JP3790194B2 - Nucleic acid probe immobilization substrate - Google Patents

Description

【図面の簡単な説明】
【図１】本発明の核酸プローブ固定化基体の１例を示す図。
【図２】スペーサ長と電流値の関係を示すグラフ。
【図３】核酸プローブと標的核酸の結合状態を示す図。
【図４】蛍光検出による標的核酸の検出の結果を示すグラフ。
【図５】電気化学的検出による標的核酸の検出の結果を示すグラフ。
【図６】エチレングリコールスペーサ長を用いた場合の結果を示すグラフ。
【図７】スペーサ構成の違いによる電流値の違いを示すグラフ。
【符号の説明】
１．核酸プローブ固定化基体 ２．基体 ３．電極 ４．スペーサ ５．核酸プローブ ６．パット[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a nucleic acid probe-immobilized substrate that is an apparatus for electrochemically detecting the presence of a target nucleic acid.
[0002]
[Prior art]
With the development of genetic engineering in recent years, it has become possible to diagnose and prevent diseases caused by genes in the medical field. These are called genetic diagnosis. For example, by detecting a defect or change in a human gene that causes a disease, it is possible to diagnose or predict the disease before the onset of the disease or at an extremely early stage. Along with the decoding of the human genome, research on the relationship between genotypes and diseases is progressing, and treatment tailored to each individual's genotype (tailor-made medicine) is becoming a reality. Therefore, it is very important to easily detect genes and determine genotypes.
[0003]
Conventional nucleic acid detection methods include those using radioactive isotopes and those using fluorescent dye labels. In the former method, the place where the detection is performed is limited, and the operation is complicated. In the case of the latter method, there is a problem that an expensive apparatus for detecting the fluorescent dye is required.
[0004]
In view of these problems, the present inventors previously disclosed a method for detecting an electrochemical nucleic acid in Japanese Patent No. 2573443. According to this method, a sample nucleic acid is reacted with a nucleic acid probe immobilized on the surface of an electrode, and the presence of hybridization occurring there is detected electrochemically using a nucleic acid recognition body. Thereby, the base sequence of the nucleic acid contained in the sample nucleic acid can be analyzed.
[0005]
However, the electrochemical nucleic acid detection device and sensor using such an electrode have a problem that the detection sensitivity is low because the hybridization efficiency of the target nucleic acid with respect to the nucleic acid probe is insufficient.
[0006]
On the other hand, in the field of genetic diagnosis in recent years, detection sensitivity has been increased. For example, in the genetic diagnosis of hepatitis C virus, a sensitivity capable of detecting a sample nucleic acid of about 10 ⁵ to 10 ⁶ copy / mL is required because it is used as an indicator of treatment or disease progress. On the other hand, it is also possible to amplify the sample nucleic acid side by a PCR method or the like. However, in order to reduce the burden on the patient, it is desired to reduce the amount of sample to be collected. In the future, it is desired that detection operations with higher sensitivity will be developed as detection operations are simplified and inspection throughput is increased.
[0007]
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to provide a nucleic acid probe-immobilized substrate that can be electrochemically detected with sufficient detection sensitivity.
[0008]
[Means for Solving the Problems]
The above object is achieved by the present invention as follows. Specifically, a nucleic acid probe fixing comprising a substrate, an electrode disposed on the substrate so as to detect an electrochemical signal, and a nucleic acid probe fixed to the electrode via a spacer having a length of 50 to 200 mm. Substrate.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
1. SUMMARY OF THE INVENTION The present invention comprises a substrate, an electrode disposed on the substrate so that an electrochemical signal can be detected, and a nucleic acid probe fixed to the electrode via a spacer having a length of 30 to 300 mm. A nucleic acid probe-immobilized substrate;
[0010]
Such a nucleic acid probe-immobilized substrate basically has a target sequence comprising a substrate, an electrode arranged on the substrate so that an electrochemical signal can be detected, and a nucleic acid probe fixed to the electrode. It is a device for detecting the presence.
[0011]
That is, the nucleic acid probe is designed to have a sequence complementary to the target sequence. When the test nucleic acid is brought into contact with such a nucleic acid probe, if the target sequence exists in the test nucleic acid, hybridization occurs on the nucleic acid probe-immobilized substrate. By detecting the presence of the double-stranded nucleic acid obtained as a result of this hybridization, the presence of the target sequence in the test nucleic acid is detected. The nucleic acid probe-immobilized substrate according to the present invention is basically a device in which detection is achieved by the principle as described above.
