JP2004170372A - Manufacturing method of biomolecule detecting apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method employed as a manufacturing method of a biomolecule detecting apparatus that detects biomolecules contained in a specimen, which can shorten the manufacturing period, simplify processes, make the object biomolecules easy to deal with and realize a small-sized biomolecule detecting apparatus. <P>SOLUTION: In the manufacturing method of the biomolecule detecting apparatus, in which two or more kinds of biomolecules capable of being charged positively or negatively are arranged at prescribed arrangement positions respectively, and the biomolecules contained in the specimen are detected by using reactions to the charged biomolecules, in the case the two or more kinds of biomolecules capable of being charged positively or negatively are arranged at the prescribed arrangement positions respectively, the prescribed arrangement position is applied with a potential value different from potential values being applied on the other arrangement positions, and thereby making a prescribed biomolecule attach electrically to the prescribed arrangement position. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for arranging a substance, particularly a biomolecule, used for chemical analysis, diagnosing a disease, and drug development, and a substance contained in a test sample, particularly, a biomolecule for detecting a substance contained in a test sample by using the substance arranging method. The present invention relates to a method for manufacturing a device. More specifically, the present invention relates to a method for arranging a probe as a substance for detection on a biochip and a method for producing a biochip using the probe arranging method.
[0002]
[Prior art]
The Human Genome Project, which has been underway since the 1990s, is an attempt by each country to share all the human genetic code, and it was announced that a draft version was completed in the summer of 2000. In the future, it is expected that the progress of functional genomics and structural genomics will clarify the function of each part of the decoded human genome sequence information.
[0003]
The Human Genome Project has brought a major paradigm shift to science and technology and industry involved in life sciences. For example, diabetes is classified based on the condition that the blood sugar level is high, and the causes of the onset are classified as types I and II based on the degree of insulin production in the patient's body. Has been done.
[0004]
On the other hand, according to the Human Genome Project, it is involved in the control of the amino acid sequence structure of proteins such as enzymes and receptors involved in the regulation of the detection, synthesis and degradation of blood sugar and insulin, and the control of the abundance of such proteins All information on the DNA sequence of the gene is presented to us.
[0005]
Using such information, diabetes, a phenomenon in which blood sugar levels are not regulated properly, depends on which of the proteins involved in a series of processes such as detection, synthesis, degradation, etc. Should be able to make the appropriate diagnosis and treatment.
[0006]
In particular, the development of genomic drug discovery drugs for specific proteins based on the human genome sequence is being vigorously pursued in the pharmaceutical industry as a future business opportunity, and it is necessary to understand the state of a series of functionally related proteins. It is expected that the time will come when genomic drug discovery drugs will be administered to alleviate or cure symptoms.
[0007]
However, a technique for easily measuring the abundance of such a series of functionally related proteins is still being developed as a proteome analysis technique.
[0008]
Currently established methods use a combination of two-dimensional electrophoresis and mass spectrometry, which requires relatively large equipment and is used in clinical settings, such as hospital laboratories and beds. In order to grasp the patient's symptoms from the side, it is necessary to develop a simpler new technique.
[0009]
Under these circumstances, interest has been gathered in researches called micro-total analysis system (μ-TAS) and Lab-on-a-chip. In this method, a micrometer-sized groove (microchannel) is formed in a glass or silicon substrate of several centimeters square to form a fine device, in which chemical analysis and reaction are performed. Flowing liquid and gas samples through microscopic channels (several hundred μm to several μm wide) offers advantages such as reduced sample and waste volume, high-speed processing, and the possibility of miniaturizing even chemical plants. Therefore, application of this technology to the biotechnology field is expected. Μ-TAS is translated as “integrated chemical analysis system”, “microchemical / biochemical analysis system”, etc., and is a chemical analysis system with downsized sensors and analyzers, and is used in analytical chemistry laboratories. It integrates the functions of devices to be implemented on a chip.
