JP2004028798A - Device and method for detecting protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To easily detect the presence of protein, the quantity and the kind thereof to be determined. <P>SOLUTION: A protein detecting element including a coupling part having a property coupling specifically to the protein, a nucleotide chain and a fluorescent dye group, and having a sensing part for detecting that the protein is coupled to the coupling part is arranged on a first electrode, the electrode is immersed into a sample liquid containing the protein, an electric field constant in the potential difference or fluctuated time-serially is applied between the first electrode and a second electrode inserted into the sample liquid, and light emission or extinction is detected by the sensing part. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a device for detecting and quantifying a protein, and more particularly, to a device capable of detecting and quantifying a protein without requiring a labeling treatment.
[0002]
[Prior art]
The Human Genome Project, which has been underway since the 1990s, is an attempt by each country to share all the human genetic code, and it was announced that a draft version was completed in the summer of 2000. In the future, it is expected that the progress of functional genomics and structural genomics will clarify the function of each part of the decoded human genome sequence information.
[0003]
The Human Genome Project has brought a major paradigm shift to science and technology and industry involved in life sciences. For example, diabetes is classified based on the condition that the blood sugar level is high, and the causes of the onset are classified as types I and II based on the degree of insulin production in the patient's body. Has been done.
[0004]
On the other hand, according to the Human Genome Project, it is involved in the control of the amino acid sequence structure of proteins such as enzymes and receptors involved in the regulation of the detection, synthesis and degradation of blood sugar and insulin, and the control of the abundance of such proteins All information on the DNA sequence of the gene is presented to us.
[0005]
Using such information, diabetes, a phenomenon in which blood sugar levels are not regulated properly, depends on which of the proteins involved in a series of processes such as detection, synthesis, degradation, etc. Should be able to make the appropriate diagnosis and treatment.
[0006]
In particular, the development of a genomic drug for a specific protein based on the human genome sequence is being vigorously pursued in the pharmaceutical industry as a future business opportunity, and it is necessary to understand the state of a series of functionally related proteins. It is expected that the time will come when genomic drug discovery drugs will be administered to alleviate or cure symptoms.
[0007]
[Problems to be solved by the invention]
However, a technique for easily measuring the abundance of such a series of functionally related proteins is still being developed as a proteome analysis technique.
[0008]
Currently established methods use a combination of two-dimensional electrophoresis and mass spectrometry, which requires relatively large equipment and is used in clinical settings, such as hospital laboratories and beds. In order to grasp the patient's symptoms from the side, it is necessary to develop a simpler new technique.
[0009]
In a so-called DNA chip, when a DNA in a sample to be measured is amplified (increased) in advance by a PCR reaction (poylmerase chain reaction), a fluorophore is introduced and bound to a complementary DNA strand arranged in an array on the chip. The purpose is to quantify the amount of DNA in a sample by fluorescence intensity.
[0010]
On the other hand, proteins cannot perform amplification corresponding to the PCR reaction in the case of DNA. In addition, when multiple types of proteins exist in a mixed state in a sample, uniform introduction of a fluorescent label cannot be used because the reactivity between each protein and the dye is different. there were.
[0011]
An object of the present invention is to solve the above-mentioned problems and to provide a simple device for detecting and quantifying a protein.
[0012]
Still other objects and advantages of the present invention will become apparent from the following description.
[0013]
[Means for Solving the Problems]
According to one aspect of the present invention, a binding portion having the property of specifically binding to a protein,
A sensing unit comprising a nucleotide chain and a fluorophore, for detecting that the protein has bound to the binding unit,
A first electrode for fixing the sensitive part,
A second electrode;
An adjusting unit configured to include the first electrode and the second electrode, and configured to change a conformation of the sensitive unit;
A detecting unit for detecting light emission or extinction by the sensitive unit;
There is provided a protein detection device comprising:
[0014]
It is preferable that an electric field whose potential difference is constant or fluctuates with time can be provided between the first electrode and the second electrode.
[0015]
According to another aspect of the present invention, a protein comprising a binding portion having a property of specifically binding to a protein and a nucleotide chain and a fluorophore, wherein the protein is bound to the binding portion Disposing a protein detector having a sensitive part for detecting that on the first electrode,
Immerse the electrode in a sample solution containing protein,
Applying a constant or time-varying electric field between the first electrode and the second electrode inserted into the sample solution;
Detecting light emission or extinction by the sensitive part
A method for detecting a protein is provided.
[0016]
In the above device or detection method, the control unit may further include a reference electrode, a natural nucleotide and / or an artificial nucleotide may be used as a nucleotide, especially a natural and / or artificial single-stranded nucleotide. It is preferred to use
[0017]
Furthermore, the presence or absence of protein binding to the binding site and / or the change in luminescence state or quenching state when the nucleotide chain in the initial state emits or quenches fluorescence by application or disappearance of an electric field, and / or It is preferred to detect the type of bound protein and / or the amount of bound protein.
[0018]
The binding portion includes an antibody that specifically binds to the protein to be measured, a product obtained by subjecting the antibody to limited digestion with a protease, an organic compound having an affinity for the protein to be measured, and an analyte. Preferably, it is composed of at least one of the group consisting of biopolymers having an affinity for a certain protein.
[0019]
In the present invention, the term “nucleotide” means any one of the group consisting of mononucleotides, oligonucleotides and polynucleotides or a mixture thereof.
[0020]
Further features of the present invention will be apparent in the embodiments and drawings of the invention described below.