[0012]
2. Explanation of Terms As used herein, the term “nucleic acid” refers to ribonucleic acid (ie RNA), deoxyribonucleic acid (ie DNA), peptide nucleic acid (ie PNA), methyl phosphonate nucleic acid, S-oligo, cDNA It is a general term for nucleic acids and nucleic acid analogues, such as and cRNA, and any oligonucleotides and polynucleotides. Moreover, such a nucleic acid may be naturally occurring or artificially synthesized.
[0013]
Here, the “nucleic acid probe” refers to a nucleic acid fragment containing a base sequence complementary to a target sequence and immobilized on a substrate. The nucleic acid probe has a sequence that is complementary to the target sequence of interest, and is thereby capable of hybridizing to the target sequence under appropriate conditions.
[0014]
Here, the “target sequence” refers to a base sequence whose presence is to be detected or a sequence to be captured by the base sequence of a nucleic acid probe. A nucleic acid containing such a target sequence is referred to as a target nucleic acid.
[0015]
The terms “complementary”, “complementary” and “complementarity” as used herein may be complementary in the range of 50% to 100%, preferably 100% complementary.
[0016]
As used herein, the term “spacer” refers to a chain substance having a certain length arranged between a nucleic acid probe and a substrate. When the substance constituting the spacer is a nucleic acid, a portion complementary or hybridized to the target sequence is classified as a probe, and the other portion is classified as a spacer.
[0017]
3. Aspects of the Invention One aspect of the present invention will be described with reference to FIG. A nucleic acid probe-immobilized substrate 1 that is one embodiment of the present invention includes a nucleic acid probe 5 that is immobilized on an electrode 3 provided on a substrate 2 via a spacer 4 (FIG. 1). The electrode 3 is connected to a pad 6 for taking out electrical information. In FIG. 1, for convenience, the spacer 4 is indicated by a thick line, and the nucleic acid probe 5 is indicated by a chain line.
[0018]
Such a nucleic acid probe-immobilized substrate 1 can be produced, for example, by placing an electrode on a silicon substrate by means known per se and immobilizing the nucleic acid probe on the electrode surface via a spacer. Is possible. The solid-phase means may be a publicly known means such as immersion, spotting or synthesis on an electrode.
[0019]
In this embodiment, the number of electrodes is six, but the number of electrodes arranged on one substrate is not limited to this. Further, the arrangement pattern of the electrodes is not limited to that shown in FIG. 1, and those skilled in the art can appropriately change the design as necessary. A reference electrode and / or a counter electrode may be provided as necessary. Such a nucleic acid probe-immobilized substrate is also within the scope of the present invention.
[0020]
4). Structure The present invention is characterized in that the nucleic acid probe is fixed via a spacer. The spacer used in the present invention may be a spacer having a length of about 30 to about 300 mm, preferably about 50 to about 300 mm, more preferably about 90 to about 110 mm (see FIG. 2). . In addition, the spacer used according to the embodiment of the present invention may have a relative current value of about 0.2 to about 1, preferably about 0.5 to about 1, when the maximum current value is 1. More preferably, from about 0.8 to about 1.
[0021]
Examples of the substance used as the spacer may be organic chain molecules, such as nucleic acids, polypeptides, alkanes, and polyethylene glycol.
[0022]
In the case of a nucleic acid, for example, the spacer is preferably about 6 bases to about 60 bases, more preferably about 10 bases to about 60 bases, more preferably about 18 bases to 22 bases or less. In the case of a nucleic acid spacer composed of a nucleic acid, the base sequence is preferably a sequence that does not bind to a target nucleic acid or a nucleic acid that can be contained in a sample. Moreover, it is preferable to set it as the arrangement | sequence which considered the coupling | bonding tendency with the nucleobase of the double strand recognition body to be used. For example, Hoechst 33258 hardly binds to cytosine and guanine, and easily binds to thymine and adenine. On the other hand, a continuous sequence of guanine is difficult to synthesize. Therefore, a base sequence containing only cytosine or containing a lot of cytosine is more preferable, a base sequence consisting of or containing a lot of thymine is preferable, and a base sequence containing only guanine or adenine or containing a lot of them is less preferable.
