WO2023075517A1 - Prussian blue nanoparticle-based optical and electrochemical biosensor - Google Patents

Abstract

The present invention relates to a Prussian blue nanoparticle-based optical and electrochemical biosensor, and, more specifically, to a biosensor capable of rapidly, simply, qualitatively and quantitatively detecting a target material in a sample by using Prussian blue nanoparticles exhibiting peroxidase-like activity and electrochemical activity.

Description

Optical and electrochemical biosensors based on Prussian blue nanoparticles

This application claims priority to Republic of Korea Patent Application No. 10-2021-0146763 filed on October 29, 2021, and the entire specification is a reference in this application.

The present invention relates to an optical and electrochemical biosensor based on Prussian blue nanoparticles, and more particularly, to quickly and conveniently detect a target substance in a sample using Prussian blue nanoparticles exhibiting peroxidase-like activity and electrochemical activity. It relates to a biosensor capable of qualitative and quantitative detection.

As the standard of living increases along with population growth and life expectancy worldwide, interest in healthy living and well-aging is increasing. In addition, medical expenses for early diagnosis and prompt prevention of diseases through steady examinations are increasing. Due to the recent emergence and spread of new viruses, the demand for diagnostic devices that can quickly and easily diagnose diseases has increased. As the healthcare paradigm changes from simple disease treatment to prevention, diagnosis, and monitoring, the need for a simple and efficient disease diagnosis system is increasing. In particular, in the field of clinical chemistry and immunology, studies for diagnosing diseases by examining various target substances with a single device are being actively conducted.

Sensors using various materials are being developed to rapidly and efficiently diagnose diseases and increase the sensitivity and selectivity of sensors, and among them, sensors using functional nanomaterials are being studied most actively. Functional nanomaterials and nanostructures are being developed to replace biological enzymes or protein enzymes that are expensive and inconvenient to use. Advantages of these materials are that they are simple to synthesize, maintain stability even when temperature and pH changes, and can be mass-produced to reduce production costs.

However, conventionally developed nanomaterial-based biosensors have limitations in that simultaneous application of qualitative and quantitative analysis is difficult.

Therefore, the present inventors can overcome the limitations of existing nanomaterial-based biosensors as well as biological enzymes or protein enzymes, can be applied to qualitative and quantitative analysis simultaneously, and can quickly detect target substances in the field. As a result of intensive research to develop a biosensor that can be used, it is very rapid and economical to use the peroxidase-like activity and electrochemical activity of the Prussian blue nanoparticle and bioreceptor complex. As a result, it was discovered that the target substance in the sample could be detected, and the present invention was completed.

Accordingly, an object of the present invention is (a) a composite of Prussian blue nanoparticles and a bioreceptor; (b) to provide a biosensor for optical detection of a target substance including hydrogen peroxide and (c) a discoloration reagent.

Another object of the present invention is (a) a working electrode to which a first bioreceptor specifically binding to a target material is fixed; (b) a complex of Prussian blue nanoparticles and a second bioreceptor specifically binding to a target substance; And (c) to provide a biosensor for electrochemical detection of a target substance containing hydrogen peroxide.

Another object of the present invention is the inlet into which the test substance is introduced (Inlet); An optical detection unit including an absorbent substrate in which the discoloration reagent is absorbed; an electrochemical detection unit including a working electrode to which a first bioreceptor specifically binding to a target substance is fixed; a first passage connecting the inlet and the optical detector; and a second flow path connecting the inlet and the electrochemical detection unit, wherein the test material is (a) Prussian blue nanoparticles and a second biosensor that specifically binds to the target material. complex of receptors; (b) to provide a biosensor that is a mixture of hydrogen peroxide and (c) a sample.

In order to achieve the above object of the present invention, the present invention (a) a complex of Prussian blue nanoparticles and bioreceptors (Bioreceptor); It provides a biosensor for optical detection of a target material, including (b) hydrogen peroxide and (c) a discoloration reagent.

In order to achieve another object of the present invention, the present invention provides (a) a working electrode to which a first bioreceptor specifically binding to a target material is fixed; (b) a complex of Prussian blue nanoparticles and a second bioreceptor specifically binding to a target substance; And (c) it provides a biosensor for electrochemical detection of a target substance containing hydrogen peroxide.

In order to achieve another object of the present invention, the present invention is an inlet into which the test substance is introduced; An optical detection unit including an absorbent substrate in which the discoloration reagent is absorbed; an electrochemical detection unit including a working electrode to which a first bioreceptor specifically binding to a target substance is fixed; a first passage connecting the inlet and the optical detector; and a second flow path connecting the inlet and the electrochemical detection unit, wherein the test material is (a) Prussian blue nanoparticles and a second biosensor that specifically binds to the target material. complex of receptors; (b) hydrogen peroxide and (c) a mixture of the sample.

Hereinafter, the present invention will be described in detail.

The present invention relates to (a) a complex of Prussian blue nanoparticles and a bioreceptor; It provides a biosensor for optical detection of a target material, including (b) hydrogen peroxide and (c) a discoloration reagent.

In the present invention, the 'Prussian blue nanoparticles' refers to a hydrate of iron ferrocyanide, and can be represented by the chemical formula of Fe ₇ (CN) ₁₈ (H ₂ O) _x , and exhibits a deep blue color. . Prussian blue nanoparticles can be prepared by adding iron chloride to a ferrocyanide salt (sodium ferrocyanide, potassium ferrocyanide, ammonium ferrocyanide, etc.) solution.

