conditions of 10 ° C. for 5 minutes, and then ultrasonic treatment is performed for 10 minutes to obtain ultra pure water. Washing was performed using As a result, an anodized alumina film having a porous layer having a thickness of about 600 nm on the upper surface of the slide glass was obtained.

Next, gold was deposited on the obtained anodized alumina film by a sputtering method. Specifically, gold was deposited around the opening of the porous layer of the anodized alumina film. As a result, a biochip substrate including a metal layer made of gold was obtained. This substrate can be stored in a desiccator until use.

(Production of biochip)
In order to immobilize the immobilized probe on the prepared substrate, first, the substrate was immersed in ethanol containing 1 mM 10-carboxyl-1-decanthiol overnight, preferably for 6 to 12 hours. Thereafter, the substrate was washed with ultrapure water and dried. Subsequently, the substrate was immersed in an aqueous solution containing 0.1 M N-hydroxysuccinimide ester (NHS) and 0.4 M water-soluble carbodiimide (WSC) for 10 minutes. Thereafter, the substrate was washed with phosphate buffered saline (PBS) and dried.

Next, an anti-TTR antibody, an anti-CRP antibody and an anti-cystatin C antibody were respectively dropped onto the substrate and allowed to stand for 30 minutes. Here, the dropping of the antibody is performed at a plurality of locations in a matrix. In this example, each antibody was arranged in 8 spots of 2 columns × 4 rows, and a total of 24 spots of 6 columns × 4 rows were arranged in total. Thereafter, the substrate was washed twice with Tween-20-containing PBS (PBST), washed once with PBS, and dried. As a result, a plurality of spot-like immobilized probes were formed.

Next, blocking was performed by immersing the substrate on which the immobilized probe was formed in 1% bovine serum albumin (BSA) for 30 minutes. Thereafter, the substrate was washed twice with PBST, once with PBS, and dried.

Next, a partition portion was provided on the substrate. A double-sided tape with a thickness of 136 μm is used for the partitioning part, and five double-sided tapes are provided in parallel between the probes and at both ends along the row of probes formed in the above process on the substrate. Stuck on.

Next, a commercially available ordinary cover glass was stuck on the substrate through a double-sided tape as a partition so as to cover the plurality of probes. Thereby, a gap corresponding to the thickness of the double-sided tape is formed between the substrate and the cover glass, and this gap becomes a flow path. In this embodiment, four flow paths are formed.

Also, a water absorption pad made of filter paper as a liquid discharge means was prepared for discharging the sample from the flow path.

The biochip of this example was completed through the above steps.

(Sample measurement)
Hereinafter, a method for measuring a sample using the biochip produced by the above process will be described. In this example, a calibration curve is prepared using a standard protein mixed solution containing three kinds of proteins TTR, CRP and cystatin C, and the results of detecting and quantifying the three kinds of proteins using serum as a sample are shown.

First, the absorbance of the region where each immobilized probe of the biochip obtained by the above method is previously measured is measured, and the absorption peak wavelength is recorded.

Next, a standard protein mixture and serum are dropped onto one end of each flow path. In this example, standard protein mixtures of 10 ng / mL and 100 ng / mL, and human serum diluted 100-fold and 1000-fold were used. These were each dropped onto one end of the flow path. Since the size of the gap between the substrate and the cover glass is the thickness of the double-sided tape and is extremely small, the human serum and standard protein mixture is developed from one end of the flow path to the other end due to capillary action. As a result, each probe in the flow path can react with the human serum and the standard protein mixture. After standing in this state for 30 minutes, a water absorption pad made of filter paper was placed on the other end of the flow channel so as to contact the sample, and the sample in the flow channel was discharged. Thereafter, the flow path was washed twice with PBST, washed once with PBS, and then dried in the same manner as the step of developing the sample in the flow path.

Next, the absorbance of the region where each immobilized probe of the biochip after washing and drying is measured, and the amount of shift from the absorption peak wavelength measured before reacting with the sample is used as an index, the standard protein mixture Was used to measure the content of each protein. The results are shown in FIGS. FIGS. 4 to 6 show the results of the present example and the results of testing by dropping a sample on each probe in sequence using a conventional biochip having no flow path.

As shown in FIGS. 4 to 6, even when the biochip according to the present invention was used, an equivalent result was obtained as compared with the case where the conventional biochip was used. That is, a measurement result equivalent to the conventional one could be obtained by a simpler method than the conventional one.

The biochip according to the present invention can easily measure a large number of samples simultaneously without using an expensive apparatus, and is particularly useful for biochips for detecting or quantifying biomolecules in a biological sample. .

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Partition part 3 Cover glass 4 Channel 5 Solid-phase probe 6 Water absorbing material 10 Biochip 20 Partition member

Claims

A substrate having a metal layer formed on the upper surface;
A plurality of partitions provided on the substrate, each extending in parallel;
A cover member provided on the plurality of partitions,
On the substrate, a plurality of flow paths extending in parallel are formed by the substrate, the partition portion, and the cover member,
A plurality of immobilized probes are formed in each flow path on the substrate,
The gap between the substrate and the cover member in the channel is a biochip having a size that allows liquid to develop from one end of the channel to the other by capillary action.
In claim 1,
The biochip further provided with the liquid discharge | emission means which is provided in the one end part or other end part of each said flow path so that attachment or detachment is possible, and discharges the liquid in the said flow path.
In claim 2,
The liquid discharging means is a biochip which is a water-absorbing material having water absorption.
The biochip according to claim 2, wherein the liquid discharging means is a suction pump capable of sucking a liquid.
In any one of claims 1 to 4,
The biochip is a biochip in which the metal layer is formed on an upper surface of the substrate via an anodized alumina film having a porous layer.
In any one of claims 1 to 4,
The substrate has an uneven structure on its upper surface,
The metal layer is a biochip formed on the concavo-convex structure.
In claim 6,
The uneven structure is a biochip formed by a nanoimprint method.
In any one of claims 1 to 4,
The biochip is a biochip formed on a plurality of silica beads arranged two-dimensionally on the upper surface of the substrate.
In any one of claims 1 to 4,
The metal layer is a biochip in which metal nanoparticles are arranged directly on the substrate.
In any one of claims 1 to 9,
The metal layer is a biochip containing gold.
In any one of claims 1 to 10,
The partition part and the cover member are biochips formed integrally.