[0021]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings, examples, and the like. It should be noted that these drawings, examples, and the like, and the description are merely examples of the present invention, and do not limit the scope of the present invention. It goes without saying that other embodiments can also belong to the category of the present invention as long as they conform to the gist of the present invention. In the following drawings, the same elements may be denoted by the same reference numerals. In these figures, elements according to the invention are not necessarily to scale, and may be significantly deformed to aid understanding of the invention.
[0022]
The protein detection device realized by the present invention corresponds to a so-called protein chip. For example, in diabetes, when hepatocytes switch intracellular glycogen metabolism in response to insulin receptivity, the fact that a part of a series of protein interaction networks from insulin receptor to glycogenase is reduced or enhanced. It is something to try to catch.
[0023]
By utilizing the present invention, it becomes possible to capture the population of proteins, including so-called post-translational modifications such as phosphorylation and glycosylation. In addition, according to the present invention, instead of diagnosing phenomena that have been manifested as symptoms as in the past as diabetes, for example, a decrease in the function of a specific protein involved in the interaction network causes a deficiency in glucose metabolism. This makes it possible to properly diagnose and treat the cause of the dysfunction and verify the treatment results.
[0024]
Of course, the same technique can be applied not only to diabetes but also to hypertension, hyperlipidemia, cancer (cell growth dysregulation) and other multifactorial diseases in general.
[0025]
The present invention provides, for example, an antibody having an affinity for a protein to be measured, a derivative thereof, and the like arranged in an array, and a sample based on a correspondence between a signal generated by binding of the target protein and an array position, and the like. It captures the presence or absence of a plurality of proteins in the protein, and the type and / or amount (population) when present.
[0026]
When one end of a single-stranded nucleotide body is fixed on an electrode and immersed in an aqueous solution, and a DC electric field is applied between the single-stranded nucleotide body and an electrode arranged in the aqueous solution, for example, the single-stranded nucleotide body chain is elongated, The phenomenon of spontaneous aggregation and contraction was observed experimentally. The present invention utilizes such properties of nucleotides represented by single-stranded nucleotides.
[0027]
That is, the function of each part of the protein detection device according to the present invention is as follows.
[0028]
The first electrode is usually installed on an electrode support having an appropriate shape, and serves to fix the sensitive portion and as a part of an adjusting portion for changing the spatial conformation of the sensitive portion. Having. The sensitive part is usually fixed to the first electrode at one end of the nucleotide body.
[0029]
For the immobilization, a known method such as a method of synthesizing a nucleotide body having a thioether group or a thiol group introduced therein and bringing the nucleotide body into contact with a polished electrode surface can be employed. Between the thioether group or thiol group and the nucleotide, for example,-(CH ₂ ) ₃ -Or-(CH ₂ ) ₆ A bond called a linker such as-may be inserted. The number of sensitive portions that can be immobilized on the electrode is often influenced by the type of the linker and the length of the bond. Generally CH ₂ As the number of units decreases, the number of sensitive parts that can be fixed on the electrode tends to decrease.
[0030]
The fixation can also be performed using a material called carbon nanotube or carbon nanofiber. Carbon nanotubes or carbon nanofibers can be formed by arranging a catalyst (eg, Ni) on the first electrode and growing it vertically from the electrode surface by thermochemical vapor deposition (CVD) or plasma CVD. it can. The five-membered ring at the tip of the formed carbon nanotube or carbon nanofiber can be easily chemically modified, and by utilizing this property, it is possible to join a predetermined nucleotide body to the tip of the carbon nanotube or carbon nanofiber. it can. Since carbon nanotubes or carbon nanofibers are hard materials, nucleotides bonded to electrodes via such materials will be more firmly fixed, which is advantageous, for example, when using a highly viscous sample solution. .
[0031]
The shape, size, number, and arrangement of the electrodes may be any, and can be freely determined according to the purpose. When it is desired to simultaneously detect a plurality of proteins, for example, the electrodes can be divided into a plurality of parts by spacers, or circular electrodes can be divided and arranged on the electrode support. However, in such a case, it is necessary to provide a function of distinguishing and detecting fluorescence emission and extinction signals from each section.
[0032]
FIG. 7 is a model diagram in which a plurality of circular electrodes 6 are separately arranged on the electrode support 2. In this case, the terminals 8 are arranged around the electrode support 2, and the corresponding electrodes 6 and the terminals 8 are electrically connected by the lead wires 17. The electrode 6 can be manufactured using a photolithography technique. The diameter of the electrode 6 can be about 1 nm to 1 cm. However, it may be larger or smaller. In FIG. 7, drawing of the lower half electrode is omitted. The electrodes 6 may not necessarily be electrically independent in some cases.
[0033]
The electrode support may be any as long as the electrodes provided thereon can be insulated from each other. Glass, ceramics, plastics, metals and the like can be considered. In the case of a conductive substance, a method in which an insulating film is disposed thereon and an electrode is disposed thereon may be considered. For example, in the case of Si, ₂ And the like.
[0034]
As a material of the first electrode, any material such as a single metal, an alloy, or a laminate thereof may be used as long as it is a conductive substance. A noble metal represented by Au is chemically stable and can be preferably used.