As a non-limiting example of the method for preparing the Prussian blue nanoparticles, a compound containing a ferricyanide ion ([Fe(CN) ₆ ] ^3- ) and a compound containing an iron ion (Fe ³⁺ ) are mixed to form a mixture. manufacturing; and preparing Prussian blue nanoparticles by introducing the mixture into a solvent and reacting the mixture. The compound containing the ferricyanide ion ([Fe(CN) ₆ ] ^3- ) may be potassium ferricyanide (K ₃ Fe(CN) ₆ ) or sodium ferricyanide (Na ₃ Fe(CN) ₆ ). The compound containing iron ions (Fe ³⁺ ) is at least one selected from the group consisting of iron chloride (FeCl ₃ ), iron perchlorate (Fe(ClO ₄ ) ₃ ) and iron nitrate (Fe(NO ₃ ) ₃ ). it could be The solvent may be water

In the present invention, the bioreceptor is a biological recognition element capable of selectively binding to a sample material to be detected, and may be an antibody, enzyme, antigen, aptamer, lectin, nucleic acid, protein, lipid, sugar, hormone receptor, or a combination thereof Yes, it may preferably be an antibody, but is not limited thereto. Binding and reactions between target substances and bioreceptors include antigen-antibody reactions, enzyme-substrate reactions, complementary binding of nucleic acids, and the like.

The 'antibody' refers to an immunoglobulin molecule and an immune active portion of an immunoglobulin molecule (ie, a molecule containing an antigen binding site that immuno-specifically binds an antigen). The term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, Humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide linked Fv, scFv, single domain antibody (dAb), diabody, multispecific antibody, bispecific antibody, anti-idiotypic antibody , bispecific antibodies, functionally active epitope-binding fragments thereof, full-length antibodies and portions thereof, including bifunctional hybrid antibodies. The antibody may be of any type (eg IgG, IgA, IgM, IgE or IgD). In addition, the antibody may be non-human (eg, from a mouse, goat or any other animal), fully human, humanized, or chimeric antibody. 'Nucleic acid' includes DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and aptamer, and forms a double helix structure through complementary bonding of base pairs. An 'aptamer' is a single-stranded nucleic acid that binds to a specific substance. It selectively binds to peptides, proteins, cells, drugs, etc. It is utilized.

In the present invention, the 'complex' can be interpreted in the same sense as a conjugate, conjugate, or complex, and means a state in which Prussian blue nanoparticles and bioreceptors are bound by an arbitrary bond . The Prussian blue nanoparticle and the bioreceptor may be indirectly bonded through a linker, polymer, or the like, or may be directly bonded.

In the present invention, the 'target substance' may be one or more selected from proteins, peptides, aptamers, nucleic acids, oligosaccharides, amino acids, carbohydrates, dissolved gases, sulfur oxide gases, nitrogen oxide gases, residual pesticides, heavy metals, and environmentally hazardous substances. , but is not limited thereto.

In the present invention, the discoloration reagent includes a color-developing substrate of oxidase (HRP, horseradish peroxidase), for example, ABTS (2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), TMB (3,3', Selected from the group consisting of 5,5'-Tetramethylbenzidine), AEC (3-Amino-9-Ethylcarbazol), DAB ((3,3'-diaminobenzidine tetrahydrochloride salt), OPD (o-phenylenediamine dihydrochloride), Luminol and Emplex Red It may be characterized as being, but is not limited thereto.

In one aspect of the present invention, the discoloration reagent may be provided in a dried state after being absorbed into an absorbent material. The absorbent material is provided in a dried state by evaporating the solvent after absorbing the discoloration reagent, and then absorbs moisture when in contact with the test substance containing the sample to be detected, and the reaction between the test substance and the discoloration reagent may occur If so, the type is not particularly limited. In the present invention, the absorbent material may be preferably a polymer film, paper, and specifically filter paper.

In the present invention, since the Prussian blue nanoparticles included in the complex exhibit a peroxidase-like activity, inducing an oxidation-reduction reaction between the hydrogen peroxide and the discoloration reagent to produce an oxidized discoloration reagent product, etc., which can be observed with the naked eye. Indicates possible color changes.

The biosensor provided by the present invention is characterized by optically detecting a target substance in a sample by detecting a discoloration reaction that occurs when the sample reacted with (a) and (b) comes into contact with (c).

In one aspect of the present invention, the biosensor may be characterized in that it determines that the target substance is present in the sample when the discoloration intensity is weak compared to a control sample containing no target substance. In the control sample without target substance, there is no target substance capable of reacting with the bioreceptor included in the complex, so that the Prussian blue nanoparticles induce a redox reaction of the discoloration reagent. Peroxidase-like activity can be sufficiently exhibited. there is. However, when the sample contains a target substance that is specifically recognized by the bioreceptor, a complex is formed between the target substance and the bioreceptor to reduce the enzyme-like activity of the Prussian blue nanoparticles, so the target substance is not included. Compared to the control group, the intensity of discoloration appears weak. With this principle, it is possible to optically or qualitatively confirm the presence or absence of a target substance in a sample to be detected.

In this case, it is preferable that the amount of the complex and the discoloration reagent mixed with the untreated control sample is the same as the amount of the complex and the discoloration reagent mixed with the sample to be detected.

