JP2016180662A - BIOSENSOR FOR ELECTROCHEMICALLY MEASURING AMYLOID β PEPTIDE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor for measuring amyloid β peptide easily, rapidly, and accurately with an electrochemical method.SOLUTION: It becomes possible to measure amyloid β peptide easily, rapidly, and accurately with an electrochemical method by using an electrode with immobilized peptide containing HDKLVFFYEVHH (sequence number 1) or an altered sequence thereof.SELECTED DRAWING: None

Description

  The present invention relates to a biosensor and method for measuring amyloid β peptide easily, in a short time, and with high accuracy by an electrochemical method.

  Various techniques have been developed for detecting amyloid β peptide (hereinafter sometimes referred to as “Aβ”), which is considered to be a causative agent of Alzheimer's disease, due to the importance of the measurement target. The most common is based on techniques such as fluorescence measurement or ELISA. Fluorescence measurement is a technique that uses a fluorescent dye thioflavin T (ThT) that specifically binds to Aβ fibrils and quantifies the fluorescence intensity proportional to the amount of fibers, and this technique is used in many academic papers on Aβ. . However, only the fibrillar Aβ can be measured by the fluorescence measurement method, and the detection sensitivity is low, and it remains at the mM level. On the other hand, the ELISA method is a method using an Aβ antibody and has high sensitivity. However, the reaction time and signal amplification time with the antibody are required for 1 to 2 days, and the antibody is required, so that it is relatively expensive. There is.

  Therefore, a method using an intermolecular interaction analyzer such as a surface plasmon resonance (SPR) method has been reported as a method for measuring Aβ based on different principles. For example, Non-Patent Document 1 discloses that 0.02 to 5 nM Aβ can be measured by using SPR together with an antibody. Non-patent document 2 reports that 3 to 30 mM Aβ was detected without using an antibody. However, since the former method uses an antibody, the cost is high, and the latter method has a drawback that the sensitivity is not so high.

Furthermore, attempts to measure Aβ by an electrochemical method have been reported. For example, Non-Patent Document 3 attempts to detect Aβ by measuring the redox of a tyrosine residue contained in Aβ. However, in Non-Patent Document 3, it is estimated that Aβ of 0.7 · g / mL can be detected, but quantitativeness is not obtained. In Non-Patent Document 4, there is a report of detecting the amount of Aβ42 and total Aβ at the pM level using redox of p-aminophenol on a gold electrode modified with an antibody. Since it is necessary to use an antibody, there is a disadvantage that the cost per measurement is high.

  Furthermore, conventionally, as a method for measuring Aβ, a method of labeling and separating Aβ with a stable isotope and quantifying it by mass spectrometry (Patent Document 1), a method using a metal colloid carrying an antibody that recognizes amyloid monomer (patent Document 2), a method using a biochip on which a β-sheet Aβ oligomer is immobilized (Patent Document 3) and the like have also been proposed.

  As described above, many methods for measuring Aβ have been reported so far, but many of the high-sensitivity measurements required special antibodies. In addition, there are many methods that require expensive devices such as SPR and mass spectrometers for measuring. On the other hand, electrochemical methods that can be measured with a relatively inexpensive device require a long measurement time and insufficient sensitivity and quantitativeness. The present condition is that the method of satisfying has not been developed.

Ning Xia et al., Anal. Chem., 82, 10151 (2010) Jungki Ryu et al., Anal. Chem., 80, 2400 (2008) M. Vestergaard et al., J. Am. Chem. Soc., 127, 11892 (2005) Lin Liu et al., Biosens. Bioelectron., 51, 208 (2014)

JP 2004-157124 A JP 2011-122928 A JP 2014-13190 A

  An object of the present invention is to provide a biosensor for measuring Aβ simply, in a short time, and with high accuracy by an electrochemical method. Another object of the present invention is to provide a method for measuring Aβ using the biosensor.

  The present inventor has conducted intensive studies to solve the above-mentioned problems. As a result, by using an electrode on which a peptide containing HDKLVFFYEVHH (SEQ ID NO: 1) or a modified sequence thereof is immobilized, it is simple, short, and highly accurate. It was found that Aβ can be measured by an electrochemical method. More specifically, when Aβ is brought into contact with the electrode, Aβ aggregates on the peptide, and when a copper divalent ion is further added, an electrochemical signal corresponding to the amount of Aβ is emitted, It has been found that Aβ can be quantified easily, in a short time, and with high accuracy by detecting a signal. The present invention has been completed by further studies based on such knowledge.