[0010]
Above all, a biochip technology represented by a DNA chip (or DNA microarray) has attracted attention as an effective means for gene analysis. A biochip is a high-density array of many different probes consisting of biopolymers such as DNA and proteins, spotted on the surface of a substrate made of glass, silicon, plastic, etc. It is characterized in that testing of nucleic acids and proteins can be simplified in the field of therapy and the like (for example, see Patent Document 1 and Non-Patent Document 1).
[0011]
As the probe, for example, DNA or nucleotide is used. When an unknown DNA fragment is flown into such a biochip, the target DNA is captured by hybridization with a probe by utilizing the property of the DNA binding to the DNA complementary thereto. . If a fluorescent label is added to the unknown DNA in advance, the captured target is detected as a fluorescent signal from each spot on the biochip. It is possible to observe the status of tens of thousands of DNAs or RNAs at once.
[0012]
[Patent Document 1]
JP 2001-235468 A (paragraph numbers 0002 to 0009)
[0013]
[Non-patent document 1]
"Journal of American Chemical Society", 1997, Vol. 119, p. 8916-8920
[0014]
[Problems to be solved by the invention]
The biochip as described above has the following problems. First, for a specific biomolecule, it is necessary to align and spot a large number of different probes at a high density, and it is necessary to arrange a device for reacting each biomolecule of interest. In addition, since each of these containers has a size that can be seen with the naked eye, it has been an obstacle to miniaturization.
[0015]
Second, after forming a fine flow path pattern on the substrate using photolithography, a groove is formed by a method such as etching, and a lid is formed to form a flow path, such as μ-TAS. This method is generally used when producing a complicated biochip, but there is no means for attaching a DNA probe after sealing. For this reason, it is necessary to perform the operation of arranging the probes at a high density and spotting them during the process of producing the biochip, and there is a problem of affecting the DNA added during the process of producing the biochip.
[0016]
Still other objects and advantages of the present invention will become apparent from the following description.
[0017]
[Means for Solving the Problems]
According to one embodiment of the present invention, two or more types of biomolecules that can be positively or negatively charged are respectively arranged at predetermined locations, and are included in a test sample by utilizing a reaction with these biomolecules. In the method of manufacturing a biomolecule detecting device for detecting a biomolecule, when arranging two or more types of biomolecules that can be positively or negatively charged at a predetermined location, another location is placed at the predetermined location. And a method for manufacturing a biomolecule detecting device, which applies a potential different from the above and electrically attaches a predetermined biomolecule to the predetermined location.
[0018]
Two or more types of biomolecules that can be positively or negatively charged include proteins, DNAs, or nucleotides, and after attaching a lid to the location, electrically attaching the predetermined biomolecule to a predetermined location. preferable.
[0019]
According to the above method, in a method for manufacturing a biomolecule detecting device that detects biomolecules contained in a test sample by arranging two or more types of biomolecules, the time is shortened, the process is simplified, and the handling of the target substance is reduced. It can be made easier. Further, the number of arrangement locations that can be arranged per predetermined area can be increased, and the biomolecule detection device can be downsized.
[0020]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings, examples, and the like. It should be noted that these drawings, examples, etc., and the description are merely examples of the present invention, and do not limit the scope of the present invention. It goes without saying that other embodiments can also belong to the category of the present invention as long as they conform to the gist of the present invention.
[0021]
The present invention has been completed as a result of a study focusing on the fact that biomolecules such as DNA are negatively charged. According to one aspect of the present invention, when two or more types of probes that can be negatively charged are arranged at predetermined probe locations on a biochip, the predetermined probe location is different from other probe locations. By applying different potentials, a predetermined probe is electrically attached to the predetermined probe location.