[0023]
When the spacer is polyethylene glycol, it may be about 5 units to about 90 units, preferably about 15 units to 90 units, and more preferably about 27 units to about 33 units.
[0024]
When the spacer is an alkane, the number of carbons contained in the main chain may be about 20 to about 300, preferably about 50 to about 300, more preferably about 90 to about 110. .
[0025]
By arranging such a spacer, efficient hybridization between the nucleic acid probe and the target nucleic acid is achieved, and nucleic acid probe immobilization capable of electrochemically detecting the presence of a double-stranded nucleic acid with high sensitivity is achieved. A substrate is provided.
[0026]
FIG. 2 is a graph showing a general relationship between the spacer length and the current value. The vertical axis of the graph indicates the relative current value measured with the nucleic acid probe-immobilized substrate according to the present invention, and the horizontal axis indicates the length of the spacer provided in the nucleic acid probe-immobilized substrate according to the present invention. A graph indicated by a black diamond shows a case of a spacer made of cytosine, which is an example of a nucleic acid spacer, and a graph shown by a black triangle shows a case of a spacer made of polyethylene glycol.
[0027]
As shown in the graph, from the relationship between the spacer length and the current value, the binding state between the nucleic acid probe and the target nucleic acid is roughly divided into five phases, namely, A, B, C, D, and E. Here, the spacer length is 0 to 10 mm in A, 10 to 50 mm in B, 50 to 90 mm in C, 90 to 110 mm in D, and 110 mm or more in E.
[0028]
A binding state between the nucleic acid probe and the sample nucleic acid when the spacers of the respective lengths are used will be described with reference to an example shown in FIG. The left side of FIG. 3 schematically shows an example of the nucleic acid probe-immobilized substrate 30 and a general target nucleic acid. A linker agent 33a and a blocking agent 33b are treated on the surface of the electrode 32 disposed on the substrate 31. The nucleic acid probe 35 is fixed to the electrode 32 through the spacer 34 by the linker agent 33a. A target nucleic acid 36 having a target sequence complementary to the base sequence of the nucleic acid probe 35 in a part thereof is shown next to it.
[0029]
A target nucleic acid containing a target sequence to be detected among a sample nucleic acid obtained from a sample obtained from a subject such as an individual, tissue or cell, or a sample obtained by processing it as desired. However, the position where the target nucleic acid exists is considered to be various. The target nucleic acid 36 in the left diagram of FIG. 3 is shown here as an average example of such various target nucleic acids.
[0030]
The state when the nucleic acid probe 35 thus immobilized and the target nucleic acid 36 are hybridized is shown on the right side of FIG. Here, the respective states A, B, C, D and E correspond to the states in the phases A, B, C, D and E shown in FIG.
[0031]
As shown in FIG. 2, in the case of A, the spacer length is 0 to 10 mm, and in the case of B, the spacer length is 10 to 50 mm. In this case, as can be seen from the right diagram of FIG. 3, when the nucleic acid probe and the target sequence are hybridized, there is an excess sequence in the portion of the target nucleic acid 36 that is present on the substrate 31 side relative to the target sequence. The length of the target nucleic acid 36, the relationship between the position of the target sequence in the target nucleic acid 36 and the nucleic acid probe 35, and the probability of encountering the target nucleic acid and the nucleic acid probe in the sample nucleic acid (not shown) Therefore, sufficient hybridization efficiency between the nucleic acid probe and the target nucleic acid cannot be obtained.
[0032]
On the other hand, as shown in FIG. 2, the spacer length is 50 to 90 mm in the case of C, 90 to 110 mm in the case of D, and 110 mm or more in the case of E. In this case, as can be seen from the right diagram of FIG. 3, when the nucleic acid probe and the target sequence are hybridized, the surplus in the portion of the target nucleic acid 36 existing on the substrate 31 side from the target sequence is short even if it is temporarily present. Or there is no such surplus. Further, the presence of the spacer 34 makes the range in which the nucleic acid probe can freely move in the reaction solvent becomes wider. Thus, the probability of encountering the target sequence during the hybridization reaction is improved.
[0033]
According to the present invention, detection is performed electrochemically. In the embodiment of the present invention, a double strand recognition body is inserted into the double strand generated by the hybridization between the nucleic acid probe 30 and the target sequence, and the electrochemical in which the double strand recognition body is generated. A signal is detected by the electrode 32. Therefore, even if the spacer 34 is too long, good detection cannot be obtained.