That is, this invention provides the invention of the aspect hung up below.
Item 1. A biosensor for measuring amyloid β peptide, comprising an electrode on which the peptide shown in (1) or (2) below is immobilized:
(1) A peptide comprising the amino acid sequence shown in SEQ ID NO: 1.
(2) Contains an amino acid sequence in which one or several amino acid residues other than the 3rd to 7th positions in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, or inserted, and has an action of aggregating Aβ peptide.
Item 2. Item 2. The biosensor according to Item 1, wherein the peptide is a peptide shown in the following (3) or (4):
(3) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(4) In the amino acid sequence shown in SEQ ID NO: 2, it consists of an amino acid sequence in which one or several amino acid residues other than the 8th to 12th positions are substituted, deleted, added, or inserted, and aggregate with Aβ. A peptide having an action.
Item 3. Item 3. The biosensor according to Item 1 or 2, which is used for diagnosis of Alzheimer-type dementia.
Item 4. A method for measuring amyloid β peptide using the biosensor according to any one of Items 1 to 3,
A first step of bringing a sample solution into contact with an electrode on which the peptide in the biosensor is immobilized;
A second step in which copper divalent ions are brought into contact with the electrode brought into contact with the sample solution in the first step and the current value is measured; and in the sample solution based on the current value measured in the second step 3rd process of calculating | requiring the amount of amyloid (beta) peptide of.
Item 5. Item 5. The measurement method according to Item 4, wherein the sample solution is plasma or a diluted solution thereof.

  According to the biosensor of the present invention, Aβ can be quantified easily and in a short time by an electrochemical method that can be measured with a relatively inexpensive apparatus without using an antibody. In addition, according to the biosensor of the present invention, even an extremely low concentration of Aβ of about 0.5 μM can be quantified, so the detection sensitivity of Aβ is extremely high and the measurement accuracy is excellent.

  In addition, since Aβ in plasma is known to be a biomarker for Alzheimer-type dementia, the biosensor of the present invention improves its diagnostic accuracy by using it for diagnosis of Alzheimer-type dementia. And can contribute to early detection.

  Since the biosensor of the present invention uses an electrochemical method, it can be spread as a home Aβ sensor by downsizing it as well as a home-use blood glucose sensor that is currently in practical use. it can.

  Furthermore, the biosensor of the present invention can be initialized by washing after use, and can maintain the measurement accuracy of Aβ even when measurement and washing are repeated. It is also possible to greatly reduce the total cost required for measurement.

In the reference example 1, it is a figure which shows the result of having evaluated the aggregation promotion effect with respect to A (beta) of AFPP and AFPP _cys . In Example 1, it is a figure which shows the result of having measured A (beta) using the AFPPcys fixed electrode. In Example 3, it is a figure which shows the result of having measured 0.1-5 micromol A (beta) using the AFPPcys fixed electrode. In Example 4, it is a figure which shows the result of having repeatedly measured and wash | cleaned A (beta) using the AFPPcys fixed electrode, and evaluated the influence which the reuse after washing | cleaning has on A (beta) measurement precision. In Example 5, it is a figure which shows the result of having measured the amylin using the AFPPcys fixed electrode.

  In the present specification, except for the sequence listing, 20 types of amino acid residues in the amino acid sequence are expressed by single letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y , Tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine (Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E Asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P.

  In the present specification, amino acid sequences are shown such that the left side is the N-terminus and the right side is the C-terminus.

1. The biosensor for measuring Aβ The present invention is a biosensor used for measuring Aβ by an electrochemical technique, wherein a peptide containing the amino acid sequence shown in SEQ ID NO: 1 or a modified sequence thereof is immobilized. It is characterized by including the electrode. Hereinafter, the biosensor of the present invention will be described in detail.

(peptide)
The peptide immobilized on the electrode in the biosensor of the present invention is the peptide shown in the following (1) or (2). The peptide shown in the following (1) or (2) has an action of efficiently capturing and aggregating Aβ. By using the peptide for a biosensor for Aβ measurement, Aβ can be quantified with high accuracy
(1) A peptide comprising the amino acid sequence shown in SEQ ID NO: 1 (HDKLVFFYEVHH).
(2) Amino acids are aggregated, including an amino acid sequence in which one or several amino acid residues other than the 3rd to 7th positions in the amino acid sequence shown in SEQ ID NO: 1 (HDKLVFFYEVHH) are substituted, deleted, or inserted. A peptide having an action.

