Novel electron transfer mediator and biosensor for detecting blood glucose comprising same

Abstract

The present invention relates to a novel electron transfer mediator and a biosensor for detecting blood glucose, comprising same and, more specifically, provides: a novel iron complex as an electron transfer mediator which can be applied to an enzyme-based biosensor for detecting blood glucose; and a biosensor for detecting blood glucose, using same. The biosensor according to the present invention has excellent sensitivity and improved performance compared to conventional blood glucose sensors, and thus can more precisely and accurately measure blood glucose.

Description

Novel electron transfer mediator and biosensor for blood glucose detection comprising the same

[0001] The present invention relates to a novel electron transfer mediator applicable to an enzyme-based blood glucose detection sensor and a blood glucose detection biosensor using the same.

[0002] Diabetes is a metabolic disorder related to the concentration of glucose in the body, primarily caused by abnormal blood sugar levels due to insufficient insulin production. Approximately 10% of the global population aged 20 to 80 has diabetes. Because this disease can lead to various complications, including heart disease, kidney dysfunction, retinopathy, obesity, and neuropathy, consistently and accurately monitoring blood glucose levels is crucial for the diagnosis and management of diabetes.

[0003] Numerous studies have been conducted to measure glucose, and since the publication of electrochemical glucose detection by Clark and Lyons in 1962, glucose sensors have been developed in three generations. First-generation glucose sensors were based on the amperometric detection of H2O2 generated during the enzymatic reaction. However, this required a high detection potential, making it susceptible to interference from various electrochemically active substances present in blood. Second-generation glucose sensors were developed to address the shortcomings of the first-generation sensors. To reduce the enzyme's dependence on oxygen, an additional electron transfer mediator was introduced to facilitate electron transfer from the enzyme surface to the measuring electrode. Third-generation glucose sensors directly transfer electrons from glucose to the measuring electrode through the enzyme's active site. Currently, most commercially available blood glucose monitoring systems are based on the second-generation approach. In second-generation glucose sensors, an electron transfer mediator transfers electrons between the enzyme's redox center and the electrode, enabling stable glucose detection.

[0004] Most glucose sensors use glucose oxidase (GOx) or glucose dehydrogenase (GDH) as the enzyme. However, GOx has the disadvantage of being vulnerable to use in commercial sensor strips due to its dependence on oxygen during the glucose oxidation process. GDH is easy to use because it can detect glucose without the need for oxygen. Among various types of GDH, FAD / GDH, an FAD-dependent GDH derived from fungi, is an enzyme that has recently attracted attention due to its low oxygen dependence and lack of electrochemical activity toward maltose. FAD / GDH-based glucose sensors exhibit high selectivity and stability, but the introduction of an additional efficient electron transfer mediator is necessary to fabricate a high-performance and stable sensor.

[0005] To date, a variety of advanced electron transfer mediators, including organic, inorganic, and organometallic compounds, have been developed. In particular, ferricyanide and ruthenium hexamine complexes are frequently used mediators. Ferricyanide has been widely used as a mediator since the 1990s due to its readily available availability, high solubility, and low cost. However, it requires a high redox potential and presents challenges during storage and distribution. Sensors using ruthenium hexamine exhibit low redox potentials, minimal interference, and smooth distribution. However, they require a co-mediator for use with FAD / GDH.

[0006] The purpose of the present invention is to provide a novel electron transfer medium for developing a blood sugar detection sensor in which the above problems are improved.

[0007] Another object of the present invention is to provide a blood sugar detection sensor with excellent performance by utilizing the novel electron transfer medium described above.

[0008] To achieve the above purpose, the present invention provides a novel iron complex represented by the following chemical formula 1 or a salt compound thereof:

[0009] [Chemical Formula 1]

[0010]

[0011] In the above chemical formula 1, R 1 , R 2 and R 3 may be the same or different, and may be selected from H, Na, Li, K, Cs, NH4, or (C1 to C2) alkyl.

[0012] The present invention provides a method for preparing the iron complex or a salt compound thereof, comprising the steps of: preparing a ligand solution by adding 3-amino-4-hydroxybenzene sulfonic acid (AHBS) to a basic solution purged with nitrogen; preparing an iron precursor solution by adding an iron precursor to distilled water purged with nitrogen; and adding the prepared ligand solution dropwise to the prepared iron precursor solution to obtain a reaction product.

