JP2007271326A - Method of manufacturing probe fixing substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of manufacturing a probe fixing substrate capable of performing the total inspection in a manufacturing process of a probe fixing substrate so that all of the manufactured probe fixing substrates have good measuring precision. <P>SOLUTION: The method of manufacturing a bio-molecule probe fixing substrate for detecting a bio-molecule includes a step of fixing the bio-molecule probe capable of interacting with a target bio-molecule to the electrode provided to the substrate and a step of electrochemically measuring the amount of the bio-molecule probe fixed to the electrode of the substrate to except electrode out of a predetermined range in fixing amount of the fixing amount of the bio-molecule probe. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for manufacturing a probe-immobilized substrate using a biomolecule as a probe, and more particularly to a method for manufacturing a probe-immobilized substrate including an inspection step in the manufacturing process.

  In recent years, chips for detecting biomolecules such as nucleic acids and proteins have been developed. In the detection method using a chip, a target substance is bound to a probe fixed to the chip and detected. Various methods are used to detect the bound target substance, one of which is a current detection method. The current detection method has an advantage that the detection device can be miniaturized, and is being developed.

  Conventionally, a chip for current detection is manufactured by preparing an electrode on a substrate and fixing a probe substance to the electrode. It is desirable that the manufactured chip should be subjected to quality inspections regarding the area of the electrode surface, the degree of contamination, the crystal orientation of the atoms constituting the electrode, the fixed amount of the probe, the coverage of the blocking agent, etc. It can be a chip capable of accurate measurement.

  However, the quality inspection of a normal chip is performed by taking out a completed chip at random and actually measuring the target substance. Accordingly, since the chips used for the inspection are discarded, there is a problem that it is difficult to inspect all the chips and it is difficult to prevent the mixture of defective products.

Patent Document 1 and Non-Patent Documents 1 and 2 disclose a method for detecting a nucleic acid immobilized on an electrode by an impedance method, but does not describe detection of a nucleic acid immobilized on an array.
JP2003-185662 J. Am. Chem. Soc. 2001, 123, 5194-5205 Biosensors and Bioelectronics 19 (2004) 1013-1019

  In view of the above-described problems, the present invention provides a method for manufacturing a probe-immobilized substrate that enables inspection of all the probe-immobilized substrates and enables all manufactured probe-immobilized substrates to have good measurement accuracy. The purpose is to provide.

  In order to solve the above problems, the present invention provides a method for producing a biomolecule probe-immobilized substrate for detecting a biomolecule, wherein the electrode comprises a biomolecule probe capable of interacting with a target biomolecule. And the step of electrochemically measuring the amount of the biomolecular probe fixed to the electrode of the substrate and excluding the electrode whose fixed amount is out of a predetermined range, A method for producing a biomolecular probe-immobilized substrate is provided.

  As another aspect of the present invention, prior to the step of immobilizing the biomolecular probe, an electrode is produced on a substrate, and the electrode is produced for quality inspection of the electrode produced on the substrate. And a method for excluding electrodes whose measured values are outside a predetermined range, and a method for producing a biomolecular probe-immobilized substrate.

  As still another aspect of the present invention, a step of treating a substrate having a biomolecular probe amount within a predetermined range with a blocking agent to coat the electrode, and a blocking agent on the electrode surface in the substrate after the blocking agent treatment The method further comprises the step of electrochemically measuring the coverage of the substrate and excluding the substrate whose coverage is outside the predetermined range.

  The amount of the immobilized biomolecular probe is preferably measured by calculating a peak potential and a peak area by an AC impedance measurement method. The electrochemical measurement of the electrode is preferably performed by calculating the peak potential and the peak area by cyclic voltammetry. Furthermore, it is preferable to measure the coverage of the blocking agent by calculating a peak potential and a peak area by an AC impedance measurement method. The biomolecule is preferably a molecule selected from the group consisting of nucleic acids, proteins or sugar chains.