[0022]
FIG. 1 is a schematic plan view of a biochip, and FIG. 2 is a schematic side sectional view thereof. When a sample is detected (qualitative or quantitative analysis) using the biochip of FIGS. 1 and 2, the sample passes through the sample introduction port 4 and is provided on the substrate 1, a sensor comprising a large number of probe placement locations 2. In general, the DNA is guided to the array unit 3 where it is hybridized with a probe to capture a target DNA or the like and detect a fluorescent signal using a fluorescent label.
[0023]
Conventionally, as a method of installing such a probe at a predetermined probe location on a biochip, a probe is manually installed at each probe location.
[0024]
On the other hand, according to the present invention, a probe is provided at a predetermined probe location on a biochip by utilizing a difference in Coulomb force when a potential difference is applied between the probe solution and the probe location. By merely pouring the probe solution onto the substrate where the probe is located, the probe can be attached to a predetermined probe location and not placed at other probe locations, and labor can be eliminated. Here, the probe solution includes not only a case where the probe itself is in a liquid state but also a solution in which the probe is diluted with a liquid medium. Dilution with a liquid medium is preferable because the concentration of a probe installed at a predetermined probe location at a predetermined area can be easily adjusted.
[0025]
In order to place a plurality of probes at different probe locations, it is necessary to provide a potential difference between the probe solution and the probe location each time a probe solution containing a different type of probe is used. A potential different from that of the other probe placement locations may be applied to the probe placement location to be used, and the predetermined probe may be electrically attached to the predetermined probe placement location.
[0026]
ADVANTAGE OF THE INVENTION According to the arrangement method concerning this invention, the structure of a biochip can be simplified and the complicated shape preparation step by photolithography etc. can be eliminated. Further, the size of the biochip can be reduced.
[0027]
In addition, since the conventional probe placement method cannot be performed after the lid is attached to the probe placement location, the probe placement must be performed before the lid is placed at the probe placement location. There was a risk that the biochip manufacturing step would alter the probe, but if the probe placement method of the present invention was employed, it would be sufficient to just pour the probe solution, and at any stage after the manufacturing step that might alter the probe. The probe can be attached and arranged, and the problem of probe deterioration can be solved. After the biochip is completed, the probe can be arranged. Further, by employing the probe arrangement method according to the present invention, the number of probe arrangement locations that can be arranged per predetermined area can be increased, and the size of the biochip can be reduced.
[0028]
Examples of the biomolecules used for the probe include proteins, DNAs, and nucleotides. Here, in the present invention, "nucleotide" means any one of the group consisting of oligonucleotides and polynucleotides or a mixture thereof. Such a substance is often negatively charged, and by hybridizing with a probe, a target substance is captured, and a technique of detecting a fluorescent signal using a fluorescent label can be used. This is because the effects of the present invention can be effectively exhibited. In addition, proteins, DNAs and nucleotides may be mixed. In addition, biomolecules include those derived from living organisms, processed ones, and synthesized molecules in addition to those derived from living organisms.
[0029]
When a probe is placed at a predetermined probe placement location on a biochip, it may be necessary to fix the probe so that the probe does not flow out when a sample is introduced. This fixing method can be arbitrarily selected without departing from the spirit of the present invention.
[0030]
For example, in the case of a protein, DNA or nucleotide, a known method such as a method of synthesizing a probe into which a disulfide group or a thiol group is introduced and bringing the probe into contact with a polished electrode surface can be employed. For example between the nucleotide binding and disulfide group and a thiol group - (CH ₂₎ n _- structure may be inserted a coupling called linker having the. The number of sensitive parts that can be immobilized at the probe location is often influenced by the type of the linker and the length of the bond. In general, when the number of CH ₂ units decreases, the number of sensitive parts that can be fixed on the electrode tends to decrease. When the number is too large, the effect is lost and it is not necessary to increase the number unnecessarily. n = 2 to 15 is preferred.
[0031]
The material of the probe placement place can be arbitrarily determined according to the purpose, but Au is particularly preferable. This is because, when a biomolecule is used as a probe, it can be easily fixed to a probe placement location.