In the present invention, the type of the 'sample' is not particularly limited as long as it is a target material for which the presence or absence of a target material is to be confirmed, and may include, for example, a biological sample, a food sample, an environmental material sample, and an air material sample. . Preferably, the sample may be a biological sample, and the biological sample includes saliva, sweat, urine, blood, plasma, serum, tears, pus, gastric fluid, intestinal fluid, ocular fluid, peritoneal fluid, vaginal fluid, cerebrospinal fluid, and body cavity fluid. can include

In the present invention, when the samples (a) and (b) are mixed, it is preferable that the (a) is mixed sufficiently to remain after reacting with all of the target substances included in the sample. Since the amount of the complex agent sufficient to remain after reacting with the target material may vary depending on the type of sample and the ratio of bioreceptors included in the complex, the amount of the complex agent mixed by the experimenter depends on the type of target material and sample to be detected. It is possible to regulate Preferably, after determining the amount of the complex to be added using a reference sample of which the absolute amount of the target substance is known, it can be standardized and used for detection and quantification of the target substance.

The biosensor provided by the present invention does not require an additional bioreceptor other than the bioreceptor bound to the Prussian blue to detect a target substance, and does not require a washing or washing step. There is a difference between technology and technology. Due to the characteristics of the present invention, the target material detection method of the present invention can be utilized to detect a target material in a sample very simply and quickly in the field.

The present invention also relates to (a) a working electrode to which a first bioreceptor specifically binding to a target material is immobilized; (b) a complex of Prussian blue nanoparticles and a second bioreceptor specifically binding to a target substance; And (c) it provides a biosensor for electrochemical detection of a target substance containing hydrogen peroxide.

In the present invention, the first bioreceptor may be directly or indirectly fixed to the working electrode. In the present disclosure, direct fixation or coupling means that two objects are fixed or coupled without the existence of a separate medium, material, or object between the two objects. Also, indirect fixation or coupling means that a separate medium, material, or object exists between two objects to fix or combine them. In the present invention, the first bioreceptor in (a) may be fixed to the working electrode by at least one of physical adsorption, entrapping, and covalent bonding.

In the present invention, the 'working electrode' may be a metal oxide electrode, a metal electrode, a conductive polymer electrode or a carbon electrode.

The metal oxide electrode is ITO (indium tin oxide), IGO (indium gallium oxide), IZO (indium zinc oxide), ATO (antimony tin oxide), AZO (antimony zinc oxide), FTO (fluorine doped tin oxide) and GIZO ( gallium indium zinc oxide), the metal electrode may include a material selected from the group consisting of mercury, amalgam, Pt, Au, Pd, and Rh, and the conductive polymer electrode polyacetylene, polypyrrole, polyaniline, poly(3,4-ethylene dioxythiophene) (PEDOT), polythiophene, poly para-phenylene, polyphenylene It may include a material selected from the group consisting of polyphenylene sulfide and polypara-phenylene vinylene, and the carbon electrode may include a noble metal electrode, a pyrolytic graphite electrode, and glassy carbon. It may be a carbon electrode, a carbon paste electrode, a carbon nanotube electrode, a graphene electrode, or a carbon glass electrode, but is not limited thereto.

Preferably, in the present invention, the working electrode may be an ITO electrode. In particular, in the case of an ITO electrode, when applied to a biosensor, noise signals can be reduced, and a substrate having an ITO thin film deposited on an organic substrate can be used as it is without a separate adhesive layer. The ITO substrate has the advantage that it can be manufactured as an electrode at a very low cost because the same process for manufacturing a thin film substrate having high and stable electrical conductivity can be used on a large substrate in the display industry.

In the present invention, the first bioreceptor and the second bioreceptor may independently apply the description of the bioreceptor described above, and are preferably bioreceptors that specifically bind to the same target material. More preferably, the first bioreceptor and the second bioreceptor specifically bind to the same target material, but the binding sites recognized by each bioreceptor are different from each other so that any one bioreceptor binds to the target material differently. It may not competitively inhibit the binding of a bioreceptor to a target substance.

The operating method of the biosensor for electrochemical detection provided by the present invention is as follows.

(b) a complex of Prussian blue nanoparticles and a second bioreceptor specifically binding to the target material; And (c) is mixed and reacted. When the target substance is present in the sample, the second bioreceptor of the complex of (b) and the target substance bind to form an antigen-antibody complex. When the reaction mixture is treated and reacted in (a), the target substance binds to the first bioreceptor in (a).

At this time, a voltage is applied to the working electrode to provide electrons to the Prussian blue nanoparticles connected to the electrode through the first bioreceptor and target material complex, or to provide electrons to the electrode, so that the electrochemical signal, current , voltage and resistance can occur.

When the target material is included in the sample, the Prussian blue nanoparticles and the second bioreceptor complex bind to the surface of the working electrode in a sandwich structure via the first bioreceptor and target material complex, and the applied Current can be measured by voltage. Since the Prussian blue nanoparticles and the second bioreceptor complex selectively bind to the target material and serve as an electron transfer medium, an additional electron transfer medium is not required.

The applied voltage may vary according to various experimental conditions. Since the oxidation-reduction voltage of water is 1.23V, the optimal oxidation-reduction voltage of the electrochemically active material in aqueous solution is determined below 1.23V. Preferably, the amount of applied voltage may be set between about 0.1 and 1V. When the voltage is less than 0.1V, a problem in that the coefficient of variation may increase due to insufficient voltage may occur. In addition, at a voltage of less than 0.1 V, since the oxidation current value may be unstable before a complete electrochemical reaction is obtained, the probability of a large error in the result values increases, which is not preferable. On the other hand, if the voltage exceeds 1V, the background signal greatly increases, making accurate measurement impossible, which is undesirable.