[0034]
The nucleic acid probe used in the present invention may have a length generally used as a probe. For example, the length of the nucleic acid probe may be about 3 bases to about 1000 bases long, and preferably about 10 bases to about 200 bases long.
[0035]
The substrate that can be used in the present invention may be any substrate on which a nucleic acid probe for hybridization with a target sequence is immobilized. Examples of such substrates may be, for example, non-porous, rigid and semi-rigid materials, even plates having wells, grooves or flat surfaces, and features having a three-dimensional shape such as spheres It may be. The substrate can be made of, but not limited to, silica-containing substrates such as silicon, glass, quartz glass, quartz, and plastics and polymers such as polyacrylamide, polystyrene, polycarbonate, and the like.
[0036]
The electrode that can be used in the present invention is not particularly limited. For example, carbon electrodes such as graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon fiber, platinum, platinum black, gold, palladium, Examples include noble metal electrodes such as rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide and lead oxide, semiconductor electrodes such as Si, Ge, ZnO, CdS, TiO ₂ and GaAs, and titanium. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with other surface treatment agents as desired.
[0037]
The nucleic acid probe may be fixed through the spacer by any means known per se. For example, the spacer may be fixed to the electrode, and then the nucleic acid probe may be further fixed to the spacer. Alternatively, a spacer may be bonded to the nucleic acid probe in advance and fixed to the electrode through the spacer. Alternatively, the spacer and the nucleic acid probe may be synthesized on the electrode by means known per se. In addition, the nucleic acid probe may be immobilized through the spacer by directly immobilizing the spacer on the treated or non-treated electrode surface by covalent bond, ionic bond, physical adsorption or the like. Alternatively, a linker agent that helps the fixation of the nucleic acid probe through the spacer may be used, and the nucleic acid probe may be fixed to the electrode through the spacer using such a linker agent. Further, a blocking agent for preventing non-specific binding of the test nucleic acid to the electrode may be treated on the electrode together with the linker agent. Moreover, the linker agent and blocking agent used here may be substances for advantageously performing electrochemical detection, for example.
[0038]
In addition, each nucleic acid probe having a different base sequence may be immobilized on a different electrode via a spacer, and a plurality of nucleic acid probes having different base sequences may be mixed with one electrode. It may be fixed via a spacer.
[0039]
5. Detection The nucleic acid probe-immobilized substrate according to the present invention is an electrochemical cell as a means for detecting the presence of a double strand resulting from a hybridization reaction between a nucleic acid probe immobilized on the substrate and a target nucleic acid. Use the method.
[0040]
Electrochemical detection of double-stranded nucleic acid may be performed using, for example, a publicly known double-stranded recognition substance. The double-stranded recognizer used here is not particularly limited. For example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator and polyintercalator Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen. In addition, any other known double-stranded recognition substance is preferably used in the present invention.
[0041]
In the nucleic acid probe-immobilized substrate according to the present invention, the nucleic acid probe is immobilized on the electrode via a spacer. In the detection of double-stranded nucleic acid using such an electrode, a counter electrode and / or a reference electrode may be further used in the same manner as other general electrochemical detection methods. When arranging the reference electrode, for example, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode may be used.
[0042]
For example, when detecting whether or not the target nucleic acid is contained in the sample nucleic acid, the test may be performed as follows. For example, a nucleic acid component is extracted as a sample nucleic acid from a sample collected from an individual such as an animal including a human, a tissue or a cell. The obtained sample nucleic acid may be subjected to treatment such as reverse transcription, extension, amplification and / or enzyme treatment, as necessary. If necessary, the sample nucleic acid pretreated is brought into contact with the nucleic acid probe immobilized on the nucleic acid probe-immobilized substrate, and the reaction is carried out under conditions that allow appropriate hybridization. Such appropriate conditions depend on various conditions such as the type of base contained in the target sequence, the type of spacer and nucleic acid probe provided on the nucleic acid probe-immobilized substrate, the type of sample nucleic acid, and the state thereof. If it is a trader, it can select suitably. Although not limited to this, for example, the reaction may be performed under the following conditions.