[0032]
In the biochip manufacturing method, when arranging two or more types of probes at respective predetermined probe placement locations, a different potential from the other probe placement locations is given to the predetermined probe placement location, and the predetermined An arrangement method of electrically attaching a predetermined probe to the probe arrangement location can be used.
[0033]
Accordingly, manual labor can be eliminated, the structure of the biochip can be simplified, and a complicated shape forming step by photolithography or the like can be eliminated. Further, the size of the biochip can be reduced. Further, after the manufacturing step that may deteriorate the probe, the probe can be arranged at an arbitrary stage, and the problem of the deterioration of the probe can be solved. After the biochip is completed, the probe can be arranged. The number of probe arrangement locations that can be arranged per predetermined area can be increased, and the size of the biochip can be reduced.
[0034]
In the above, the method of arranging probes and the method of manufacturing a biochip in the biochip have been described, but the present invention is not limited to these, and has a wider application range. In other words, without being limited to the concept of a probe or a biomolecule, generally, two or more types of substances that can be positively or negatively charged are respectively attached to predetermined locations, and a substance placement method or a positively or negatively charged substance is used. The present invention can be applied to a method of manufacturing a substance detection device that arranges two or more types of substances that can be used at predetermined locations and detects a substance contained in a test sample by using a reaction with these substances. it is conceivable that. That is, the ranges shown in the following supplementary notes are also included in the scope of the present invention.
[0035]
In the above description, "two or more types of substances" means that there are two or more substances to be detected and distinguished by the present invention, for example, "different types of DNA". The reaction used can be freely determined according to the purpose of use. The substance contained in the test sample can be analyzed qualitatively or quantitatively according to the purpose of use. That is, the substance detection according to the present invention includes detecting the presence of the specific substance and analyzing how much the specific substance is contained in the test sample.
[0036]
By adopting the method of manufacturing the substance arrangement and the substance detection device, it is possible to eliminate the manual work of arranging the substance at a predetermined location, thereby shortening the production time, simplifying the process, and facilitating the handling of the target substance. Can be realized.
[0037]
Further, since there is no restriction that the target substance cannot be placed after the lid is attached to the predetermined placement place as in the case of manual work, the predetermined placement can be performed at any time after the manufacturing step of altering the target substance. The target substance can be attached to the place. Even after the completion of the substance detection device, it is possible to perform the attachment and placement on the predetermined placement place.
[0038]
Further, since the probe can be easily arranged in a minute area or an area having a complicated shape which is physically difficult to access, the degree of freedom in designing the substance detecting device is increased. This makes it possible to increase the number of probe arrangement locations that can be arranged per predetermined area, and to reduce the size of the substance detection device as compared with the related art.
[0039]
The substance detection device realized by the present invention is, for example, in the case where hepatocytes switch intracellular glycogen metabolism in response to insulin receiving state in diabetes, for example, a series of protein interaction networks from insulin receptors to glycogenases. It can be used as a protein detection device that detects that a part is decreasing or increasing.
[0040]
By utilizing such a protein detection device, it becomes possible to capture the population of proteins, including post-translational modifications such as phosphorylation and sugar chain addition. Also, instead of diagnosing the phenomenon that appeared as a symptom as in the past as diabetes, for example, it is possible to grasp that a decrease in the function of a specific protein involved in the interaction network is causing the dysfunction of glucose metabolism. As a result, appropriate diagnosis and treatment corresponding to the cause of the dysfunction and verification of the treatment result can be performed. The same technique can be applied not only to diabetes but also to hypertension, hyperlipidemia, cancer (cell growth dysregulation) and other multifactorial diseases in general.
[0041]
【Example】
Next, embodiments of the present invention will be described in detail.
[0042]
[Example 1]
A biochip having the sensor array unit 3 as shown in FIGS. In FIGS. 1 and 2, the probe arrangement location 2 is made of Au.