[0035]
The binding portion includes an antibody that specifically binds to the protein to be measured, a product obtained by subjecting the antibody to limited digestion with a protease, an organic compound having an affinity for the protein to be measured, and an analyte. And has a property of binding specifically to a protein. Different materials may be used for each electrode. There are no particular restrictions on the type of binding to the protein and the binding site, but it is better to avoid binding with particularly weak binding strength. Here, the “product” is obtained by subjecting an antibody to limited digestion with a protease, and as long as the spirit of the present invention is met, a Fab fragment of the antibody or a fragment derived from the Fab fragment of the antibody, Can include any derivative thereof.
[0036]
As the antibody, for example, a monoclonal immunoglobulin IgG antibody can be used. Further, as a fragment derived from an IgG antibody, for example, a Fab fragment of an IgG antibody can be used. Furthermore, a fragment derived from such a Fab fragment can also be used. Examples of usable organic compounds having an affinity for the protein to be measured include enzyme substrate analogs such as adenosine-5′-O- (3-thiotriphosphate) (also known as ATP-γ-S); There are enzyme activity inhibitors, neurotransmission inhibitors (antagonists) and the like. Examples of the biopolymer having an affinity for the protein to be measured include a protein serving as a substrate or a catalyst of the protein, and elemental proteins constituting a molecular complex.
[0037]
It is useful to use, as the binding portion, a product obtained by limited digestion with a monoclonal antibody or a protease, since a bond produced by a reaction similar to an antigen-antibody reaction can be used.
[0038]
It is preferable to use a monoclonal antibody, a monoclonal antibody Fab fragment, or a fragment derived from the monoclonal antibody Fab fragment as the binding portion. The fragment derived from the Fab fragment of the monoclonal antibody refers to a fragment obtained by subdividing the Fab fragment of the monoclonal antibody or a derivative thereof.
[0039]
Furthermore, it is more preferable to use an IgG antibody, an antibody Fab fragment, an IgG antibody or a fragment derived from an IgG antibody Fab fragment as the binding portion. The fragment derived from the IgG antibody Fab fragment means a fragment obtained by subdividing the IgG antibody Fab fragment or a derivative thereof. It is also preferred that the binding moiety is a nucleotide aptamer.
[0040]
In general, those having a smaller molecular weight have better detection sensitivity, which is the reason why they are preferred.
[0041]
The binding part is bound to the nucleotide chain constituting the sensitive part. There are no particular restrictions on the type and location of the bond to the nucleotide chain, but it is better to avoid bonds with particularly weak binding power. In addition, since it is generally preferable that the stretch of the polynucleotide be large at the binding site, it is often preferable that the terminal side is different from the terminal fixed to the electrode or in the vicinity thereof. When the above-mentioned substance constituting the binding portion cannot be directly linked to and immobilized on the nucleotide body chain constituting the sensitive portion, the substance may be immobilized by interposing an effective connecting portion therefor. .
[0042]
The sensitive part contains a nucleotide chain and a fluorophore, and the function of the regulatory part changes its conformation, causing the fluorophore to emit or quench, and the protein is bound to the binding part. That can be detected. Specifically, for example, a direct-current electric field is applied to extend the nucleotide chain, and the electric field is cut off to cause aggregation and contraction. This change is a conformational change. The essence of the sensitive part lies in detecting that the protein has bound to the binding part. Therefore, if the binding of the protein to the binding portion can be detected, the sensitive portion may be sufficient even when the protein does not contain a nucleotide chain or a fluorophore as described above.
[0043]
As the nucleotide body for this purpose, a natural nucleotide body or an artificial nucleotide body can be used. Artificial nucleotides include both fully artificial and those derived from natural nucleotides. Use of an artificial nucleotide form may be advantageous because it may increase detection sensitivity or improve stability.
[0044]
Further, it may be a single-stranded nucleotide or a double-stranded nucleotide which is a pair of single-stranded nucleotides complementary to each other. From the viewpoint of easy extension and contraction, single-stranded nucleotides are often preferred. Different nucleotide bodies can be used for each electrode. The length of the nucleotide body chain may be one or more residues. That is, it may be a mononucleotide chain.
[0045]
The fluorophore may be covalently attached to the nucleotide body, or may be contained within the nucleotide body, as in instances where it is inserted (intercalated) between adjacent complementary bonds. Or it may be incorporated into a part of the nucleotide chain by substitution. Preferably, the fluorophore is present near the tip of the nucleotide chain on the binding side.
[0046]
The fluorophore is selected from substances that are excited by the action of light to generate fluorescence. Fluorescein maleimide Cy3 (trademark) is an example of a fluorophore that can be suitably used in the present invention.
[0047]
The adjusting unit has a role of changing the conformation of the sensitive unit, and includes the first electrode and a second electrode inserted into the sample solution.
[0048]
As the sample solution used in the present invention, an aqueous solution containing a protein is appropriate, and is often used as a buffer solution whose pH has been adjusted.
[0049]
It is preferable that an electric field having a constant potential difference or an electric field having a time-varying potential difference can be applied between the first electrode and the second electrode. It is preferable to provide a reference electrode for compensating for a change in the potential difference in addition to the first electrode and the second electrode, so that a detected signal is preferably stable.
[0050]
An electric field having a constant potential difference can be obtained by using a direct current. The potential difference whose value changes with time is, for example, a DC component, an AC component, and a pulse current component that is a combination of a case where a potential difference exists and a case where a potential difference does not exist or a case where a potential difference changes stepwise. It can be obtained by arbitrarily combining and modulating. In this case, the AC component may have a very low frequency of 1 second or more. In addition, as the material of the second electrode and the reference electrode, any material may be used as long as a stable detection result is obtained.