  The peptide of (1) may be composed of the amino acid sequence shown in SEQ ID NO: 1, but one or several amino acid residues may be added to the N-terminus and / or C-terminus.

  In the peptide of (1), when amino acid residues are added to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1, the number of added amino acid residues is not particularly limited. In the case of the N-terminal side, 1 to 10, preferably 1 to 6, more preferably 4 to 6 are mentioned; in the case of the C-terminal side, 1 to 10, preferably 1 to 5, More preferably, 1-3 are mentioned.

  In the peptide (2), the amino acid residues 3 to 7 (KLVFF) in the amino acid sequence shown in SEQ ID NO: 1 greatly contribute to the aggregation promoting action of Aβ, so these amino acid residues are conserved. .

  In the peptide of (2), the substituted, deleted, or inserted amino acid residues may be the first, second, and 8-12 amino acid residues in the amino acid sequence shown in SEQ ID NO: 1. Since the histidine residues at positions 1, 11, and 12 can contribute to the role of inducing structural changes in the peptide, the histidine residues at positions 1, 11, and 12 can be maintained in order to maintain high measurement accuracy of Aβ. Desirably the group is not substituted, deleted or inserted.

  In the peptide of (2) above, the number of amino acid residues introduced into the amino acid sequence shown in SEQ ID NO: 1 (substitution, deletion, addition, or insertion) is within one or several ranges. Although it is not particularly limited as long as it has an action of aggregating with Aβ, specifically 1 to 4, preferably 1 to 3, more preferably 1 or 2, particularly preferably 1 is mentioned. It is done.

  In the peptide of (2), when an amino acid substitution is made on the amino acid sequence shown in SEQ ID NO: 1, the amino acid substitution is preferably a conservative substitution. As amino acid substitution, specifically, if the amino acid before substitution is a nonpolar amino acid, the substitution to another nonpolar amino acid, and if the amino acid before substitution is an uncharged amino acid, the substitution to another uncharged amino acid Substitution, if the amino acid before substitution is an acidic amino acid, substitution with another acidic amino acid, and substitution of another basic amino acid if the amino acid before substitution is a basic amino acid.

  The peptide of (2) above may be composed of an amino acid sequence that is substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO: 1, but the N-terminal and / or C of the amino acid sequence. One or several amino acid residues may be further added to the terminal.

  In the peptide (2), when an amino acid residue is added to the N-terminus and / or C-terminus of the amino acid sequence substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO: 1, the added amino acid The number of residues is not particularly limited. For example, in the case of the N-terminal side, 1 to 10, preferably 1 to 6, more preferably 4 to 6 are mentioned; 1 to 10, preferably 1 to 5, and more preferably 1 to 3.

  The peptide used in the present invention is not particularly limited as to the number of amino acid residues constituting the peptide, as long as it has the amino acid sequence described above and has an action of aggregating Aβ. The number is, for example, 14 to 30, preferably 17 to 25, and more preferably 18 to 20.

In addition, as a suitable example of the peptide used in the present invention (the peptide of the above (1) or (2)), the peptide shown in the following (3) or (4) may be mentioned.
(3) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(4) In the amino acid sequence shown in SEQ ID NO: 2, it consists of an amino acid sequence in which one or several amino acid residues other than the 8th to 12th positions are substituted, deleted, added, or inserted, and aggregate with Aβ. A peptide having an action.

  The peptide (3) is a peptide consisting of an amino acid sequence in which DAEFR is added to the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 and QK is added to the C-terminal side.

  In the peptide of the above (4), the substituted, deleted, added or inserted amino acid residues are the 8th to 12th amino acids in the amino acid sequence shown in SEQ ID NO: 2 (that is, 3-7 of the amino acid sequence shown in SEQ ID NO: 1). The histidine residues at positions 6, 16, and 17 can contribute to the role of inducing structural changes in the peptide. In order to maintain high measurement accuracy, it is desirable that the histidine residues at positions 6, 16, and 17 are not substituted, deleted, added, or inserted. In the peptide of (4) above, the amino acid residues at positions 6 to 17 of the amino acid sequence shown in SEQ ID NO: 2 (corresponding to the amino acid residues at positions 1 to 12 of the amino acid sequence shown in SEQ ID NO: 1) In order to maintain the high measurement accuracy of Aβ, it is desirable that no substitution, deletion, addition or insertion is present.