[0013] The present invention provides a biosensor for blood sugar detection, comprising: a substrate; a working electrode and an auxiliary electrode disposed on the substrate; and an enzyme layer formed on the electrode and including an enzyme and an electron transfer mediator, wherein the electron transfer mediator is the iron complex or a salt compound thereof.

[0014] In addition, the present invention provides a blood sugar detection method, including a step of contacting the above-described blood sugar detection biosensor with a blood sample.

[0015] The biosensor for blood sugar detection according to the present invention utilizes a novel iron complex as an electron transfer medium, thereby enabling precise blood sugar measurement with superior sensitivity and improved performance compared to conventional blood sugar sensors.

[0016] In addition, the biosensor can measure blood sugar more accurately by suppressing the interference effects of various substances in the blood and increasing reproducibility and long-term stability, and can stably maintain sensing sensitivity and performance.

[0017] Figure 1 schematically illustrates a synthesis process of a novel electron transfer mediator (FeAHBS) according to one embodiment of the present invention.

[0018] Figure 2 shows (a) the Fourier transform-infrared spectroscopy (FT-IR) analysis results and (b) the X-ray photoelectron spectroscopy (XPS) analysis results of a novel electron transfer mediator synthesized according to Figure 1.

[0019] Figure 3 shows the element-specific XPS analysis results of the novel electron transfer mediator synthesized according to Figure 1, where (a) shows C 1s, (b) shows N 1s, (c) shows O 1s, and (d) shows Fe 2p.

[0020] Figure 4 is a schematic diagram of a blood sugar detection sensor using a novel electron transfer medium synthesized according to Figure 1.

[0021] Figure 5 shows the change in redox current according to the injection speed of a novel electron transfer medium according to an experimental example of the present invention.

[0022] FIG. 6 shows (a) the redox characteristics and (b) the oxidation change according to glucose concentration of a blood sugar detection sensor manufactured using a novel electron transfer medium according to another embodiment of the present invention.

[0023] Figure 7 shows a performance evaluation of a sensor for blood sugar detection according to the present invention under optimized glucose detection conditions, where (a) shows a change in the current method signal over time according to glucose concentration, and (b) shows a corresponding calibration curve.

[0024] Figure 8 shows an evaluation of (a, b) the interference effect, (c) stability, and (d) reproducibility of a blood sugar detection sensor according to the present invention.

[0025] Figure 9 shows the results of evaluation using actual samples.

[0026] Hereinafter, the present invention will be described in detail.

[0027]

[0028] In order to improve the above-described problems, the present inventor synthesized a novel electron transfer mediator capable of detecting glucose even at low redox potentials without problems during storage and distribution by forming a complex between a 3-amino-4-hydroxybenzene sulfonic acid (AHBS) ligand and Fe(III), and confirmed that a sensor strip using the synthesized novel electron transfer mediator exhibited excellent glucose detection performance even in the presence of interfering substances, thereby completing the present invention.

[0029]

[0030] The present invention provides a novel iron complex or a salt compound thereof.

[0031] Preferably, the iron complex or its salt compound can be represented by the following chemical formula 1:

[0032] [Chemical Formula 1]

[0033]

[0034] In the above chemical formula 1, R 1 , R 2 and R 3 may be the same or different, and may be selected from hydrogen, alkali metal, ammonium or (C1 to C4) alkyl, preferably, but not limited to, H, Na, Li, K, Cs, NH4, -CH3, or -CH2CH3.

[0035] The above iron complex or its salt compound may be in a form in which a ligand derived from 3-amino-4-hydroxy benzene sulfonic acid (AHBS) is bound to iron (Fe(III)), which is a central metal ion.

[0036] The above iron complex or its salt compound can be utilized as an electron transfer mediator that assists electron transfer between the electrode and the enzyme of an enzyme-based biosensor. The above iron complex or its salt compound can be used by being physically or chemically fixed on the electrode of the enzyme-based biosensor.