[0051]
The detection unit detects that the protein has bound to the binding unit. This is by detecting light emission or extinction by the sensitive part. As the detection unit, any known detection device can be used as long as it can detect fluorescence and does not violate the gist of the present invention.
[0052]
The protein detection device and the mechanism of light emission and quenching according to the present invention will be described with reference to FIGS. FIG. 1 shows a cross-sectional view of an example of the protein detection device according to the present invention.
[0053]
In FIG. 1, a sample 7 is placed in a container comprising an electrode support 2 and a wall 3 placed on a table 1, and the space between them is sealed with an O-ring 4, and is covered with a lid 5. An electrode 6 is provided on the electrode support 2 and is connected to an electrode 9 via a terminal 8 outside the container. The electrode 6 corresponds to the first electrode, and the electrode 9 corresponds to the second electrode. A plurality of electrodes 6 can be provided. The adjusting unit includes the electrode 6 and the electrode 9. The configuration may be any known configuration as long as it conforms to the gist of the present invention.
[0054]
Using the protein detection device having such a configuration, light emission or quenching by the sensitive part is detected. Note that the coupling section and the sensitive section are not shown.
[0055]
For example, an excitation light source 10 using a laser oscillator 11 is prepared. The measurement unit 13 at the detection end 12 of the protein detection device is preferably connected to a data processing device 14 for processing measurement data. The processing device 14 includes a measurement result display device 15, an electrode array arrangement and a detection unit. A storage device 16 storing the calibration values of the above is attached.
[0056]
FIG. 2 is a model diagram showing a manner in which the nucleotide body chain emits fluorescence on application of an electric field on the electrode 6 in FIG. 1 or quenches by the disappearance of the electric field.
[0057]
The left side of FIG. 2 shows that the protein bound to the binding portion, which is composed of the binding portion 23 having the property of specifically binding to the protein 24 and the nucleotide chain 21 and the fluorophore 22, is shown. The protein detector 27 comprising the sensitive part 26 for detection is shown standing on the electrode 6. This standing state is due to the fact that an electric field is applied to the system to cause an electric repulsion (Coulomb force) between the nucleotide chain 21 and the electrode 6 to expand the nucleotide chain 21. .
[0058]
With this extension, the fluorescent chromophore 22 becomes less or less affected by the electrical influence of the electrode 6, and can emit light 28.
[0059]
On the other hand, the right side of FIG. 2 shows a state in which the protein detector 27 including the binding part 23 and the sensitive part 26 composed of the nucleotide chain 21 and the fluorophore 22 is near the electrode 6. This is because the nucleotide chain 21 has an energetically stable structure when the electric field is removed from the system, so that the nucleotide chain 21 has aggregated and contracted. In addition, it is also possible to positively apply an electric field opposite to that in the past, to apply an electric attractive force (Coulomb force) between the nucleotide chain 21 and the electrode 6 to cause the nucleotide chain 21 to aggregate and contract. In the case of single-stranded nucleotides, the flexibility of the molecule is large, and it is often sufficient without actively applying an opposite electric field. However, in the case of double-stranded nucleotides, the rigidity of the molecule increases, and the electric field is increased. If there is no, it is close to a standing state, so it is often better to apply an opposite electric field positively.
[0060]
When the aggregation and contraction occur in this manner, the fluorescent chromophore 22 is electrically affected by the electrode 6 and can be extinguished. For this purpose, it is not always necessary for the protein detector 27 to be in contact with the electrode 6, and it may be sufficient if the protein detector 27 is close to the vicinity of the electrode 6.
[0061]
Furthermore, if the quencher 25 is bonded to the surface of the electrode 6 as shown in FIG. 2, the quenching speed and efficiency can be changed. Quenchers are selected from those that cause effective quenching to the fluorophore used. For example, when fluorescein maleimide or Cy3 (trademark) is used as a fluorescent chromophore, a quencher such as D-damine B sulfonyl chloride can be used as a quencher. More preferably, a combination of a fluorescent chromophore and a quenching chromophore that produces a fluorescence energy transfer (FRET) effect is preferred.
[0062]
In addition, as the excitation light source 10 that emits the irradiation light 29 that excites the light-emitting atomic group, for example, a generally available light source such as a visible light or an ultraviolet lamp can be used.
[0063]
This light emission or extinction is detected by the detection end 12 that detects the light emission 28. In addition, in this specification, although the case where the elongation and the aggregation and contraction behavior are exhibited as described above is mainly described, on the contrary, when the nucleotide body chain 21 is quenched when it is elongated, and when the nucleotide body chain 21 is aggregated and contracted. It can be made to emit light and belongs to the category of the present invention. In this case, no quencher is used. A luminescence aid that promotes luminescence can be used.
[0064]
On the other hand, looking at the relationship between the application or disappearance of the electric field and the elongation / aggregation / shrinkage behavior of the nucleotide chain, when the electric field is applied, the elongation or aggregation / shrinkage of the nucleotide chain may occur depending on the direction of the electric field. In contrast, when the electric field is extinguished, aggregation contraction of the nucleotide body chain may occur.
[0065]
Therefore, from the above, the relationship between the application or disappearance of the electric field and the emission or quenching of the fluorescence is the case where the light is emitted when the electric field is applied, the case where the light is quenched when the electric field is applied, and the case where the electric field is extinguished. Various combinations of a case where light is emitted when the electric field is emitted and a case where light is extinguished when the electric field is lost are possible. In the present invention, "the nucleotide body chain in the initial state emits or quenches fluorescence by application or disappearance of an electric field" includes all the above-mentioned states.