  In the peptide of the above (4), the number of amino acid residues into which alteration (substitution, deletion, addition, or insertion) is introduced into the amino acid sequence shown in SEQ ID NO: 2 is in the range of 1 or several, And it is not particularly limited as long as it has an action of aggregating with Aβ. Specifically, 1 to 10, 1 to 9, 1 to 8, or 1 to 7, preferably 1 to 6, more preferably Preferably 1 to 5, more preferably 1 to 4, particularly preferably 1 to 3, and most preferably 1 or 2.

  In the peptide (4), when an amino acid substitution is made with respect to the amino acid sequence shown in SEQ ID NO: 2, the amino acid substitution is preferably a conservative substitution. Specific embodiments of conservative substitution are as described above.

  The peptide used in the present invention can be produced by a known peptide synthesis method based on its amino acid sequence.

(electrode)
The constituent material of the electrode used in the biosensor of the present invention is not particularly limited as long as it exhibits conductivity and can immobilize the peptide. For example, gold, platinum, palladium, nickel, ruthenium, iridium, etc. Metal oxide electrodes such as ITO (Tin-doped indium oxide); Carbon electrodes such as glassy carbon. Among these, a metal electrode is preferable, and a gold electrode is more preferable.

(Immobilization of peptide to electrode)
In the biosensor of the present invention, the peptide can be immobilized on the electrode by a known peptide immobilization method depending on the electrode material.

  For example, if the peptide is immobilized on a metal electrode such as gold or platinum, a compound having a thiol group such as cysteine is bonded to the terminal of the peptide, and the thiol group and the metal atom constituting the electrode are combined. A method of forming a self-integrated (SAM) film by forming a metal-S bond (for example, an Au-S bond in the case of a gold electrode) by bonding is used. In addition, the peptide can be immobilized on the metal electrode by using a compound having a disulfide group instead of the compound having a thiol group.

  Also, for example, when the peptide is immobilized on a glassy carbon electrode, the surface of the glassy carbon electrode is subjected to air oxidation or chromic acid solution treatment at a high temperature, and then the carboxyl group is chlorinated. Examples include a method of converting to an acid chloride with thionyl to form an amide bond with the amino group of the peptide.

In the biosensor of the present invention, the amount of the peptide immobilized on the electrode is not particularly limited. For example, the peptide is about 0.5 to 40 μg, preferably about 1 to ²⁰ μg, more preferably about 1 cm ^{2 of the} electrode. The quantity which becomes about 1-10 micrograms is mentioned.

  In the biosensor of the present invention, the peptide may be immobilized in such a manner that the N-terminal side of the peptide binds to the electrode, or may be immobilized in such a manner that the C-terminal side of the peptide binds to the electrode. From the viewpoint of measuring Aβ with higher accuracy, it is preferable that the N-terminal side of the peptide is immobilized in such a manner that it binds to the electrode. That is, as a preferred embodiment of the present invention, cysteine is bonded to the N-terminus of the peptide, and the thiol group of the cysteine forms a metal-S bond on the electrode, whereby the peptide is immobilized on the electrode. The aspect currently performed is mentioned.

(Other components)
The biosensor of the present invention may be configured as an electrochemical analyzer using the electrode on which the peptide is immobilized as a working electrode. That is, the biosensor of the present invention may be provided with an electrode (working electrode) on which the peptide is immobilized, a counter electrode, and a current measuring device for measuring the generated current value.

  In addition, the biosensor of the present invention may be configured to include a working electrode and a counter electrode so that measurement can be performed by the two-electrode method, but further includes a reference electrode to be configured to perform measurement by the three-electrode method. It may be.

  In the biosensor of the present invention, the electrode on which the peptide is immobilized is provided with a cell for holding the sample solution on the electrode on which the peptide is immobilized so that the electrode and the sample solution can be easily contacted. It may be done.

2. Method for Measuring Aβ The method for measuring Aβ according to the present invention is a method for measuring Aβ using the biosensor, and includes the following first to third steps.
1st process: A sample solution is made to contact with the electrode by which the said peptide of the said biosensor was fix | immobilized.
Second step: Copper divalent ions are brought into contact with the electrode brought into contact with the sample solution in the first step, and the current value is measured.
Third step: The amount of Aβ in the sample solution is obtained based on the current value measured in the second step.