The voltage may be applied for 1 to 200 seconds, preferably for 5 to 80 seconds, and more preferably for 10 to 60 seconds. The application time is proportional to the measurement time, and the shorter it is, the better it is from the viewpoint of the purpose of on-site self-diagnosis. However, an application time of less than 1 second may have a large coefficient of variation and a large variation in S/N ratio during repeated experiments under the same conditions, which may be undesirable from the viewpoint of reproducibility.

The S/N ratio is an indicator of signal-to-noise of a current or charge value measured under the same conditions, and from the viewpoint of reproducibility, the more constant the S/N ratio is, the more preferable it is.

Electrochemical detection using the biosensor of the present invention can detect the target substance in the sample by comparing the current value measured in response to the applied voltage with a control sample that does not contain the target substance.

In measuring the current, a known electrochemical measurement method may be used. For example, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and amperometry may be used. The cyclic voltammetry is a method of measuring current by circulating a voltage applied to a working electrode at a constant speed, and through this method, a cyclic voltammetry curve can be obtained. DPV is a method of measuring current by applying a change in voltage to an electrode in the form of a pulse and obtaining a graph of the current value according to the changing voltage. The voltammetry is a method of measuring the current generated when a constant voltage is applied to an electrode, and the current value over time is obtained as a graph. Here, the electric signal to be measured means the maximum value of the current values shown in the respective CV and DPV curves or the current value stably obtained for a certain period of time in the voltammetric method.

In the present invention, when the measured current value is higher than the current value of the control sample without the target substance, it can be determined that the target substance is present in the sample.

In another aspect of the present invention, it can be determined that the higher the current value measured using the biosensor, the higher the concentration of the target substance in the sample.

Specifically, a standard sample of which the absolute amount of the target substance is known is detected by using the biosensor of the present invention, and a standard curve for the concentration and current value of the target substance is prepared based on the result. It may be possible to quantify the concentration of a target substance contained in a sample of

The present invention also relates to a substrate comprising a working electrode on which a first bioreceptor is fixed; Prussian blue nanoparticles and a second bioreceptor complex; And it provides a target material detection kit comprising a current meter.

In the kit, any one selected from the group consisting of an internal control that does not bind to the first bioreceptor and the second bioreceptor and does not affect the electrochemical signal, a target substance by concentration required for preparing a standard curve, and a combination thereof is added. can be included as

As described above, the kit provided by the present invention can also be used for quantifying a target substance in a sample using the standard curve.

The present invention is also an inlet into which the test substance is introduced; An optical detection unit including an absorbent substrate in which the discoloration reagent is absorbed; an electrochemical detection unit including a working electrode to which a first bioreceptor specifically binding to a target substance is fixed; a first passage connecting the inlet and the optical detector; and a second flow path connecting the inlet and the electrochemical detection unit, wherein the test material is (a) Prussian blue nanoparticles and a second biosensor that specifically binds to the target material. complex of receptors; (b) hydrogen peroxide and (c) a mixture of the sample.

The biosensor of the present invention may further include an ammeter connected to the electrochemical detection unit.

The method of operating the biosensor provided by the present invention is as follows.

The test material may include (a) a complex of Prussian blue nanoparticles and a second bioreceptor specifically binding to a target material; (b) a mixture of hydrogen peroxide and (c) a sample, and when a target substance exists in the sample, the second bioreceptor binds to the target substance.

When the test material is injected into the inlet, the test material reaches the optical detection unit and the electrochemical detection unit along the first and second passages according to the capillary phenomenon.

When the target material combined with the Prussian Blue nanoparticle-second bioreceptor complex present in the test material is absorbed into the absorbent material of the optical detection unit, the discoloration reagent contained in the absorbent material and hydrogen peroxide and Prussian Blue in the test material It reacts to show a discoloration reaction, and the presence or absence of the target substance in the sample can be confirmed by visually observing the degree of discoloration.

In addition, when the target substance bound to the Prussian blue nanoparticle-second bioreceptor complex present in the test substance reaches the electrochemical detection unit and binds to the first bioreceptor, a change in current occurs according to the applied voltage. and the presence and concentration of the target substance in the sample can be confirmed by measuring the change in current through an ammeter.

That is, the biosensor provided by the present invention detects the target in the sample only by injecting one test substance, that is, a mixture of a sample to be checked for the presence of a target substance, hydrogen peroxide, and a Prussian blue-bioreceptor complex into an inlet. It has the advantage of being able to check the presence or absence of a substance and its amount very easily and conveniently.

In addition, the present invention,

(a) adding a sample to a working electrode on which a first bioreceptor specifically binding to a target substance to be detected is immobilized;

(b) adding a complex of Prussian blue nanoparticles and a second bioreceptor to the sample;

(c) applying a voltage to the working electrode; and

(d) a method for detecting a target substance in a sample is provided, comprising the step of comparing the current value measured corresponding to the voltage with the current value of an untreated control sample.

In the method of the present invention, a quantification step of calculating the concentration of the target substance in the sample using a standard curve of the concentration-current value of the target substance may be additionally performed after step (d), It can be determined that the higher the current value measured according to the above method, the higher the concentration of the target substance in the sample.

The present invention also relates to (i) a complex of Prussian blue nanoparticles and a bioreceptor; and a first kit including a discoloration reagent and (ii) a substrate including a working electrode on which the first bioreceptor is immobilized; Prussian blue nanoparticles and a second bioreceptor complex; And it provides a target material qualitative and quantitative analysis kit including a second kit including an ampereometer.