[0066]
When such a device is used, a nucleotide body chain in an initial state emits or quenches fluorescence by application or disappearance of an electric field, and thus changes in luminescence state or quenching state causes the protein to bind to the binding site. The presence or absence of binding and / or the type of bound protein and / or the amount of bound protein can be detected.
[0067]
FIG. 2 shows a state where the protein 24 is bound to the binding part 23 on both the right and left sides. When the protein 24 binds to the binding portion 23 in this manner, it takes longer time to extend and aggregate / shrink the nucleotide chain 21 than when the protein 24 is not bound. Therefore, it takes a longer time to change between light emission and extinction. Here, in the present invention, the initial state before the application of the electric field means a state where the electric field is not applied or a state where the electric field in which the potential difference is applied in the opposite direction, and the initial state before the disappearance of the electric field, It means a state in which an electric field is applied.
[0068]
Therefore, first, the presence or absence of the protein binding to the binding portion can be detected by the change of the light emission state or the change of the quenching state when fluorescence is emitted or quenched due to the application or disappearance of the electric field. Then, if it is known what kind of protein the binding portion used binds, it becomes possible to know the type of the protein. Since the binding portion according to the present invention has a property of specifically binding to a protein, such detection can be easily performed.
[0069]
Then, the amount of the protein bound to the binding portion can be detected based on the change in the light emitting state or the change in the quenching state.
[0070]
Specifically, for example, it can be detected by the luminescence intensity and / or the rate of change of the luminescence intensity when fluorescence is emitted or quenched.
[0071]
In addition, when an electric field having a potential difference whose value changes with time is applied, it can be detected by the peak intensity of fluorescence emission and its change or change rate.
[0072]
FIG. 3 is a diagram showing a temporal change in the luminescence intensity when no protein is bound, and FIG. 4 is a diagram showing a temporal change in the luminescence intensity when the protein is bound. The horizontal axis represents the time from the start of applying the electric field. The vertical axis represents the light emission intensity, and the unit can be arbitrarily determined, but is a common unit in FIGS.
[0073]
FIGS. 3 and 4 show a specific example of the change in the light emission state, which is a time required until a point Y indicating a half value of the emission intensity at the point X indicating the peak value of the fluorescent light emission. When FIG. 4 is compared with FIG. 3, it is understood that the time of FIG. 4 is longer. In FIG. 4, the time from the point Y to the point X is shorter than that in FIG. 3, but this is because the peak value is greatly reduced. Seen in FIG. 4 is longer.
[0074]
The reason why the time is long seems to be primarily related to the type, size, charge state and the like of the protein. For example, in the case of a protein having a large size, it is considered that the elongation of the nucleotide body takes a long time due to its mass effect and shape effect.
[0075]
Furthermore, this time also depends on the amount of protein bound to the binding site. This is considered to be because, for example, the binding of the protein to the binding portion causes the protein detector on the electrode to be crowded, thereby inhibiting the extension of the nucleotide body.
[0076]
The emission intensity at the time of generating the fluorescence emission is the emission intensity (peak value) at the point X, and the change rate of the emission intensity is the average value of the emission change speed from the start of the emission to the point Y or the point Y to the point X. And the average value of the light emission change speed up to.
[0077]
When FIG. 4 is compared with FIG. 3, the peak value, the average value of the light emission change speed from the start of light emission to the point Y, and the average value of the light emission change speed from the point Y to the point X are clearly shown in FIG. Is getting smaller. This is presumably because the binding of the protein to the binding portion increases the resistance to the solvent during the movement, and makes it difficult for the nucleotide body to be extended.
[0078]
3 and 4 can be obtained by applying an electric field having a constant potential difference or an electric field having a time-varying potential difference between the first electrode and the second electrode.
[0079]
When an electric field having a potential difference whose value changes with time, such as an alternating current, is applied, the potential difference fluctuates with time or the direction of the electric field changes, so that light emission repeatedly increases and decreases.
[0080]
FIG. 5 is a diagram showing a temporal change in luminescence intensity when no protein is bound when a pulse waveform voltage is applied, and FIG. 6 shows a protein bound when a pulse waveform voltage is applied. FIG. 7 is a diagram showing a temporal change of the light emission intensity when the light emission is performed.
[0081]
In FIGS. 5 and 6, the horizontal axis represents time when a certain electric field is applied. The vertical axis on the right side represents the potential difference between the first electrode and the second electrode. The given potential difference 51 has a rectangular wave shape. The vertical axis on the left represents the light emission intensity. The unit of the light emission intensity is arbitrarily determined, but is a common unit in FIGS. The change 52 in the emission intensity is saw-toothed.
[0082]
5 and 6, even when the same potential difference waveform is applied, the peak value becomes smaller when the protein is bound, and even when the same potential difference waveform is maintained, for example, the region indicated by Z It can be understood that the difference in the decrease of the peak value occurs with time as shown in FIG.
[0083]
Generally speaking, when an electric field whose potential difference varies with time is applied, the peak intensity and / or the rate of change thereof change depending on the presence or absence of protein binding. It also varies depending on the type and amount of the bound protein. The electric field whose potential difference varies with time can be obtained by arbitrarily combining and modulating an AC component or a pulse current component.
[0084]
Therefore, utilizing such a change, it is possible to detect the presence / absence of protein binding to the binding site and / or the type of bound protein and / or the amount of bound protein.
[0085]
The enlarged portions of the light emission in FIGS. 5 and 6 give curves corresponding to the diagrams in FIGS.