[0037] The above biosensor may be, but is not limited to, a sensor for detecting blood sugar.

[0038]

[0039] The present invention provides a method for producing the novel iron complex or its salt compound.

[0040] A method for producing an iron complex or a salt compound thereof according to the present invention may include the steps of: preparing a ligand solution by adding 3-amino-4-hydroxy benzene sulfonic acid (AHBS) to a basic solution purged with nitrogen; preparing an iron precursor solution by adding an iron precursor to distilled water purged with nitrogen; and adding the prepared ligand solution dropwise to the prepared iron precursor solution to obtain a reaction product.

[0041] The step of preparing the above ligand solution can be performed by adding AHBS to a basic solution, wherein the basic solution can be at least one selected from the group consisting of NaOH, KOH, CsOH and NH4OH, and preferably NaOH, but is not limited thereto.

[0042] The step of preparing the above iron precursor solution can be performed by adding a precursor capable of providing iron (III) to distilled water, wherein the iron precursor may be at least one selected from the group consisting of iron (III) nitrate, iron (III) chloride, iron (III) citrate, iron (III) acetylacetonate, iron (III) pyrophosphate, iron (III) tartrate, iron (III) bromide, and iron (III) sulfate, but is not limited thereto, and may include any precursor material capable of providing iron ions.

[0043] The step of obtaining the above reaction product can be performed by adding the prepared ligand solution dropwise to the prepared iron precursor solution at a reaction temperature of 0 to 100°C, stirring at the same temperature range for 2 to 24 hours to complete the reaction, and then recovering the reaction product by a method such as chromatography, centrifugation, freeze-drying, or reduced pressure evaporation.

[0044]

[0045] The present invention provides a biosensor for blood sugar detection comprising the novel iron complex or a salt compound thereof.

[0046] More specifically, a biosensor according to the present invention may include a substrate; a working electrode and an auxiliary electrode disposed on the substrate; and an enzyme layer formed on the electrode and including an enzyme and an electron transfer mediator, wherein the electron transfer mediator may include the novel iron complex described above or a salt compound thereof.

[0047] The above electrode may be selected from the group consisting of a carbon electrode, a gold electrode, a silver electrode, and a copper electrode, but is not limited thereto, and an electrode commonly used in the art may be used.

[0048] The enzyme may be changed depending on the target to be measured by the biosensor, and in the case of the biosensor for blood sugar detection according to the present invention, the enzyme may be at least one selected from the group consisting of glucose oxidase (GOx), glucose dehydrogenase (GDH), flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (FAD / GDH), glucose hexokinase, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase, and preferably FAD / GDH, but is not limited thereto.

[0049] The above biosensor uses Ru as the electron transfer medium. 3+ / Ru 2+ , Os 3+ / Os 2+ and Fc + / Fc may further include one or more selected from the group consisting of, and by additionally including this, blood sugar detection performance may be improved.

[0050]

[0051] The biosensor for blood sugar detection according to the present invention can suppress the interference effects caused by various substances in blood.

[0052] The various substances in the blood may be, but are not limited to, one or more selected from the group consisting of monosaccharides including mannose, lactose, xylose, and fructose; ascorbic acid (AA), uric acid (UA), dopamine (DA), and acetaminophen (AP).

[0053] In addition, the biosensor has high reproducibility and shows stable performance even after 6 months, so it may have improved reproducibility and long-term stability.

[0054]

[0055] In addition, the present invention provides a blood sugar detection method, including a step of contacting the above-described blood sugar detection biosensor with a blood sample.

[0056] The above blood sugar detection method can evaluate whether or not blood sugar is detected or the degree thereof using cyclic voltammetry or chronoamperometry.

[0057] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0058]

[0059] <Example 1> Synthesis of a novel electron transfer mediator (FeAHBS)

[0060] Through the following synthetic process, a novel electron transfer mediator (FeAHBS) was synthesized as shown in Fig. 1.