  Hereinafter, the method for measuring Aβ according to the present invention will be described.

(Sample solution)
In the Aβ measurement method of the present invention, the sample solution to be measured is not particularly limited as long as Aβ measurement, particularly quantification, is required. For example, tissue extract, culture supernatant, cerebrospinal fluid And biological samples such as plasma; reagents containing Aβ; and dilutions thereof. As described above, since Aβ in plasma is a biomarker for Alzheimer-type dementia, the sample solution to be measured is preferably plasma or a diluted solution thereof.

(First step)
In the method for measuring Aβ of the present invention, first, a sample solution is brought into contact with an electrode on which the peptide of the sensor is fixed (first step). By this first step, Aβ contained in the sample solution aggregates on the peptide immobilized on the electrode.

  In order to bring the sample solution into contact with the electrode in the first step, for example, a sample sample may be added to the electrode on which the peptide is immobilized, and the electrode is covered with the sample sample.

  In the first step, the sample solution may be left still or shaken at about 37 ° C. for about 30 to 60 minutes with the sample solution in contact with the electrode.

(Second step)
After the first step, a copper divalent ion is brought into contact with the electrode brought into contact with the sample solution, and the current value is measured (second step). When a copper divalent ion is brought into contact with the electrode on which Aβ is aggregated after the first step, the Aβ is bonded to the copper divalent ion, and a current is generated between the copper divalent ion and the electrode.

  Before contacting the copper divalent ions in the second step, it is desirable to remove Aβ that has not been aggregated on the electrode by washing the electrode after the first step.

  In order to contact the copper divalent ions in the second step, specifically, an aqueous copper divalent ion solution in which a water-soluble divalent copper compound is dissolved may be added onto the electrode.

  Examples of the water-soluble divalent copper compound used for the preparation of the copper divalent ion aqueous solution include copper sulfate, copper chloride, and copper acetate. These water-soluble divalent copper compounds may be used individually by 1 type, and may be used in combination of 2 or more type. Moreover, what is necessary is just to set suitably about the copper divalent ion concentration in copper divalent ion aqueous solution in the range which can satisfy the contact amount of the copper divalent ion mentioned later, for example, 0.1-1 mM, Preferably it is 0.1 What is necessary is just to adjust so that it may become 0.2 mM.

  About the solvent used for the said copper divalent ion aqueous solution, pH should just be adjusted to about 7 so that the precipitation by formation of copper hydroxide may not arise, for example, a Tris / HCl buffer solution is used suitably. .

In the second step, the amount of copper divalent ions to be contacted is not particularly limited. For example, the copper divalent ions are 0.1 to 1 micromole, preferably 0.1 to 0 per 1 cm ^{2 of} the electrode. The amount which becomes 3 micromol is mentioned.

  By contacting the copper divalent ions in this manner, the copper divalent ions bind to Aβ aggregated on the peptide, and a current flows between the copper divalent ions and the electrode. In the second step, the current value thus generated is measured.

(Third step)
Based on the current value measured in the second step, the amount of Aβ in the sample solution is determined (third step). Since the amount of Aβ in the sample solution and the current value measured in the second step are in a proportional relationship, the amount of Aβ in the sample solution is quantified from the current value measured in the second step.

  In the third step, specifically, a standard curve showing a correlation between the Aβ concentration and the current value is prepared in advance using a standard sample solution with a known concentration, and the standard curve is used to generate a standard curve in the second step. What is necessary is just to quantify the amount of Aβ in the sample solution from the measured current value.

  In addition, since the copper divalent ions can bind to the peptide itself immobilized on the electrode to generate a weak current, the same operation as described above is performed without adding a sample solution separately in the second step. It is desirable to measure the background current value when contacting with copper divalent ions. In this case, the amount of Aβ in the sample solution is obtained by using the value obtained by subtracting the background current value from the current value measured when the sample solution is brought into contact with the sample solution. Can do.

(Initialize)
The biosensor can remove Aβ aggregated and copper divalent ions bound to the Aβ by washing the electrode on which the peptide is immobilized after once measuring Aβ. So it can be used repeatedly.

  As a method of washing after the peptide-immobilized electrode is used once, a method of washing with an aqueous solution of a chelating agent containing a chelating agent and then washing with an aqueous alkaline solution can be mentioned.