  According to the present invention, by including an inspection step in the manufacturing process of the probe-immobilized substrate, 100% inspection can be performed, and all of the manufactured probe-immobilized substrates can have good measurement accuracy. .

  The present invention is a method of manufacturing a probe-immobilized substrate for detecting biomolecules, and realizes a total inspection of probe-immobilized substrates by including an inspection step in the manufacturing process.

  In the present invention, the “biomolecule” is a molecule selected from nucleic acids, proteins and sugar chains, preferably nucleic acids. The probe immobilized on the substrate is a molecule that interacts and preferably binds to the biomolecule to be detected. For example, when the biomolecule to be detected is a nucleic acid, a nucleic acid complementary to the nucleic acid can be used as a probe.

  As used herein, the term “nucleic acid” includes ribonucleic acid (RNA), deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), methyl phosphonate nucleic acid, S-oligo, cDNA and cRNA, and any oligonucleotide. And a general term for nucleic acids and nucleic acid analogs such as polynucleotides. Moreover, such a nucleic acid may be naturally occurring or artificially synthesized. The nucleic acid sequence may be a genomic DNA sequence or a fragmented sequence in which the entire genome is not secured.

  The term “protein” as used herein is a general term for polymers of amino nucleic acids and amino acid analogs. The amino acid may be naturally occurring or artificially synthesized.

  Further, the term “sugar chain” as used herein is a term indicating a substance in which monosaccharides such as glucose and galactose are linked in a chain. The sugar chain may be naturally occurring or artificially synthesized. Moreover, the thing couple | bonded with protein etc. may be sufficient.

  The substrate used in the present invention may be a substrate on which electrodes can be arranged. For example, a plate-like shape having wells, grooves or flat surfaces, or a solid body such as a sphere made of a non-porous, hard and semi-hard material. It may have a shape. 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.

  The electrode 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 , Noble metal electrodes such as rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide, lead oxide, semiconductor electrodes such as Si, Ge, ZnO, CdS, TiO2, GaAs, titanium, etc. Can do. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with other surface treatment agents as desired.

  In the first aspect of the present invention, a probe is first fixed to an electrode produced on a substrate. Different types of probes are immobilized on different electrodes, but a plurality of electrodes can be arranged on the same substrate.

  A method for fixing the probe to the substrate may be performed by a known means. For example, the probe may be fixed to the electrode via the spacer by fixing the spacer to the electrode and fixing the probe to the spacer. Alternatively, a spacer may be bonded to the probe in advance, and the probe may be fixed to the electrode via the spacer. Alternatively, the spacer and the probe may be synthesized on the electrode by a known means. In addition, the probe may be immobilized via a spacer by directly immobilizing the spacer on a treated or untreated electrode surface by covalent bond, ionic bond or physical adsorption. Alternatively, a linker agent that helps fix the probe through a spacer may be used.

  Next, the amount of the probe fixed to the electrode of the substrate is electrochemically measured. When the amount of the immobilized probe is outside the predetermined range, it can be said that the measurement accuracy when detecting the biomolecule is poor and the electrode has low reliability. Therefore, it is unsuitable for use in detection measurement, and the electrode can be regarded as a defective product. In this step, the electrodes for which the probe fixing amount is determined to be outside the predetermined range are excluded. When a plurality of electrodes are formed on the substrate, only the electrodes determined to be defective may be excluded or unusable, or the entire substrate having a defective electrode may be excluded.

  As for the appropriate range of probe immobilization amount, the correlation between the amount of biomolecular probe immobilization and the measured values such as peak potential and peak area is clarified in advance, and a desirable range is set.

  The measurement of the amount of the immobilized biomolecular probe is preferably performed by an AC impedance measurement method, and can be performed by calculating a peak potential and a peak area.