[0061] First, 0.1 to 5 g of 3-amino-4-hydroxybenzene sulfonic acid (AHBS) was prepared in 50 to 200 mL of a 1 to 50 mM NaOH solution sufficiently purged with nitrogen, and 0.1 to 5 g of a metal precursor capable of providing Fe(III) (including at least one of Iron(III) nitrate, Iron(III) chloride, Iron(III) citrate, Iron(III) acetylacetonate, Iron(III) pyrophosphate, Iron(III) tartrate, Iron(III) bromide, and Iron(III) sulfate) was prepared in 50 to 200 mL of distilled water sufficiently purged with nitrogen. A reactor was prepared by attaching a dropping funnel to a Schlenk flask, and then the Fe(III) solution was injected into the Schlenk flask and the ligand into the dropping funnel. The ligand solution was slowly added dropwise at a reaction temperature of 0 to 100°C. After the addition, the reaction was terminated by stirring at the same temperature for 2 to 24 hours, and the mediator was recovered by a method such as chromatography, centrifugation, freeze-drying, or reduced pressure evaporation.

[0062]

[0063] The novel electron transfer mediator synthesized by the above method was confirmed using FT-IR and XPS.

[0064] Referring to Figure 2(a), as can be seen from the black line, the ligand itself has a peak at 3260 cm -1 and 2940 cm -1In the case of the new mediator complexed with Fe, the -NH and -OH peaks were broadened and the overall FT-IR band positions shifted due to the coordination between the metal and the ligand. In addition, both species showed C=C stretching and S=O stretching bands at 1638 and 1357 cm, respectively. -1 . Referring to Fig. 2(b), the synthesized medium showed Fe, C, N, O, S, and Na as a result of XPS analysis, which was consistent with the element types of the synthesized medium.

[0065] Referring to Figure 3, in the C 1s drawing of (a), the CC sp of the ligand 2 (284.46 eV), CC sp 3 (285.11 eV), CN / CS (285.96 eV), CO (286.77 eV) bonds were observed, and in the N 1s diagram of (b), it was confirmed that the peaks of -NH2 = 398.40 eV, CN = 400.01 eV shown in the ligand itself shifted to -NH peak = 399.01 eV, CN peak = 400.13 eV after the complex. In the case of O 1s of (c), the COH shown in the ligand disappeared after the complex formation. Therefore, it can be inferred that Fe in (d) is a compound coordinated to O and N of the ligand. In the case of Fe, Fe (III) 2p 3 / 2 (712.15 eV), Fe (III) satellite, Fe (III) 2p 1 / 2 (725.54 eV) was observed.

[0066]

[0067] <Example 2> Fabrication of a blood glucose detection sensor using a novel electron transfer medium.

[0068] A blood glucose detection sensor was fabricated using the novel electron transfer mediator synthesized according to Example 1 (Fig. 4). The fabrication process is as follows:

[0069] First, the synthesized novel electron transfer mediator was prepared in 0.1 to 5 mL of PBS solution to be 1 to 40 mM, and 1 to 30 mg of fungal-derived FAD (flavin adenine dinucleotide)-dependent GDH (glucose dehydrogenase) (FAD / GDH) enzyme was added to the prepared solution, followed by 1 to 100 mmol of Ru 3+ / Ru 2+ A final enzyme solution was prepared by injecting a couple. 0.1–5 μL of the prepared final enzyme solution was dropped onto screen-printed electrodes, dried at 0–40°C for 1–36 hours, and a blood glucose sensor was fabricated. The electrodes of the blood glucose sensor used screen-printed electrodes consisting of a working electrode and an auxiliary electrode, and carbon ink was used for both electrodes.

[0070]

[0071] <Comparative Example 1>Ru 3+ / Ru 2+ Development of a blood sugar detection sensor utilizing a couple

[0072] In the above Example 2, Ru as a secondary electron transfer mediator was used in the manufacture of the blood glucose sensor. 3+ / Ru 2+ A blood glucose sensor was manufactured using the same method without inserting a couple.

[0073]

[0074] <Experimental Example 1> Measurement of the oxidation / reduction current of a novel electron transfer mediator according to the electrochemical scan rate.