Using the kit, it is possible to quickly and conveniently check the presence and amount of a target substance to be detected in a sample through optical and electrochemical methods, and the same Prussian blue nanoparticle and bioreceptor complex are used in the first kit. And it can be used in both kits, so it has a very high utilization feature.

The present invention also provides an optical target sample detection method using the peroxidase-like activity of Prussian blue and an electrochemical target sample detection method using the activity of Prussian blue as an electron transport medium, thereby detecting the target substance in the sample. It provides a method for qualitative and quantitative analysis simultaneously by optical and electrochemical methods.

Using a biosensor based on Prussian blue nanoparticles and bioreceptors, it is possible to qualitatively and quantitatively analyze the target substances included in the sample to be detected very quickly and conveniently using optical and electrochemical methods, which is used for research and development, diagnosis of diseases, etc. can be very useful in the field of

1 is a view showing an image of Prussian blue nanoparticles of the present invention.

2 is a view showing the results of X-ray diffraction analysis of Prussian blue nanoparticles and a substrate.

3 is a view for explaining the peroxidase-like activity of Prussian blue nanoparticles and the resulting color change according to the presence or absence of a target substance. It has a structure in which an antibody is bound to the surface of Prussian blue nanoparticles, where the antibody, which is a bioreceptor, selectively binds to a target substance.

4a and 4b are diagrams showing the results of optical detection of two types of proteins using the peroxidase-like activity of Prussian blue nanoparticles. In FIG. 4a, protein A (CD9) is the target material. When there is no protein A in the diagnostic sample, when TMB and hydrogen peroxide are added by peroxidase-like activity, oxidation of TMB occurs and the color is displayed. However, when protein A is present, the antibody and protein A bind on the surface of the Prussian blue nanoparticles, blocking the peroxidase-like activity of the Prussian blue nanoparticles, so that the oxidation reaction of TMB does not occur, and the color reaction is suppressed. Figure 4b is the result of observing the color change using protein B (neuropeptide FF receptor 2; NPFFR2) as a target material, and it was confirmed that the color faded as the concentration of protein B increased.

5 is a view for explaining the electrochemical oxidation-reduction reaction of Prussian blue nanoparticles and the detection of a target material resulting therefrom.

6a and 6b are views showing results of electrochemical detection of two types of proteins using electrochemical oxidation-reduction of Prussian blue nanoparticles. 6a shows the comparison of oxidation current according to the presence or absence of protein A. When protein A is not present in the electrolyte (1 mM potassium chloride, pH 7.0), oxidation of Prussian blue nanoparticles does not occur on the surface of the electrode. No current is observed. However, when a target substance is present, antibody and protein are bound by an antigen-antibody reaction on the surface of the working electrode, and when an additional Prussian blue nanoparticle-antibody complex is added, a sandwich structure is formed and Prussian blue nanoparticles are formed. bonded to the surface of the working electrode. As a result, the Prussian blue nanoparticles are oxidized on the surface of the electrode, resulting in a high current value. 6B shows the result of measuring the change in current according to the concentration of protein B. As the concentration increased, the number of Prussian blue nanoparticles bonded to the surface of the working electrode in a sandwich structure increased, confirming that the oxidation current increased.

7 is an exploded perspective view of a biosensor according to an embodiment of the present invention. The plurality of film layers may be formed by alternately stacking hydrophobic films and hydrophilic films having adhesive surfaces. A liquid sample may be injected from the inlet and moved to the detection unit by capillary action by being exposed to the hydrophilic film. It includes an inlet and an optical detection unit treated with a TMB color reagent, a working electrode treated with a first antibody, an electrochemical detection unit formed with a counter electrode and a reference electrode, and an absorption pad.

8 is a view for explaining a method of optically detecting CA19-9 and CEA as target substances using a paper layer treated with a TMB color developing reagent. The peroxidase-like activity of Prussian blue nanoparticles and the resulting color change depending on the presence or absence of target substances (tumor markers, CA19-9 and CEA) are shown. It has a structure in which an antibody is bound to the surface of Prussian blue nanoparticles, where the antibody selectively binds to a tumor marker (antigen). When there is no tumor marker in the diagnostic sample, when TMB and hydrogen peroxide are added by a peroxidase-like activity, an oxidation reaction of TMB occurs, resulting in a color. However, when the tumor marker is present, it binds to the antibody on the surface of the Prussian blue nanoparticles, blocks the peroxidase-like activity of the Prussian blue nanoparticles, and prevents the oxidation reaction of TMB, suppressing the color reaction.

9 is a view for explaining the electrochemical oxidation-reduction reaction of Prussian blue nanoparticles and the resulting detection of tumor markers on the surface of the working electrode of the biosensor of the present invention. When a tumor marker is present in the sample, the tumor marker bound to the Prussian blue nanoparticle-antibody complex contained in the electrolyte (1 mM potassium chloride containing 1% hydrogen peroxide) reacts with the first antibody on the surface of the working electrode and the antigen-antibody are combined to form a sandwich structure. Due to the voltage applied to the working electrode, Prussian blue nanoparticles are oxidized on the surface of the electrode, resulting in a high current value.

10A and 10B are CV and DPV graphs according to the composition of the electrolyte solution. Current values observed when the Prussian blue nanoparticle-antibody complex and the tumor marker are combined in a sandwich structure by an antigen-antibody reaction on the surface of the working electrode on which the first antibody is formed are shown. The result shows the difference in oxidation current values of Prussian blue nanoparticles between a 1 mM potassium chloride solution containing 1% hydrogen peroxide and a 1 mM potassium chloride solution without hydrogen peroxide. When 1% hydrogen peroxide is included in the electrolyte, a high oxidation peak is observed around 0.15 V. also. When hydrogen peroxide is included, an improved current value can be obtained by the catalytic activity of Prussian blue nanoparticles against hydrogen peroxide.