  A schematic diagram of a series of operations in the first aspect is shown in FIG. FIG. 1 shows an example in which a nucleic acid is used as a biomolecule and the probe-immobilized substrate is an array. First, nucleic acids are immobilized on the electrode array. Next, the electrode array on which the nucleic acid is immobilized is set in, for example, a rack and installed in a test container. The container is filled with a test solution and the electrode portion of the array is immersed. At this time, the electrode pad for detecting the current value is exposed outside the liquid. An electrode probe connected to a potentiostat is brought into contact with the exposed electrode pad to perform electrochemical measurement. For example, AC impedance measurement is performed, and the peak potential and the peak area are calculated.

  Electrodes whose numerical values such as peak potential and peak area obtained by measurement are outside a predetermined range set in advance are excluded as defective products. The remaining electrode array is pure, washed and dried. Although it may be used as an array for inspection as it is, it may be used for further processes.

  Examples of the inspection solution used for electrode inspection in the present invention include, but are not limited to, sulfuric acid, perchloric acid, potassium hydroxide, sodium hydroxide, and the like. Further, the use concentration is not limited.

  The electrochemical measurement method is not limited to the AC impedance measurement method, and any method such as cyclic voltammetry, pulse voltammetry, or chronoamperometry may be used.

  Next, the second aspect of the present invention will be described. In the second aspect of the present invention, in addition to the first aspect, a step of producing an electrode on the substrate is also included. In this aspect, prior to the step of immobilizing the biomolecular probe, the method includes a step of producing an electrode on a substrate, and further, quality inspection of the produced electrode is performed.

  The electrode may be produced by a conventionally known method. Inspection of the produced electrode is performed by electrochemically measuring the electrode. Electrodes whose measured values are outside the predetermined range are excluded as defective products, and the remaining electrodes are transferred to the next probe immobilization step. The appropriate measurement value range is set in advance by clarifying the correlation with measurement values such as peak potential and peak area in advance.

  FIG. 2 shows an outline of the inspection process taking the electrode array as an example in the second embodiment. The prepared electrode array is set in a rack and installed in a test container. The container is filled with the same test solution as described above, and the electrode portion of the array is immersed. An electrode probe connected to a potentiostat is brought into contact with the exposed electrode pad to perform electrochemical measurement. In the electrochemical measurement, for example, the crystal orientation, contamination, effective area, etc. of the electrode surface can be inspected, but not limited thereto. For the measurement, for example, cyclic voltammetry can be used. Similarly to the above, a suitable range of values obtained by measurement is set in advance, and electrode arrays whose measured values are outside the set range are excluded as defective products. The electrode array that has passed the inspection is pure, washed and dried, and proceeds to the next step.

  Next, the third aspect of the present invention will be described. The third aspect of the present invention includes a step of treating the electrode with a blocking agent in addition to the first aspect. Moreover, you may add said 2nd aspect arbitrarily. In this embodiment, following the step of immobilizing the biomolecular probe, the electrode is subjected to blocking treatment, and further quality inspection of the treated electrode is performed.

  The blocking agent covers the electrode surface and can prevent the biomolecule to be detected and other biomolecules from directly binding nonspecifically to the electrode. What is necessary is just to perform the process by a blocking agent according to a well-known method.

  After the substrate on which the probe is immobilized is treated with a blocking agent, the coverage of the electrode surface with the blocking agent is electrochemically measured. The coverage can be measured by calculating the peak potential and the peak area by an AC impedance measurement method.

  In this step, electrodes whose coverage is outside the predetermined range are excluded. When a plurality of electrodes are formed on the substrate, only the electrodes determined to be defective may be excluded or unusable, or the entire substrate having a defective electrode may be excluded.

  FIG. 3 shows an outline of the inspection process taking the electrode array as an example in the third aspect. The prepared electrode array is immersed in a solution containing a blocking agent, and blocking treatment is performed. Next, the processed electrode array is set in a rack and placed in a test container. The container is filled with a test solution and the electrode portion of the array is immersed. An electrode probe connected to a potentiostat is brought into contact with the exposed electrode pad to perform electrochemical measurement. For example, the peak potential and the peak area are calculated by AC impedance measurement. Similarly to the above, a suitable range of values obtained by measurement is set in advance, and electrode arrays whose measured values are outside the set range are excluded as defective products. The electrode array that has passed the inspection is pure, washed and dried to obtain a completed electrode array.