[0075] In order to confirm the electron transfer characteristics of the novel electron transfer mediator (hereinafter, FeAHBS) synthesized according to Example 1 of the present invention, the novel electron transfer mediator was dissolved in 1 M KCl to a concentration of 1 mM, and then evaluated by cyclic voltammetry using a glassy anode as a working electrode. The electrochemical potential measurement range was from -150 mV to 150 mV, and the change in oxidation / reduction current was measured as the scan rate changed from 20 mV / s to 200 mV / s, and the results are shown in Fig. 5. As a result according to this experimental example, it was confirmed that FeAHBS is a chemical species with high electrochemical reversibility, and the electron transfer rate at this time was calculated using the following Equation 1:

[0076] [Formula 1]

[0077]

[0078] In the above equation, ψ is the displacement parameter, D ox Wow D red is the diffusion coefficient, n is the number of electrons involved in the process, υ is the scan rate, α is the transport coefficient, R is the gas constant, and T is the temperature. k o For the calculation of , the median values ​​of ΔEp, Epa, and Epc were found to be 66 mV at 100 mV / s. The Randles-Sevcik equation is D ox Wow D red The values ​​of each are 1.5×10 -5 and 1.53×10 -5 cm 2 / s was calculated. Taking all of this into account, the heterogeneous rate constant k of 1 mM FeAHBS o is 1.18×10 -4 It is cm / s.

[0079]

[0080] <Experimental Example 2> Performance Evaluation of a Modified Sensor for Glucose Oxidation

[0081] The results of oxidation / reduction of the electron transfer mediator were confirmed by measuring a solution containing 10 mM glucose dissolved in 0.1 M PBS (pH 7.4) in a potential range of -0.4 to 0.3 V and a scan rate of 50 mV / s using cyclic voltammetry in the blood glucose sensor manufactured according to the above Example 2 and Comparative Example 1.

[0082] As a result, referring to Fig. 6(a), FeAHBS showed Epa = 62.25 mV, Epc = -51.62 mV, and low reversibility was confirmed due to reasons such as decreased electrical conductivity in the electrode coated with FAD / GDH, which is a glucose oxidase. Afterwards, Ru, which is a secondary electron transfer mediator 3+ / Ru 2+ When Ru is introduced, the conductivity of the electrode surface increases threefold, and 3+ / Ru 2+ The redox peaks of the couple were identified at Epa = 46.24 mV and Epc = -43.15 mV, respectively. Cyclic voltammetry was used to observe the signal amplitude as the glucose solution concentration varied from 0 to 50 mM using the final blood glucose sensor, and the signal amplitude changed linearly, as shown in Fig. 6(b).

[0083]

[0084] <Experimental Example 3> Current Evaluation of a Blood Glucose Sensor Based on Glucose Concentration

[0085] The performance of the sensor was evaluated under optimized glucose detection conditions for the blood glucose sensor manufactured according to the above Example 2 and Comparative Example 1. The glucose solution was prepared in a 0.1 M PBS (pH 7.4) buffer solution, and the detection voltage was fixed at one of the potentials of 100 to 300 mV and the current value was measured at one of the time ranges of 1 to 10 seconds.

[0086] As a result, as shown in Fig. 7(a), the response current of the time-dependent current method increased as the glucose concentration increased, and referring to Fig. 7(b), which is a calibration curve, the detection limit of the final sensor is 0.023 mM, the correlation coefficient of the calibration curve is 0.999, and the dynamic range of the calibration curve is 0.05 to 50 mM. (S / N = 3)

[0087]

[0088] <Experimental Example 4> Evaluation of the Interference Effect, Stability, and Reproducibility of a Blood Glucose Sensor

[0089] Interfering molecules in the blood that interfere with glucose detection include monosaccharides, including ascorbic acid (AA), uric acid (UA), dopamine (DA), acetaminophen (AP), mannose, lactose, xylose, and fructose. Since their blood concentrations are very low compared to glucose, the selectivity of the sensor manufactured according to the above example was evaluated using cyclic voltammetry in the presence of 0.2 mM AA, UA, DA, and AP relative to 5 mM glucose. As a result, as shown in Figs. 8(a) and (b), the final sensor did not exhibit any oxidation / reduction current in the presence of interfering substances.

[0090] In order to evaluate the reproducibility of the sensor manufactured according to the above example, five sensors were measured with a glucose solution having a concentration of 10 mM. As a result, as shown in Fig. 8(c), the relative standard deviation (RSD) of the oxidation current was approximately 1.5%, confirming that it was a highly reproducible sensor.