[0043]
3 to 7 are schematic diagrams in which the sensor array unit 3 which is a set of a plurality of probe arrangement locations is enlarged. FIG. 3 shows a state before installing the probe, FIGS. 4 and 6 show a state where the probe A is installed at the probe arrangement location a, FIGS. 5 and 7 show a state after installing the probes A to F at the probe arrangement places a to f, Is shown.
[0044]
Probes are installed at individual probe arrangement locations a to f of the sensor array unit 3 in FIG. The arrangement places a to f can be electrically independent from each other, and are wired so that a potential is given from each electrode. When the probes A to F are arranged in the order of the probe arrangement locations a to f, the operation is performed as follows.
[0045]
(1) After completion of the biochip, a probe solution is injected from the sample inlet 4 in FIGS. At this time, a voltage is applied between the probe solution and each probe location by setting the potentials b to f to a minus side from the potential a. In this state, the probe solution containing the probe A is caused to flow through the sensor array. By this operation, A is attached to a as shown in FIGS.
[0046]
(2) Next, the potentials of a, c to f are set to be more negative than the potential of b, and a voltage is applied between the probe solution and each probe arrangement location. In this state, the probe solution containing the probe B is caused to flow through the sensor array. By this operation, B is attached to b. At this time, A set in a may flow out a little, but it is easy to leave a necessary amount of A.
[0047]
(3) Similarly, a probe is arranged at each of the probe arrangement locations c to f. In this way, as shown in FIGS. 5 and 7, the probes A to F are attached to the probe placement locations a to f.
[0048]
(4) The optimum values of the applied voltage and the potential at the probe arrangement location can be appropriately determined by experiments and the like.
[0049]
FIGS. 4 and 5 and FIGS. 6 and 7 are obtained by estimating the difference in the state of immobilization when the probes A to F are DNA.
[0050]
FIGS. 4 and 5 are model diagrams in which a probe is fixed to a probe installation location by using the entire charged DNA molecule. In such a case, for example, in the above (1), the potential of a is set to 0 (zero) V and the potential of b to f is set to minus several hundred volts between the probe solution and the probe installation location. , A voltage is applied.
[0051]
6 and 7, the following formula ^{RSH + Au → AuSR + H +} + e -
(Here, R means the nucleotide structure of DNA and SH means a thiol group.) FIG. 7 is a model diagram illustrating a case where a probe is fixed to a probe installation location using a probe. For example, in the above (1), a case where a voltage is applied between the probe solution and the probe installation location such that a becomes a positive potential and b to f become negative potentials can be exemplified.
[0052]
As a result of preparing the biochip in this way, the manual probe placement operation can be eliminated, and the manufacturing time can be reduced, the process can be simplified, and the handling of the target substance can be facilitated. In addition, the probe can be arranged in a minute area or an area having a complicated shape that is physically difficult to access, and the number of probe arrangement places that can be arranged per a predetermined area can be increased. The device can be made smaller than before.
[0053]
From the contents disclosed above, the inventions shown in the following supplementary notes can be derived.
[0054]
(Supplementary Note 1) In a substance arranging method of arranging two or more types of substances that can be positively or negatively charged at respective predetermined locations,
Applying a different potential to the predetermined location to another location to electrically attach a predetermined substance to the predetermined location,
Material placement method.
[0055]
(Supplementary Note 2) The substance disposing method according to Supplementary Note 1, wherein the two or more types of substances that can be positively or negatively charged include biomolecules.
[0056]
(Supplementary note 3) The substance arrangement method according to supplementary note 1, wherein the two or more types of substances that can be positively or negatively charged include proteins, DNAs, or nucleotides.
[0057]
(Supplementary Note 4) The protein, DNA, or nucleotide body has a disulfide or thiol group, between the nucleotide binding and disulfide group and a thiol group - a bond _{(n = 2~15) - (CH} 2) n 4. The method for arranging a substance according to claim 3, comprising:
[0058]
(Supplementary note 5) The substance disposing method according to any one of Supplementary notes 1 to 4, wherein the disposing place is made of Au.