[0086]
Thus, according to the present invention, the presence or absence of a protein and the type and amount of the protein can be easily detected and determined. Moreover, in the present invention, there is no need to perform labeling treatment on the protein.
[0087]
【Example】
Next, examples and comparative examples of the present invention will be described in detail.
[0088]
[Example 1]
The device shown in FIGS. 1 and 2 was used.
[0089]
Using a natural single-stranded oligonucleotide and an artificial single-stranded oligonucleotide, a single-stranded oligonucleotide having a thiol group introduced at the 3 ′ end via a spacer was synthesized, and the polished gold electrode was synthesized at room temperature. After a reaction for 24 hours, a natural single-stranded oligonucleotide and an artificial single-stranded oligonucleotide were bound to a gold electrode provided on sapphire, as shown in FIG. In addition, the fluorophore was previously introduced into the single-stranded oligonucleotide. The thiol group and fluorophore may be introduced at the end of a single strand, or may be introduced at the 5 'end of the strand.
[0090]
The oligonucleotide chain was immobilized on a 1 mm circular Au electrode. No quencher was used in this example.
[0091]
Furthermore, a monoclonal Fab fragment of immunoglobulin IgG was immobilized at the end of the oligonucleotide chain. At this time, Fab fragments having different specificities were immobilized for each of the population sections of the oligonucleotide separated by the spacer.
[0092]
A sample solution containing the protein to be detected was brought into contact with the device, and kept at room temperature for 10 minutes, which is a sufficient time for forming a bond between the detection portion and the protein.
[0093]
The electrode on which the oligonucleotide chain having been subjected to the above treatment is formed is immersed in an aqueous solution, and a two-electrode method including a first electrode and a second electrode or a three-electrode method including a first electrode, a second electrode, and a reference electrode is used. A direct current or alternating current was applied to the oligonucleotide chain by an electrode method to apply an electric field, and a fluorescent lamp on the oligonucleotide chain was excited by an ultraviolet lamp to emit fluorescence, and the time course of the fluorescence intensity was measured. The three-electrode method gave more stable results.
[0094]
FIGS. 3, 4, 5, and 6 are obtained as a result of this experiment.
[0095]
When an electric field is applied so that the first electrode has a negative potential, the oligonucleotide, which is a negative ion, is extended by Coulomb repulsion, so that the fluorophore bound to the oligonucleotide leaves the electrode. As a result, the fluorescent chromophore that had been quenched because it was near the first electrode began to emit light.
[0096]
At this time, if the protein to be evaluated is present in the sample solution, the Fab fragment binds to the protein, so that the mass at the tip of the single-stranded oligonucleotide increases or the shape becomes large, so that the fluorescence intensity is reduced. An increase in the time taken for the increase was observed.
[0097]
When the electric field disappears or when the electric field is applied so that the first electrode has a negative potential, the oligonucleotide chain is spontaneously aggregated or contracted by the Coulomb force, and as a result, the fluorescent dye It was observed that the closer the distance between the group and the electrode surface, the lower the fluorescence intensity.
[0098]
At this time, it is observed that when the protein to be evaluated is present in the sample solution as in the case where the electric field is applied so that the first electrode has a negative potential, the time required for decreasing the fluorescence intensity increases. Was. The degree of increase in time changes depending on the amount of protein present in the sample, and this is taken into account, and the amount of protein in the sample is quantified. Could be identified.
[0099]
When an electric field is applied so that the first electrode has a negative potential, the fluorescence intensity increases. However, when no protein is bound to the oligonucleotide, the single-stranded oligonucleotide is not sufficiently elongated. However, while the distance between the electrode and the fluorophore increases and the emission intensity increases, when a protein is bound to the oligonucleotide, the protein becomes resistant and the single-stranded oligonucleotide does not extend sufficiently, and the fluorescence intensity also increases. It was shown to be smaller.
[0100]
Further, as shown in FIGS. 5 and 6, even when the waveforms of the same potential difference are maintained, the peak value decreases with time, for example, as shown in a region indicated by Z. As described above, such behavior can also be used to determine the amount of protein in a sample and to specify the type of protein.
[0101]
FIGS. 5 and 6 show examples of the voltage of the pulse waveform. Even when the voltage has an AC component such as a sine wave, the amount of the protein in the sample is quantified from the data on the emission intensity, and the type of the protein is determined. Can be identified.
[0102]
Since the degree of fluorescence intensity changes depending on the amount of protein present in the sample, this is captured, the amount of protein in the sample is quantified, and the type of protein is identified based on which oligonucleotide chain signal. It was possible to do.
[0103]
The detection sensitivity of the protein varies depending on the molecular weight (size) of the protein to be bound, and also varies depending on the binding constant between the protein and, for example, a monoclonal IgG antibody. That is, for example, by arranging a plurality of monoclonal antibodies having different binding constants on an array, it is possible to cover a wide measurement range.
[0104]
From the contents disclosed above, the inventions shown in the following supplementary notes can be derived.
[0105]
(Supplementary Note 1) a binding portion having a property of specifically binding to a protein,
A sensing unit for detecting that the protein has bound to the binding unit;
A first electrode for fixing the sensitive part,
A second electrode;
An adjusting unit configured to include the first electrode and the second electrode, and configured to change a conformation of the sensitive unit;
A detecting unit for detecting light emission or extinction by the sensitive unit;
And a protein detection device.