[0091] In addition, to evaluate long-term stability, the electrode was stored at room temperature after manufacturing the sensor, and the signal for a 10 mM glucose solution was examined for 180 days. As shown in Fig. 8(d), even after 6 months, the performance of the sensor was maintained at 99.09%, which is very stable.

[0092]

[0093] <Experimental Example 5> Actual Sample Evaluation

[0094] For actual sample analysis, a single donor's whole blood (Na Heparin) sample was purchased and evaluated using the standard addition method. Blood samples containing glucose concentrations of 0.5, 5, 10, 20, 30, 40, and 50 mM were prepared by diluting the sample solution with a glucose solution, and the results were measured using the same method as in Experimental Example 3.

[0095] As a result, the time-dependent current method results shown in Fig. 9(a) were obtained, and the current response according to concentration was plotted as in Fig. 8(b). The correlation coefficient of the calibration curve for glucose measurements in actual blood samples containing interfering substances was 0.997, and the dynamic range of the calibration curve was 0.05 to 50 mM, demonstrating excellent performance. (S / N = 5)

[0096]

[0097] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An iron complex represented by the following chemical formula 1 or a salt compound thereof: [Chemical Formula 1] In the above chemical formula 1, R 1 , R 2 and R 3 may be the same or different, and are selected from hydrogen, H, Na, Li, K, Cs, NH4, or (C1 to C2) alkyl.

2. In paragraph 1, The above iron complex or its salt compound, An iron complex or a salt thereof, characterized in that it serves as an electron transfer mediator for an enzyme-based biosensor. A step of preparing a ligand solution by adding 3-amino-4-hydroxy benzene sulfonic acid (AHBS) to a basic solution purged with nitrogen; A step of preparing an iron precursor solution by adding an iron precursor to distilled water purged with nitrogen; and A method for producing an iron complex or a salt compound thereof according to claim 1, comprising a step of adding the prepared ligand solution dropwise to the prepared iron precursor solution to obtain a reaction product.

4. In paragraph 3, The above basic solution is, A manufacturing method characterized in that at least one selected from the group consisting of NaOH, KOH, CsOH and NH4OH.

5. In paragraph 3, The above iron precursor is, A manufacturing method characterized in that at least one selected from the group consisting of iron (III) nitrate, iron (III) chloride, iron (III) citrate, iron (III) acetylacetonate, iron (III) pyrophosphate, iron (III) tartrate, iron (III) bromide, and iron (III) sulfate.

6. Substrate; Working electrodes and auxiliary electrodes arranged on the substrate; and An enzyme layer formed on the electrode and including an enzyme and an electron transfer mediator, A biosensor for blood sugar detection, characterized in that the electron transfer mediator is an iron complex according to claim 1 or a salt compound thereof.

7. In paragraph 6, The above enzyme is, A biosensor for detecting blood glucose, characterized in that at least one enzyme is selected from the group consisting of glucose oxidase (GOx), glucose dehydrogenase (GDH), flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (FAD / GDH), glucose hexokinase, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase.

8. In paragraph 6, The above biosensor, Ru as the above electron transfer medium 3+ / Ru 2+ , Os 3+ / Os 2+ A biosensor for blood sugar detection, characterized in that it further comprises at least one selected from the group consisting of Fc+ / Fc.

9. In paragraph 6, The above biosensor, A biosensor for blood glucose detection, characterized in that it suppresses the interfering effect of one or more substances in the blood selected from the group consisting of monosaccharides including mannose, lactose, xylose, and fructose; ascorbic acid (AA), uric acid (UA), dopamine (DA), and acetaminophen (AP).

10. In paragraph 6, The above biosensor, A biosensor for blood glucose detection characterized by improved reproducibility and long-term stability.

11. A method for detecting blood sugar, comprising the step of contacting a biosensor for detecting blood sugar according to Article 6 with a blood sample.

12. In paragraph 11, The above method, A method for detecting blood sugar, characterized in that blood sugar detection is evaluated using cyclic voltammetry or chronoamperometry.

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