[0043]
That is, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The sample nucleic acid obtained here is added and heat denatured at 90 ° C. or higher. The nucleic acid probe-immobilized substrate may be inserted into the heat-denatured sample nucleic acid immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate.
[0044]
During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, the electrode is washed. For washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 may be used. When the target nucleic acid containing the target sequence is present in the sample nucleic acid, it hybridizes with the nucleic acid probe, thereby generating a double-stranded nucleic acid.
[0045]
Subsequently, the double-stranded nucleic acid generated by the following procedure is detected by electrochemical means. In general, after the hybridization reaction, the substrate is washed, and a double-stranded recognizer is allowed to act on the double-stranded portion formed on the electrode surface, and the resulting signal is electrochemically measured.
[0046]
The concentration of the double-stranded recognizer varies depending on the type, but is generally used in the range of 1 ng / mL to 1 mg / mL. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.
[0047]
For example, the electrochemical measurement may be performed by applying a potential equal to or higher than the potential at which the double-stranded recognizer reacts electrochemically and measuring the reaction current value derived from the double-stranded recognizer. At this time, the potential may be swept at a constant speed, applied with a pulse, or a constant potential may be applied. In the measurement, for example, the current and voltage may be controlled by using a device such as a potentiostat, a digital multimeter, and a function generator. For example, the concentration of the target nucleic acid may be calculated from a calibration curve based on the obtained current value.
[0048]
The nucleic acid analysis method according to the present invention includes analysis of a sample nucleic acid contained in a sample, for example, detection and quantification of the presence of a target sequence, expression analysis such as the disappearance of gene expression, single nucleotide polymorphism in a genome (Single Nucleotide Polymorphism, That is, when conducting polymorphism analysis such as SNP) and microsatellite sequences, disease diagnosis and disease risk prediction by analysis of disease-related genes, detection of the presence of infection, virus type analysis, and toxicity testing It can be used for such as. Therefore, it can be used for various clinical purposes such as clinical diagnosis and onset prediction. Further, it can be widely used for various basic researches and applied researches such as food inspection, quarantine, pharmaceutical inspection, forensic medicine, agriculture, livestock, fishery and forestry.
[0049]
【Example】
Example 1
In this example, the relationship between the length of the spacer of the nucleic acid probe and the hybridization efficiency of the sample nucleic acid with respect to the probe was examined. As the spacer, base sequences composed of cytosine having different lengths, spacers composed of 0, 2, 4, 10, 20 and 30 cytosines, respectively, were used. E. coli rDNA was used as the sample nucleic acid.
[0050]
(1) Immobilization of nucleic acid probe on Au electrode surface The nucleic acid probe was immobilized by immersing the Au electrode surface in a single-stranded nucleic acid probe solution having the following base sequence for 1 hour. The single-stranded nucleic acid probe used here is complementary to the target sequence contained in the sample nucleic acid, and the sequence is as follows. Here, “C” is cytosine and a spacer portion, and “n” is 0, 2, 4, 10, 20 and 30.
[0051]
C _n -CTGGACGAAGACTGA (SEQ ID NO: 1)
(2) Detection of sample nucleic acid using nucleic acid probe-immobilized surface Sample nucleic acid was extracted and then amplified by PCR. At this time, two types were prepared: a sample in which the 5 ′ end on the (−) chain side was labeled with Cy-5 and an unlabeled sample. The hybridization reaction was performed by immersing the nucleic acid probe-immobilized surface prepared in (1) in a 2 × SSC solution containing each nucleic acid and allowing to stand at 35 ° C. for 60 minutes. Next, the fluorescence intensity derived from the sample nucleic acid hybridized with the probe was measured with a fluorescence detection apparatus. The electrode annealed with the unlabeled sample was immersed in a solution containing 50 μM Hoechst 33258 solution as an intercalating agent for 15 minutes, and then the oxidation current response of Hoechst 33258 molecule was measured. The results of fluorescence measurement and current measurement are shown in FIGS. When fluorescence detection was performed, the fluorescence intensity increased as the spacer length increased. On the other hand, when current detection was performed, the current value reached the maximum at about 20 bases, and the current value decreased at longer lengths.
[0052]
Example 2
In this example, ethylene glycol (hereinafter referred to as PEG) composed of 6, 18, 30, 60, and 120 chains was used as the spacer.