[0106]
(Supplementary Note 2) a binding portion having a property of specifically binding to a protein,
A sensing unit comprising a nucleotide chain and a fluorophore, for detecting that the protein has bound to the binding unit,
A first electrode for fixing the sensitive part,
A second electrode;
An adjusting unit configured to include the first electrode and the second electrode, and configured to change a conformation of the sensitive unit;
A detecting unit for detecting light emission or extinction by the sensitive unit;
And a protein detection device.
[0107]
(Supplementary Note 3) The protein detection device according to Supplementary Note 1 or 2, wherein the adjustment unit further includes a reference electrode.
[0108]
(Supplementary note 4) The protein detection device according to any one of Supplementary notes 1 to 3, wherein an electric field with a constant or time-varying electric potential is applied between the first electrode and the second electrode.
[0109]
(Supplementary Note 5) The protein detection device according to any one of Supplementary Notes 2 to 4, wherein a natural nucleotide body and / or an artificial nucleotide body is used as the nucleotide body.
[0110]
(Supplementary note 6) The protein detection device according to any one of Supplementary notes 2 to 4, wherein a natural and / or artificial single-stranded nucleotide body is used as the nucleotide body.
[0111]
(Supplementary Note 7) The presence or absence of binding of the protein to the binding site is determined by the change in the luminescence state or the change in the quenching state when the nucleotide chain in the initial state emits or quenches fluorescence by application or disappearance of an electric field, and 7. The protein detection device according to any one of supplementary notes 4 to 6, wherein the device detects the type of bound protein and / or the amount of bound protein.
[0112]
(Supplementary Note 8) Presence or absence of protein binding to the binding site depending on the luminescence intensity and / or the rate of change in luminescence intensity when the nucleotide chain in the initial state emits or quenches fluorescence by application or disappearance of an electric field 8. The protein detection device according to any one of supplementary notes 4 to 7, wherein the type of the bound protein and / or the amount of the bound protein are detected.
[0113]
(Supplementary Note 9) The presence or absence of protein binding to the binding site and / or the type and / or binding of the bound protein depending on the peak intensity of fluorescence emission and / or the rate of change when an electric field whose potential difference varies with time is applied. 9. The protein detection device according to any one of Supplementary Notes 4 to 8, wherein the detected protein amount is detected.
[0114]
(Supplementary Note 10) An antibody whose binding portion specifically binds to a protein to be measured, a product obtained by subjecting the antibody to limited digestion with a protease, and an organic substance having an affinity for the protein to be measured. 10. The protein detection device according to any one of supplementary notes 1 to 9, comprising at least one of a group consisting of a compound and a biopolymer having an affinity for a protein to be measured.
[0115]
(Supplementary Note 11) The supplementary note according to any one of Supplementary Notes 1 to 9, wherein the binding portion is composed of at least one of a group consisting of a monoclonal antibody, a Fab fragment of the monoclonal antibody, and a fragment derived from the Fab fragment of the monoclonal antibody. Protein detection device.
[0116]
(Supplementary note 12) The protein detection according to any one of Supplementary notes 1 to 9, wherein the binding portion is composed of at least one of a group consisting of an IgG antibody, an IgG antibody Fab fragment, and a fragment derived from the IgG antibody Fab fragment. device.
[0117]
(Supplementary Note 13) The protein detection device according to any one of Supplementary Notes 1 to 9, wherein the binding part is a nucleotide aptamer.
[0118]
(Supplementary Note 14) A protein detector having a binding portion having a property of specifically binding to a protein and a sensitive portion for detecting that the protein has bound to the binding portion is disposed on the first electrode. ,
Immerse the electrode in a sample solution containing protein,
Applying a constant or time-varying electric field between the first electrode and the second electrode inserted into the sample solution;
Detecting light emission or extinction by the sensitive part
A method for detecting a protein.
[0119]
(Supplementary Note 15) Sensitivity for detecting that a protein has bound to a binding portion, comprising a binding portion having a property of specifically binding to a protein and a nucleotide chain and a fluorophore. Disposing a protein detector having a portion on the first electrode;
Immerse the electrode in a sample solution containing protein,
Applying a constant or time-varying electric field between the first electrode and the second electrode inserted into the sample solution;
Detecting light emission or extinction by the sensitive part
A method for detecting a protein.
[0120]
(Supplementary Note 16) The protein detection method according to Supplementary Notes 14 and 15, further using a reference electrode.
[0121]
(Supplementary Note 17) The protein detection method according to Supplementary Note 15 or 16, wherein a natural nucleotide body and / or an artificial nucleotide body is used as the nucleotide body.
[0122]
(Supplementary note 18) The method for detecting a protein according to Supplementary note 15 or 16, wherein a natural and / or artificial single-stranded nucleotide is used as the nucleotide.
[0123]
(Supplementary Note 19) The presence or absence of protein binding to the binding site is determined by the change in luminescence state or change in quenching state when the nucleotide chain in the initial state emits or quenches fluorescence by application or disappearance of an electric field. 19. The method for detecting a protein according to any one of supplementary notes 15 to 18, wherein the type of the bound protein and / or the amount of the bound protein is detected.
[0124]
(Supplementary Note 20) Presence or absence of protein binding to the binding site depending on the luminescence intensity and / or the rate of change of luminescence intensity when the nucleotide chain in the initial state emits or quenches fluorescence by application or disappearance of an electric field 19. The method for detecting a protein according to any one of supplementary notes 15 to 18, wherein the type of bound protein and / or the amount of bound protein are detected.