  The blocking agent may be treated on the electrode together with the linker agent when fixing the probe. The linker agent and blocking agent used herein may be substances that favor the electrochemical detection of biomolecules.

  Information obtained by electrochemical measurement in each of the above aspects includes the effective area of the electrode, the degree of contamination, the crystal orientation of the atoms constituting the electrode, the fixed amount of molecules fixed to the electrode, and the coverage of the electrode with the clocking agent However, the present invention is not limited to these, and any factor may be used as long as it has an influence on the use of the electrode.

  As described above, the probe-immobilized substrate manufactured according to the method of the present invention can be inspected simply and in total for quality in the manufacturing process. Moreover, since defective products can be excluded in the manufacturing process, manufacturing efficiency can be improved. Furthermore, it is possible to prevent a defective product from being mixed in the completed probe-immobilized substrate, and a highly reliable product with stable measurement accuracy can be manufactured.

  Another aspect of the present invention provides a test container used for the electrochemical test as described above. The inspection container of the present invention has a configuration in which the electrode portion in the substrate is immersed in the solution, and the electrode pad portion is held outside the solution. It is preferable that a plurality of substrates can be stored. The inspection container may be made of any material as long as it is suitable for holding the inspection solution used for the inspection.

  The probe-immobilized substrate produced according to the present invention is used for detection of biomolecules such as nucleic acids, proteins and sugar chains. For example, when detecting a nucleic acid, a nucleic acid probe immobilized on a substrate is hybridized with a target nucleic acid, and the resulting double strand is detected. As a detection means, an electrochemical method can be used, but it is not limited to this.

  The detection of double-stranded nucleic acid by an electrochemical method may be performed using, for example, a known double-stranded recognition substance. The double-stranded recognition substance is not particularly limited. For example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator and polyintercalator are used. Is possible. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen. Other known double-stranded recognition substances can also be used.

  Furthermore, as in other general electrochemical detection methods, a counter electrode and / or a reference electrode may be provided on the substrate. When installing the reference electrode, for example, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode can be used.

Below, the example of the manufacturing process of a gold electrode array is described.
First, an electrode was produced. After the substrate was washed with a sulfuric acid / hydrogen peroxide solution, titanium was deposited by an electron beam method so as to have a thickness of 500 angstroms. Alternatively, chromium was deposited to a thickness of 500 angstroms by sputtering. Similarly, gold was deposited or deposited to a thickness of 5000 angstroms. For details of the electrode manufacturing method, refer to JP-A-10-146183.

  Next, the prepared gold electrode array was set in a rack, set in a container for inspection, and immersed in perchloric acid (100 mM) and covered. An electrode probe connected to a potentiostat was brought into contact with the electrode pad exposed from the perchloric acid solution. For all the arrays, cyclic voltammetry was performed on all the gold electrodes, and the peak potential and the peak area were calculated. An electrode array in which the peak potential and the peak area were out of the set range was excluded as a defective product.

  After cyclic voltammetry, the rack on which the electrode array was set was immersed in pure water for washing and vibrated. The rack was taken out from the pure water, and the pure water adhering to the electrode surface was dried.

Subsequently, the nucleic acid was immobilized on the electrode array produced as described above.
A solution containing a thiol-labeled nucleic acid chain was spotted on each gold electrode of the inspected gold electrode array, and the nucleic acid chain was bonded to the gold electrode via a bond between thiol and gold. The electrode array on which the nucleic acid was immobilized was set in a rack, set in a test container, immersed in perchloric acid (100 mM), and capped. An electrode probe connected to a potentiostat was brought into contact with the electrode pad exposed from the perchloric acid solution. For all the arrays, AC impedance measurement was performed on all the gold electrodes, and the peak potential and the peak area were calculated. An electrode array in which the peak potential and the peak area were out of the set range was excluded as a defective product.