11a and 11b are views showing the detection results of CA19-9 using an optical and electrochemical sensor based on Prussian blue nanoparticles. In FIG. 11a , it can be confirmed that the color reaction generated in the optical detector decreases as the concentration of CA19-9 increases, and when the color change of the optical detector according to the concentration of CA19-9 is shown, a decreasing graph can be obtained. The linear interval is 2.3-37.5 U/mL, and the detection limit is 1.37 U/mL. In Figure 11b, the change in current value according to the concentration of CA19-9 was measured. When a voltage of 0.2 V was applied to the working electrode for 120 seconds using the amperometry method, the value of the oxidation current of the Prussian blue nanoparticles measured on the electrode surface was shown as a graph according to the concentration of CA19-9. As the concentration of CA19-9 increased, the measured oxidation current also increased linearly, and the linear range at this time was 1-75 U/mL, and the detection limit was 0.78 U/mL.

12a and 12b are diagrams showing CEA detection results using an optical and electrochemical sensor based on Prussian blue nanoparticles. In FIG. 12a, the color reaction of the optical detector was measured as the concentration of CEA was changed from 0 to 1000 pg/mL. As the concentration increased, the color change observed in the optical detection unit became lighter, and the detection limit obtained when this was depicted was 5.66 pg/mL. 12B shows the result of electrochemical detection of CEA, and shows the current value measured when a voltage of 0.2 V was applied to the working electrode for 100 seconds using the Amperometry method. As the concentration of CEA increased from 0 to 500 pg/mL, the current value increased linearly, and the detection limit obtained at this time was 7.5 pg/mL.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1: Synthesis of Prussian Blue nanoparticle-antibody complex

1-1: Solution used for nanoparticle-antibody complex synthesis

① 0.05% Chitosan in 0.2 M HCl: 1 mL

② 4% FeCl3 in solution in ①: 100 uL

③ 2% Potassium ferrocyanide in 0.2 M HCl: 100 uL

④ 2% FeCl2 in 0.2 M HCl: 100 uL

⑤ 0.2 M NaOH: 1 mL

⑥ 4% Glutaraldehyde in sodium phosphate buffer (pH 7.0): 100 uL

⑦ 10 μg/mL Anti-CD9 antibody solution

⑧ 0.1% skimmilk

1-2: Nanoparticle-antibody complex synthesis method

4% iron(III) chloride and 2% potassium ferrocyanide were prepared using a 0.2 M HCl solution containing 0.05% chitosan (② and ③), and each solution was mixed in a 1:1 volume ratio. React for 20 minutes at 60°C. Then, 2% (v/v) iron chloride (±) (④) was added in a 1:1 volume ratio and reacted at 60°C for 10 minutes. Then, a 0.2M NaOH solution (⑤) was slowly added in a volume ratio of 1:3 while ultrasonicating to the solution, reacted at 60° C. for 10 minutes, and then washed 5 times with distilled water. Through this, Prussian blue nanoparticles were synthesized.

The prepared Prussian blue nanoparticles were reacted with 4% (v/v) glutaraldehyde (⑥) at room temperature for 2 hours to treat the aldehyde on the surface, and then washed three times with distilled water. Then, it is mixed with 10 μg/mL CA19-9 antibody, 2 μg/mL CEA antibody solution, or 10 μg/mL Anti-CD9 antibody solution (⑦) at a volume ratio of 1:1, and reacted at 4° C. for 30 minutes. After washing three times with distilled water, a 0.1% skimmilk solution was added to prevent non-specific binding, followed by treatment at 4° C. for 30 minutes. After washing three times with distilled water, it was refrigerated at 4°C. Through this, antibody-coupled Prussian blue nanoparticle-antibody complexes were synthesized.

Example 2: Optical detection method using peroxidase-like activity of Prussian blue nanoparticles

2-1: Solution for measuring peroxidase-like activity

① 0.15 mg/mL TMB in PC buffer: 100 uL

② 0.5% H ₂ O ₂ : 100 uL

③ CD9 antigen solution (0, 1, 10 μg/mL)

2-1: Method for measuring peroxidase-like activity

(1) To investigate the peroxidase-like activity of Prussian blue nanoparticles, a reaction solution containing 0.15 mg/mL TMB (①) and 0.5% hydrogen peroxide (②) was prepared. Protein A (2.417 μg/mL) (CD9 antigen solution (③)) was mixed with the Prussian blue nanoparticle-antibody complex in a volume ratio of 1:1 and reacted at room temperature for 30 minutes. At the same time, a blank solution that does not contain protein A (CD9 antigen solution) is mixed with the Prussian blue nanoparticle-antibody complex in a volume ratio of 1:1 and reacted at room temperature for 30 minutes. The above solution was mixed with the substrate solution (①+②) in a volume ratio of 1:10, and the color change after 1 minute was measured. When protein A (CD9 antigen) binds to the surface of the Prussian blue nanoparticle-antibody complex, the peroxidase-like activity of the Prussian blue nanoparticles is lowered, thereby inhibiting the color reaction caused by TMB oxidation. As a result, as shown in FIG. 4a, a clear difference in color development according to the presence or absence of protein A can be confirmed.