  The type of chelating agent contained in the aqueous chelating agent solution is not particularly limited as long as it can remove copper divalent ions. For example, sodium ethylenediaminetetraacetate, sodium diethylenetriaminepentaacetate, sodium nitrilotriacetate, Citric acid and the like can be mentioned. These chelating agents may be used individually by 1 type, and may be used in combination of 2 or more type. Moreover, as a density | concentration of the chelating agent in chelating agent aqueous solution, 0.1-1 mM, for example, Preferably 0.1-0.3 mM is mentioned.

  After washing with the chelating agent aqueous solution and before washing with the alkaline aqueous solution, the aqueous chelating agent solution remaining on the electrode may be washed away by washing the electrode as necessary.

  The cleaning method using the chelating agent aqueous solution is not particularly limited, and examples thereof include a method of rinsing the electrode with the chelating agent solution and a method of immersing the electrode in the chelating agent solution.

  Moreover, as said alkaline aqueous solution, alkalis, such as sodium hydroxide and potassium hydroxide, are added, What is necessary is pH adjusted to 9-12, Preferably it is 10-11.

  The washing method using the alkaline aqueous solution is not particularly limited, and examples thereof include a method of rinsing the electrode with the alkaline solution and a method of immersing the electrode in the alkaline solution.

  An electrode washed with an alkaline aqueous solution can be reused by washing with water and thoroughly washing away the alkaline aqueous solution.

  Next, although an Example is given and this invention is demonstrated concretely, this invention is not limited to the following.

Reference Example 1: Confirmation of Aggregation Promoting Effect on Aβ A peptide comprising the amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as “AFPP”) and a peptide in which cysteine is bound to the N-terminus of the AFPP (hereinafter referred to as “AFPP”) _cys ")) and these peptides were used to confirm the aggregation promoting effect of Aβ. Specifically, with respect to an N-ethylmorpholine buffer solution adjusted to pH 7.0, Aβ is 10 μM, AFPP is 10 μM, Aβ is 5 μM, AFPP is 10 μM, AFPP _cys is 10 μM, or Aβ is 5 μM and AFPP _cys. Was added to a concentration of 5 μM, and at the same time, a fluorescent dye thioflavin T (ThT) used for amyloid fiber detection was added to a concentration of 50 μM. This was allowed to stand at room temperature for 8 hours, and the fluorescence intensity (487 nm) was measured over time.

The obtained results are shown in FIG. As is clear from FIG. 1, increase in fluorescence intensity was not observed with Aβ alone, but when Aβ and AFPP or AFPP _cys were allowed to coexist, increase in fluorescence intensity was observed in a short time. That is, from this result, it was clarified that AFPP and AFPP _cys have an action of promoting Aβ aggregation and can aggregate Aβ in a short time.

Example 1: Measurement of Aβ with an AFPPcys immobilized electrode
Aβ was measured using a gold electrode on which AFPPcys had been immobilized. Specifically, first, the electrode was immersed in 400 μl of N-ethylmorpholine buffer solution (pH 7.0) containing 50 μM AFPPcys and allowed to stand overnight to fix AFPPcys on the gold electrode. Next, 400 μl of N-ethylmorpholine buffer solution (pH 7.0) containing 5 μM Aβ was added onto AFPPcys immobilized on the metal electrode, and allowed to stand at room temperature for 60 minutes. After adding 400 μl of a Tris / HCl buffer solution (pH 7.0) containing copper chloride, cyclic voltammetry was measured, and current values were measured. Further, the measurement was performed under the same conditions as described above except that the liquid containing Aβ was not added, and the background current value was also measured.

  The obtained results are shown in FIG. As shown in FIG. 2, since divalent copper ions were also bound to AFPPcys, a background current value was observed, but when Aβ was present, an increase in current value was clearly observed. That is, it was clarified from this result that electrochemical Aβ measurement can be performed by using an electrode fixed with AFPPcys and copper divalent ions.

Example 2: Quantification of Aβ with AFPPcys immobilized electrode
Aβ was quantified using a gold electrode on which AFPPcys had been immobilized. Specifically, first, a gold electrode on which AFPPcys was fixed was produced in the same manner as in Example 1. Next, 400 μl of N-ethylmorpholine buffer solution (pH 7.0) containing 0 to 5 μM Aβ was added onto AFPPcys immobilized on the metal electrode, and allowed to stand at room temperature for 60 minutes. Next, the electrode was washed with an N-ethylmorpholine buffer solution (pH 7.0). On the washed electrode, 400 μl of Tris / HCl buffer solution (pH 7.0) containing 200 μM copper chloride was added, and excess copper divalent ions were washed with Tris / HCl buffer solution (pH 7.0). Click voltammetry was performed, and current values were measured.