[0059]
(Supplementary Note 6) A substance detection device that arranges two or more types of substances that can be positively or negatively charged at predetermined locations, and detects a substance contained in a test sample by using a reaction with these substances. In the manufacturing method of
When arranging two or more types of substances that can be positively or negatively charged at the respective predetermined locations, a different potential is applied to the predetermined location from the other locations, and the predetermined substance is applied to the predetermined location. To electrically attach the
A method for manufacturing a substance detection device.
[0060]
(Supplementary Note 7) The substance detection device according to Supplementary Note 6, wherein a predetermined substance is electrically attached to a predetermined location after the step of possibly altering two or more types of substances that can be positively or negatively charged. Production method.
[0061]
(Supplementary Note 8) The method for manufacturing a substance detection device according to Supplementary Note 6, wherein a predetermined substance is electrically attached to a predetermined location after the lid is attached to the location.
[0062]
(Supplementary note 9) The method for manufacturing a substance detection device according to any one of supplementary notes 6 to 8, wherein the substance detection device is a biochip.
[0063]
(Supplementary note 10) The method for manufacturing a substance detection device according to any one of Supplementary notes 6 to 9, wherein the two or more types of substances that can be positively or negatively charged include biomolecules.
[0064]
(Supplementary Note 11) The method for producing a substance detection device according to any one of Supplementary Notes 6 to 9, wherein the two or more types of substances that can be positively or negatively charged include proteins, DNAs, or nucleotides.
[0065]
(Supplementary Note 12) The protein, DNA, or nucleotide body has a disulfide or thiol group, between the nucleotide binding and disulfide group and a thiol group - a bond _{(n = 2~15) - (CH} 2) n 12. The method for producing a substance detection device according to supplementary note 11, wherein the method comprises:
[0066]
(Supplementary Note 13) The method for manufacturing a substance detection device according to any one of Supplementary Notes 6 to 12, wherein the arrangement place is made of Au.
[0067]
【The invention's effect】
According to the present invention, in a method for arranging two or more types of substances, particularly a substance for arranging a biomolecule, and a method for manufacturing a substance detection apparatus for detecting a substance contained in a test sample, particularly, a biomolecule, the time can be reduced and the number of steps can be reduced. Simplification and easy handling of the target substance can be realized. Further, the number of arrangement places that can be arranged per predetermined area can be increased, and the substance detection device can be downsized.
[Brief description of the drawings]
FIG. 1 is a schematic plan view of a sensor array portion of a biochip.
FIG. 2 is a schematic side sectional view of a sensor array part of FIG. 1;
FIG. 3 is an enlarged schematic view of a sensor array unit.
FIG. 4 is a model diagram showing how a probe is installed in a probe placement field.
FIG. 5 is a model diagram illustrating a state where a probe is installed at a probe installation location.
FIG. 6 is another model diagram showing how a probe is installed in a probe placement field.
FIG. 7 is another model diagram illustrating a state where a probe is installed at a probe installation location.
[Explanation of symbols]
1 Substrate 2 Probe placement location 3 Sensor array section 4 Sample inlet

Claims (3)

A biomolecule detection device that arranges two or more types of biomolecules that can be positively or negatively charged at predetermined locations, and detects biomolecules contained in a test sample by utilizing a reaction with these biomolecules. In the manufacturing method of
When arranging two or more types of biomolecules that can be positively or negatively charged at each predetermined location, a different potential from the other location is applied to the predetermined location, and a predetermined potential is applied to the predetermined location. Electrically attach biomolecules,
A method for manufacturing a biomolecule detection device. The method for producing a biomolecule detecting device according to claim 1, wherein the two or more types of biomolecules that can be positively or negatively charged include proteins, DNAs, or nucleotides. The method for producing a biomolecule detecting device according to claim 1, wherein a predetermined biomolecule is electrically attached to a predetermined location after attaching a lid to the location.

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