(2) As shown in FIG. 4B, the concentration of protein B (NPFFR2 antigen) was changed to 0, 1, and 10 μg/mL, and mixed with the Prussian blue nanoparticle-antibody complex at a volume ratio of 1:1, followed by mixing at room temperature for 30 μg/mL. Respond in minutes. The above solution was mixed with the substrate solution at a volume ratio of 1:10, and the color changed after 1 minute was measured. It was confirmed that the higher the concentration of protein B bound to the surface of the Prussian blue nanoparticle-antibody complex, the lower the intensity of color development by TMB oxidation.

Example 3: Fabrication of a working electrode (WE) for electrochemical detection

3-1: Solution for electrode fabrication

① 1% Chitosan containing in 1X acetate buffer: 1 mL

② 2% Glutaraldehyde in sodium phosphate buffer (pH 7.0): 1 mL

③ 10 μg/mL Anti-CD9 antibody solution

3-2: Method for fabricating electrodes

ITO-coated PET (ITO-PET) was purchased from Sigma-aldrich. ITO-PET is prepared so that the area of WE is 0.5 cm ² . Prepare a 1% chitosan solution (①) using 1X acetate buffer, drop 40 uL on the surface of each working electrode, and treat at room temperature for 1 hour. The working electrode whose surface is treated with amine using 1% chitosan solution is washed with distilled water and nitrogen gas. React with 2% glutaraldehyde (②) at room temperature for 2 hours, and wash with distilled water and nitrogen gas. Antibodies are formed on the surface of the working electrode by reacting with the antibody solution at 4° C. for 12 hours. After washing with distilled water and nitrogen gas, a 0.1% skimmilk solution was added and treated at 4° C. for 30 minutes. Thereafter, the working electrode was washed with distilled water and nitrogen gas and stored in a refrigerator at 4°C.

Example 4: Electrochemical Detection Method Using Electrochemical Oxidation-Reduction Characteristics of Prussian Blue Nanoparticles

4-1: Solution for electrochemical redox measurement

① 1% H ₂ O ₂ in 1 mM KCl solution (PBS buffer pH 4.0)

4-2: Method for measuring electrochemical redox

(1) Treat the antibody-formed working electrode with protein A (2.417 μg/mL) (CD9 antigen) at room temperature for 30 minutes, and then wash with distilled water and nitrogen gas. Then, the Prussian blue nanoparticle-antibody complex was treated for 30 minutes to form a sandwich structure, the Prussian blue nanoparticle-antibody-protein A-antibody-electrode structure (FIG. 5). After washing with distilled water and nitrogen gas, the oxidation-reduction current of the Prussian blue nanoparticles was measured using a 1 mM potassium chloride solution (①). 0.2 V applied to the working electrode using the Amperometry method. A voltage is applied for 120 seconds to obtain a current value. When protein A binds to the surface of the working electrode to form a sandwich structure with the Prussian blue nanoparticle-antibody complex, the Prussian blue nanoparticles are oxidized on the surface of the electrode to show a high current value. In Figure 6a, the change in current value according to the presence or absence of protein A was confirmed. The current value in the presence of protein A (CD9 antigen) shows a peak value at 0.2V.

(2) In FIG. 6B , protein B (NPFFR2) is treated at a concentration of 0, 1, or 10 µg/mL at room temperature for 30 minutes to bind to the antibody on the working electrode. After washing with distilled water and nitrogen gas, the Prussian blue nanoparticle-antibody complex was treated for 30 minutes to form a sandwich structure of Prussian blue nanoparticle-antibody-protein B-antibody-electrode structure. Subsequently, the oxidation-reduction current of the Prussian blue nanoparticles was measured using a 1 mM potassium chloride solution. A voltage of 0.2V is applied to the working electrode for 120 seconds using the amperometry method to obtain the current value. Through this, the change in the oxidation current value of the Prussian blue nanoparticles according to the change in the protein B concentration was confirmed.

Example 5: Fabrication of optical and electrochemical sensors

(1) FIG. 7 is an exploded perspective view of an optical and electrochemical sensor for detecting multiple tumor markers according to an embodiment of the present invention.

The sensor in the present invention includes an inlet and an optical detection unit treated with TMB color reagent, a working electrode treated with the first antibody, an electrochemical detection unit formed with a counter electrode and a reference electrode, and an absorption pad.

In one embodiment, the plurality of film layers may be formed by alternately stacking hydrophobic films and hydrophilic films having adhesive surfaces. A liquid sample may be injected from the inlet and moved to the detection unit by capillary action by being exposed to the hydrophilic film.

(2) FIG. 8 is a picture of a paper layer treated with a TMB coloring reagent located in the optical detection unit.

A circular filter paper with a diameter of 0.6 cm was prepared, 10 μL of TMB color reagent was added, and then dried in an oven at 30 ° C for 30 minutes. The concentration of the TMB color reagent is prepared as 0.15 mg/mL for optical detection of CA19-9 or 0.6 mg/mL for optical detection of CEA. A completely dry TMB paper layer is used by attaching it to the optical detection part of the sensor.

(3) FIG. 9 is a drawing of a working electrode located in an electrochemical detector in the sensor of the present invention.

ITO-coated PET (ITO-PET) is prepared so that the area of the exposed portion is 0.5 cm ² . Prepare a 1% chitosan solution using 1 X acetate buffer solution, and treat the surface of each working electrode at room temperature for 1 hour. The working electrode whose surface is treated with amine using 1% chitosan solution is washed with distilled water and nitrogen gas. React with 2% glutaraldehyde at room temperature for 2 hours, and wash with distilled water and nitrogen gas. The antibody is formed on the surface of the working electrode by reacting the first antibody solution at 4° C. for 12 hours. The concentration of the first antibody solution is 0.12 μg/mL CA19-9 antibody or 4 μg/mL CEA antibody solution. Thereafter, the working electrode is washed with distilled water and nitrogen gas and attached to the electrochemical detection unit of the sensor of the present invention.