[0125]
(Supplementary Note 21) The presence or absence of protein binding to the binding site and / or the type and / or binding of the bound protein depends on the peak intensity of fluorescence emission and / or the rate of change when an electric field whose potential difference fluctuates with time is applied. 19. The method for detecting a protein according to any one of supplementary notes 15 to 18, wherein the amount of the detected protein is detected.
[0126]
(Supplementary Note 22) An antibody whose binding portion specifically binds to a protein to be measured, a product obtained by subjecting the antibody to limited digestion with a protease, and an organic compound having an affinity for the protein to be measured 22. The method for detecting a protein according to any one of appendices 14 to 21, comprising at least one selected from the group consisting of biopolymers having an affinity for a protein to be measured.
[0127]
(Supplementary note 23) Any one of Supplementary notes 14 to 21, wherein the binding portion is constituted by at least one of a group consisting of a monoclonal antibody, a Fab fragment of a monoclonal antibody, and a fragment derived from the Fab fragment of the monoclonal antibody. A method for detecting a protein.
[0128]
(Supplementary note 24) The protein according to any one of Supplementary notes 14 to 21, wherein the binding portion is composed of at least one of a group consisting of an IgG antibody, an IgG antibody Fab fragment, and a fragment derived from the IgG antibody Fab fragment. Detection method.
[0129]
(Supplementary note 25) The method for detecting a protein according to any one of supplementary notes 14 to 21, wherein the binding part is a nucleotide aptamer.
[0130]
【The invention's effect】
The presence or absence of the protein and its type and amount can be easily detected and determined.
[Brief description of the drawings]
FIG. 1 shows a cross-sectional view of an example of a protein detection device according to the present invention.
FIG. 2 shows a state in which a protein detector emits or quenches fluorescence upon application or disappearance of an electric field on an electrode.
FIG. 3 is a diagram showing a temporal change in luminescence intensity when no protein is bound.
FIG. 4 is a diagram showing a temporal change in luminescence intensity when a protein is bound.
FIG. 5 is a diagram showing a temporal change in luminescence intensity when a protein is not bound when a pulse waveform voltage is applied.
FIG. 6 is a diagram showing a temporal change in luminescence intensity when a protein is bound when a pulse waveform voltage is applied.
FIG. 7 is a model diagram in which a plurality of circular electrodes are separately arranged on an electrode support.
[Explanation of symbols]
One
2 Electrode support
3 wall
4 O-ring
5 Lid
6 electrodes
7 samples
8 terminals
9 electrodes
10 Excitation light source
11 Laser oscillator
12 Detection end
13 Measurement unit
14 Data processing device
15 Measurement result display device
16 Storage device
17 Lead wire
21 nucleotide chain
22 Fluorophore
23 Joint
24 proteins
25 Quencher
26 Sensitive part
27 Protein detector
28 luminescence
29 Irradiation light
51 Square wave
52 Change in luminescence intensity

Claims (10)

A binding portion having a property of specifically binding to a protein,
A sensing unit comprising a nucleotide chain and a fluorophore, for detecting that the protein has bound to the binding unit,
A first electrode for fixing the sensitive part,
A second electrode;
An adjusting unit configured to include the first electrode and the second electrode, and configured to change a conformation of the sensitive unit;
A detection unit for detecting light emission or quenching by the sensitive unit. The protein detection device according to claim 1, wherein the adjustment unit further includes a reference electrode. The protein detection device according to claim 1, wherein an electric field having a constant or time-varying potential difference can be applied between the first electrode and the second electrode. The protein detection device according to any one of claims 1 to 3, wherein a natural and / or artificial single-stranded nucleotide is used as the nucleotide. The nucleotide body chain in the initial state has the presence or absence of protein binding to the binding site and / or has been bound by the change in the luminescence state or the change in the quenching state when emitting or quenching fluorescence by application or disappearance of an electric field. 5. The protein detection device according to claim 3, wherein the type of protein and / or the amount of bound protein is detected. The presence or absence and / or binding of the protein to the binding site depends on the luminescence intensity and / or the rate of change of the luminescence intensity when the nucleotide chain in the initial state emits or quenches fluorescence by application or disappearance of an electric field. The protein detection device according to any one of claims 3 to 5, wherein the type of the detected protein and / or the amount of the bound protein are detected. The presence or absence of protein binding to the binding site and / or the type of bound protein and / or the amount of bound protein can be determined by the peak intensity of fluorescence emission and / or the rate of change when an electric field whose potential difference varies with time is applied. The protein detection device according to any one of claims 3 to 6, which performs detection. An antibody whose binding portion specifically binds to a protein to be measured, a product obtained by subjecting the antibody to limited digestion with a protease, an organic compound having an affinity for the protein to be measured, and an analyte The protein detection device according to any one of claims 1 to 7, comprising at least one of a group consisting of a biopolymer having an affinity for a protein. The protein detection device according to any one of claims 1 to 8, wherein the binding portion is composed of at least one of a group consisting of an IgG antibody, an IgG antibody Fab fragment, and a fragment derived from the IgG antibody Fab fragment. A protein having a binding portion having a property of specifically binding to a protein and a sensitive portion for detecting that the protein has bound to the binding portion, comprising a nucleotide chain and a fluorophore. Placing the detector on the first electrode;
Immerse the electrode in a sample solution containing protein,
Applying a constant or time-varying electric field between the first electrode and the second electrode inserted into the sample solution;
A method for detecting a protein that detects light emission or quenching by the sensitive part.

Images