  After the AC impedance measurement, the rack on which the electrode array was set was immersed in pure water for cleaning and vibrated. The rack was taken out from the pure water, and the pure water adhering to the electrode surface was dried.

  The electrode array obtained in the above steps was subsequently treated with a blocking agent. The gold electrode array on which the probe was fixed was immersed in a solution containing a blocking agent, and the gold electrode surface was covered with the blocking agent.

  Next, the prepared probe-immobilized electrode array was set in a rack and set in an inspection container, and immersed in perchloric acid (100 mM) and covered. An electrode probe connected to a potentiostat was brought into contact with the electrode pad exposed from the perchloric acid solution. AC impedance measurement was performed on all the gold electrodes of all the gold electrode arrays, and the peak potential and the peak area were calculated. An electrode array in which the peak potential and the peak area were out of the set range was excluded as a defective product.

  After the AC impedance measurement, a rack including a plurality of electrode arrays was immersed in cleaning pure water and vibrated. The rack was taken out from the pure water, and the pure water adhering to the electrode surface was dried. Thus, an electrode array on which the nucleic acid probe was immobilized was obtained.

  Next, the measurement accuracy of the produced electrode array was tested. A solution containing the target nucleic acid was applied to the nucleic acid-immobilized gold electrode array obtained by the above method and reacted. After the reaction, an electrochemically active intercalating agent (Hoechst 33258) was allowed to act.

The potential was swept with respect to each electrode after the reaction to oxidize the intercalating agent bound to the double strand consisting of the nucleic acid probe and the target nucleic acid, and the current value was detected.
Moreover, the target nucleic acid was made to react also with the electrode array manufactured by the conventional manufacturing process, and it tested similarly.

  When the standard deviation was calculated and compared based on the current values obtained from each, the standard deviation of the electrode array produced according to the present invention was lower than that of the electrode array produced by the conventional method. Thus, according to the method of the present invention, the average measurement accuracy of the manufactured electrode array can be increased.

The schematic diagram of the manufacturing process of a probe fixed base | substrate. The schematic diagram of the manufacturing process of the electrode for probe fixed bases. The schematic diagram of the blocking agent processing process of a probe fixed base.

Claims (7)

A method for producing a biomolecular probe-immobilized substrate for detecting a biomolecule,
Immobilizing a biomolecule probe capable of interacting with a target biomolecule to an electrode provided on a substrate;
And a step of electrochemically measuring the amount of the biomolecular probe immobilized on the electrode of the substrate and excluding the electrode whose amount of immobilization is outside a predetermined range. Manufacturing method. The method of claim 1, prior to the step of immobilizing the biomolecular probe.
Producing an electrode on a substrate;
For the quality inspection of the electrode produced on the substrate, electrochemically measuring the electrode, and excluding the electrode whose measured value is outside a predetermined range;
A method for producing a biomolecular probe-immobilized substrate, further comprising: The method according to claim 1 or 2, wherein
Treating the substrate having the biomolecular probe amount within a predetermined range with a blocking agent and coating the electrode;
A step of electrochemically measuring the coverage of the blocking agent on the electrode surface in the substrate after the treatment with the blocking agent, and excluding the substrate whose coverage is outside a predetermined range; and
A method for producing a biomolecular probe-immobilized substrate, further comprising:   The production method according to any one of claims 1 to 3, wherein the amount of the immobilized biomolecular probe is measured by calculating a peak potential and a peak area by an AC impedance measurement method.   The manufacturing method according to any one of claims 2 to 4, wherein the electrochemical measurement of the electrode is performed by calculating a peak potential and a peak area by cyclic voltammetry.   The manufacturing method according to claim 3, wherein the covering ratio of the blocking agent is measured by calculating a peak potential and a peak area by an AC impedance measurement method.   The said biomolecule is a manufacturing method as described in any one of Claims 1-6 which is a molecule | numerator selected from the group which consists of a nucleic acid, protein, or a sugar chain.

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