[0053]
(1) Immobilization of nucleic acid probe on Au electrode surface Immobilization was performed by immersing the Au electrode surface in a thiol-modified nucleic acid probe solution for 1 hour. The single-stranded nucleic acid probe used here is complementary to the sequence contained in the sample nucleic acid, and the sequence is as follows. Here, “(PEG) n” is a spacer portion, and “n” is 6, 18, 30, 60 or 120.
[0054]
(PEG) _n -GTTTCTGCGCCCGA (SEQ ID NO: 2)
(2) Detection of sample nucleic acid using nucleic acid probe-immobilized surface Sample nucleic acid was amplified by PCR. The hybridization reaction was carried out by immersing the nucleic acid probe-immobilized surface prepared in (1) in a buffer containing this nucleic acid and allowing it to stand at 35 ° C. for 60 minutes. Thereafter, washing was performed with a buffer. The result of the current measurement is shown in FIG. The current value increased with increasing ethylene glycol length. When the ethylene glycol length was about 30 (that is, 30 units), the maximum was obtained, and when the length was longer than that, a decrease in the current value was confirmed.
[0055]
Example 3
In this example, the difference in signal amount in current detection due to the configuration of the spacer of the nucleic acid probe was confirmed. As the spacers, three types of base sequences consisting of 20 cytosines, straight alkanes containing 96 C, and ethylene glycol chains consisting of 30 ethylene glycol were used so as to have substantially the same length.
[0056]
(1) Immobilization of nucleic acid probe on Au electrode surface Immobilization was performed by immersing the Au electrode surface in a single-stranded nucleic acid probe solution for 1 hour. The single-stranded probe used here was prepared with a base sequence complementary to the target sequence contained in the sample nucleic acid and a base sequence different from the target sequence as a control. Each sequence is as follows. Here, “S” is a spacer portion.
[0057]
S-ATGCTTTCGGTGGCA (SEQ ID NO: 3)
S-GTTTCTGCCGCCCGGA (control, SEQ ID NO: 4)
(2) Detection of sample nucleic acid using nucleic acid probe-immobilized surface Sample nucleic acid was amplified by PCR. The nucleic acid probe-immobilized surface prepared in (1) was immersed in a buffer containing this nucleic acid, and allowed to stand at 35 ° C. for 60 minutes to carry out a hybridization reaction. Thereafter, washing was performed with a buffer. The result of the current measurement is shown in FIG. Compared with the case where the spacer is a base sequence, the background current decreased when the spacer consisted of a linear alkane and the ethylene glycol chain, resulting in an improved S / N ratio. In the case of the ethylene glycol straight chain, the decrease in the background current value was particularly large.
[0058]
【The invention's effect】
According to the present invention as described above, a nucleic acid probe-immobilized substrate capable of electrochemical detection with sufficient detection sensitivity is provided.
[0059]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a view showing an example of a nucleic acid probe-immobilized substrate of the present invention.
FIG. 2 is a graph showing a relationship between a spacer length and a current value.
FIG. 3 is a view showing a binding state between a nucleic acid probe and a target nucleic acid.
FIG. 4 is a graph showing the results of target nucleic acid detection by fluorescence detection.
FIG. 5 is a graph showing the results of target nucleic acid detection by electrochemical detection.
FIG. 6 is a graph showing the results when ethylene glycol spacer length is used.
FIG. 7 is a graph showing a difference in current value due to a difference in spacer configuration.
[Explanation of symbols]
1. Nucleic acid probe-immobilized substrate Substrate 3. Electrode 4. Spacer 5. Nucleic acid probe Pat

Claims (5)

Nucleic acid probe-immobilized substrate comprising a substrate, an electrode disposed on the substrate so as to detect an electrochemical signal, and a nucleic acid probe fixed to the electrode via a spacer having a length of 30 to 300 mm . 2. The nucleic acid probe-immobilized substrate according to claim 1, wherein the spacer has a length of 50 to 300 mm. 2. The nucleic acid probe-immobilized substrate according to claim 1, wherein the spacer has a length of 90 to 110 mm. The nucleic acid probe-immobilized substrate according to any one of claims 1 to 3, wherein the spacer is an organic chain molecule. The nucleic acid probe-immobilized substrate according to any one of claims 1 to 3, wherein the spacer is selected from the group consisting of a nucleic acid, ethylene glycol, and alkane.

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