Example 6: Detection of CA19-9 and CEA using optical and electrochemical sensors based on Prussian blue nanoparticles

(1) A solution containing a tumor marker is mixed with a Prussian blue nanoparticle-antibody complex in a volume ratio of 1:1 and reacted at room temperature for 30 minutes. The above solution was mixed with a 1 mM potassium chloride solution containing 1% hydrogen peroxide at a volume ratio of 1:1 to prepare a 1% hydrogen peroxide solution containing a tumor marker bound to the Prussian blue nanoparticle-antibody complex, and add to

(2) The added sample reaches the optical detection unit, and in order to measure the color change, the optical detection unit is photographed after 1 minute. The image taken at this time is converted into a black and white image using the ImageJ program, and then the intensity of the color is measured. When a tumor marker binds to the surface of the Prussian blue nanoparticle-antibody complex, the peroxidase-like activity of the Prussian blue nanoparticles is lowered, thereby inhibiting the color reaction caused by TMB oxidation. As shown in FIGS. 11A and 12A, it was confirmed that there was a clear difference in color development according to the presence or absence of the tumor marker, and the higher the concentration of the tumor marker, the lower the intensity of color development due to TMB oxidation.

(3) The sample reaching the electrochemical detector is reacted on the surface of the electrode for 30 minutes to form a sandwich structure, Prussian Blue nanoparticle-antibody-protein A-antibody-electrode structure. Using the amperometry method, a voltage of 0.2 V is applied to the working electrode for 120 seconds to obtain a current value. 11b and 12b, when the oxidation-reduction current of the Prussian blue nanoparticles sandwiched on the surface of the working electrode was measured, the oxidation current value of the Prussian blue nanoparticles according to the concentration change of the tumor marker Changes were confirmed.

Using a biosensor based on Prussian blue nanoparticles and bioreceptors, it is possible to qualitatively and quantitatively analyze the target substances included in the sample to be detected very quickly and conveniently using optical and electrochemical methods, which is used for research and development, diagnosis of diseases, etc. It can be used very usefully in the field of industrial use.

Claims (15)

1. (a) a composite of Prussian blue nanoparticles and a bioreceptor; (b) hydrogen peroxide and (c) a color changing reagent, a biosensor for optical detection of a target substance.
2. The biosensor according to claim 1, wherein the color changing reagent comprises a color-developing substrate of horseradish peroxidase (HRP).
3. The method of claim 2, wherein the oxidase chromogenic substrate is ABTS (2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), TMB (3,3',5,5'-Tetramethylbenzidine), AEC (3- A biosensor, characterized in that it is selected from the group consisting of Amino-9-Ethylcarbazol), DAB ((3,3'-diaminobenzidine tetrahydrochloride salt), OPD (o-phenylenediamine dihydrochloride), luminol and Emplex Red.
4. The biosensor according to claim 1, wherein the discoloration reagent is absorbed into an absorbent material and provided in a dried state.
5. The biosensor according to claim 1, wherein the target substance in the sample is optically detected by detecting a discoloration reaction that occurs when the sample reacted with (a) and (b) comes into contact with (c).
6. 6. The biosensor according to claim 5, wherein it is determined that the target substance is present in the sample when the discoloration intensity of (c) is weak compared to the control sample without the target substance.
7. The biosensor according to claim 5, wherein the sample is a biological sample.
8. The biosensor according to claim 1, wherein the bioreceptor is an antibody, enzyme, antigen, aptamer, lectin, nucleic acid, protein, lipid, sugar, hormone receptor, or a combination thereof.
9. (a) a working electrode to which a first bioreceptor that specifically binds to a target substance is fixed; (b) a complex of Prussian blue nanoparticles and a second bioreceptor specifically binding to a target substance; And (c) a biosensor for electrochemical detection of a target material containing hydrogen peroxide.
10. The method of claim 9, wherein the target substance in the sample is measured by measuring the change in the current value of the working electrode that appears when the target substance in the sample reacted with (b) and (c) is captured by the first bioreceptor of (a). A biosensor characterized in that for electrochemically detecting.
11. 10. The biosensor according to claim 9, wherein the working electrode is a metal oxide electrode, a metal electrode, a conductive polymer electrode or a carbon electrode.
12. 10. The method of claim 9, wherein the first bioreceptor is fixed to the working electrode by at least one method selected from the group consisting of physical adsorption, entrapping method, and covalent bonding method. A biosensor with
13. 11. The biosensor according to claim 10, wherein when the measured current value is higher than that of a control sample containing no target substance, it is determined that the target substance is present in the sample.
14. Inlet through which the test substance is introduced;

    An optical detection unit including an absorbent substrate in which the discoloration reagent is absorbed;

    an electrochemical detection unit including a working electrode to which a first bioreceptor specifically binding to a target substance is fixed;

    a first passage connecting the inlet and the optical detector; and

    A biosensor including a second flow path connecting the inlet and the electrochemical detection unit,

    The test material may include (a) a complex of Prussian blue nanoparticles and a second bioreceptor specifically binding to the target material; A biosensor that is a mixture of (b) hydrogen peroxide and (c) a sample.
15. 15. The biosensor according to claim 14, further comprising an ammeter connected to the electrochemical detection unit.

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