  The obtained results are shown in FIG. As shown in FIG. 3, since the current value observed in the range of Aβ 0.1 to 5 μM is proportional to the concentration of Aβ, by using a gold electrode with AFPPcys fixed, It was found that even with a concentration of Aβ as low as 5 μM, highly accurate quantification is possible.

Example 3: Evaluation of electrode initialization by cleaning
In order to evaluate the influence of repeated use of a gold electrode with AFPPcys on the measurement accuracy, the following tests were conducted. First, a gold electrode to which AFPPcys was fixed was produced in the same manner as in Example 1. Subsequently, the following operation 1, washing 1, operation 2, and washing 2 were taken as one cycle, and a total of 6 cycles were repeated. Note that only the operation 1 was performed in the last one cycle.
Procedure 1: After 400 μl of Tris / HCl buffer solution (pH 7.0) containing 200 μM copper chloride is placed on AFPPcys immobilized on the metal electrode, cyclic voltammetry is measured and current value is measured. It was.
Washing 1: The gold electrode used to measure the current value in operation 1 was immersed in a Tris buffer containing 20 μM sodium ethylenediaminetetraacetate for 10 minutes, and then washed with ultrapure water. Further, the washed electrode was immersed in a 10 mM aqueous sodium hydroxide solution for 10 minutes and then washed with ultrapure water.
Operation 2: 400 μl of N-ethylmorpholine buffer solution (pH 7.0) containing 5 μM Aβ was added on AFPPcys of the metal electrode washed in Wash 1 and allowed to stand at room temperature for 60 minutes. Thereafter, 400 μl of a Tris / HCl buffer solution (pH 7.0) containing 200 μM copper chloride was added on the electrode, and then cyclic voltammetry was measured to measure the current value.
Wash 2: The gold electrode used for the current value measurement in operation 2 was washed with Tris buffer containing sodium ethylenediaminetetraacetate and 10 mM aqueous sodium hydroxide solution under the same conditions as in wash 1. It was.

  FIG. 4 shows the current values measured in operations 1 and 2 when a total of 6 cycles were repeated. From this result, it was found that the gold electrode with AFPPcys fixed had a current response with good reproducibility even after washing, and that Aβ can be quantified with high accuracy even after washing and repeated use.

Example 4: Evaluation of the effect of a similar peptide (amylin) Similar to Aβ, amylin known to form amyloid fibrils was used as a sample to examine the aggregation selectivity of AFPPcys. Specifically, the current value was measured under the same conditions as in Example 1 except that amylin was used instead of Aβ.

  The obtained results are shown in FIG. Like Aβ, amylin is known to bind to copper divalent ions. However, even when it was brought into contact with a gold electrode on which AFPPcys was immobilized, the observed current value was not affected. This indicates that AFPPcys immobilized on the gold electrode did not aggregate amylin or aggregated, but the copper divalent ions were not coordinated to the aggregate.

Claims (5)

A biosensor for measuring amyloid β peptide, comprising an electrode on which the peptide shown in (1) or (2) below is immobilized:
(1) A peptide comprising the amino acid sequence shown in SEQ ID NO: 1.
(2) Contains an amino acid sequence in which one or several amino acid residues other than the 3rd to 7th positions in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, or inserted, and has an action of aggregating Aβ peptide. The biosensor according to claim 1, wherein the peptide is a peptide shown in the following (3) or (4):
(3) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(4) In the amino acid sequence shown in SEQ ID NO: 2, it consists of an amino acid sequence in which one or several amino acid residues other than the 8th to 12th positions are substituted, deleted, added, or inserted, and aggregate with Aβ. A peptide having an action.   The biosensor according to claim 1 or 2, which is used for diagnosis of Alzheimer-type dementia. A method for measuring amyloid β peptide using the biosensor according to claim 1,
A first step of bringing a sample solution into contact with an electrode on which the peptide in the biosensor is immobilized;
A second step in which copper divalent ions are brought into contact with the electrode brought into contact with the sample solution in the first step and the current value is measured; and in the sample solution based on the current value measured in the second step 3rd process of calculating | requiring the amount of amyloid (beta) peptide of.   The measurement method according to claim 4, wherein the sample solution is plasma or a diluted solution thereof.