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

Abstract

The present invention relates to a novel electron transfer medium and a biosensor for blood glucose detection comprising the same. More specifically, the invention provides a novel iron complex electron transfer medium applicable to an enzyme-based blood glucose detection biosensor and a biosensor for blood glucose detection using the same. The biosensor according to the present invention has superior sensitivity and improved performance compared to conventional blood glucose sensors, enabling more precise and accurate measurement of blood glucose.

Description

Technology Field

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

[0002] Diabetes is a metabolic disorder related to glucose levels in the body, primarily caused by blood glucose abnormalities resulting from insufficient insulin production. Globally, approximately 10% of the population aged 20 to 80 suffers from diabetes. Since this disease can lead to various complications, including heart disease, kidney dysfunction, retinopathy, obesity, and neuropathy, consistently and accurately measuring glucose levels is crucial for the diagnosis and management of diabetes.

[0003] Numerous studies have been conducted for glucose measurement, and particularly following the 발표 of electrochemical glucose detection by Clark and Lyons in 1962, glucose sensors have been developed into three generations. First-generation glucose sensors were based on the amperometric detection of H2O2 generated during enzymatic reactions; however, because this required a high detection potential, it had the disadvantage of being vulnerable to interference effects caused by various electrochemically active substances present in the blood. Second-generation glucose sensors were developed to address the shortcomings of the first generation; additional electron transfer mediators were introduced to reduce the enzyme's oxygen dependence and facilitate the smooth transfer of electrons from the enzyme surface to the measurement electrode. Third-generation glucose sensors utilize a method that directly transfers electrons from glucose to the measurement electrode through the enzyme's active site. Currently, most commercially available blood glucose monitoring systems are based on the second generation. In second-generation glucose sensors, electron transfer mediators facilitate the movement of electrons between the enzyme's redox center and the electrode, enabling stable glucose detection.

[0004] Most glucose sensors utilize glucose oxidase (GOx) or glucose dehydrogenase (GDH) as the enzyme. However, GOx has the disadvantage of being unsuitable for use in commercial sensor strips due to its oxygen dependence during the glucose oxidation process. GDH, on the other hand, is easy to use because it enables glucose detection without the need for oxygen. Among the various types of GDH, FAD / GDH, a FAD-dependent GDH derived from fungi, is an enzyme that has recently garnered attention due to its low oxygen dependence and lack of electrochemical activity toward maltose. While FAD / GDH-based glucose sensors exhibit high selectivity and stability, an additional efficient electron transfer medium must be introduced to fabricate high-performance, stable sensors.

[0005] To date, various types of developed electron transport media, including organic, inorganic, and organometallic materials, have been developed. In particular, ferricyanide and ruthenium hexamine complexes are frequently used media. Ferricyanide has been widely used as a mediator since the 1990s due to its availability, high solubility, and low cost; however, it requires a high redox potential and presents issues regarding storage and distribution. Sensors using ruthenium hexamine have a low redox potential, minimal interference, and no issues during distribution, but they have the disadvantage of requiring an auxiliary mediator for use with FAD / GDH. Prior art literature

[0006] Republic of Korea Registered Patent No. 10-0729307 (Published June 15, 2007) The problem to be solved

[0007] The objective of the present invention is to provide a novel electron transfer medium for the development of a blood glucose detection sensor with improved problems as described above.

[0008] Another objective of the present invention is to provide a high-performance blood glucose detection sensor by utilizing the novel electron transfer medium described above. means of solving the problem

[0009] To achieve the above objective, the present invention provides a novel iron complex represented by the following chemical formula 1 or a chloride compound thereof:

[0010] [Chemical Formula 1]

[0011]

[0012] In the above chemical formula 1, R 1 , R 2 and R 3 Each may be the same or different and may be selected from H, Na, Li, K, Cs, NH4, or (C1 to C2) alkyl.

[0013] The present invention provides a method for preparing the iron complex or its chloride compound, 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 obtaining a reaction product by adding the prepared ligand solution dropwise to the prepared iron precursor solution.

[0014] The present invention provides a biosensor for blood glucose detection, comprising: a substrate; an operating electrode and an auxiliary electrode disposed on the substrate; and an enzyme layer formed on the electrodes and comprising an enzyme and an electron transfer medium, wherein the electron transfer medium is the iron complex or a salt compound thereof.

[0015] In addition, the present invention provides a blood glucose detection method comprising the step of contacting the above-described blood glucose detection biosensor with a blood sample. Effects of the invention

[0016] The biosensor for blood glucose detection according to the present invention utilizes a novel iron complex as an electron transfer medium, thereby providing superior sensitivity and improved performance compared to conventional blood glucose sensors, enabling precise blood glucose measurement.

[0017] In addition, the biosensor can suppress interference effects caused by various substances in the blood and increase reproducibility and long-term stability, thereby measuring blood glucose more accurately and maintaining sensing sensitivity and performance stably. Brief explanation of the drawing

[0018] Figure 1 schematically illustrates the synthesis process of a novel electron transfer medium (FeAHBS) according to one embodiment of the present invention. Figure 2 shows (a) Fourier transform-infrared spectroscopy (FT-IR) analysis results and (b) X-ray photoelectron spectroscopy (XPS) analysis results of a novel electron transfer medium synthesized according to Figure 1. Figure 3 shows the elemental XPS analysis results of the novel electron transfer media synthesized according to Figure 1, where (a) is C 1s, (b) is N 1s, (c) is O 1s, and (d) is Fe 2p. Figure 4 shows a schematic diagram of a blood glucose detection sensor using a novel electron transfer medium synthesized according to Figure 1. Figure 5 shows the change in redox current according to the scanning rate of a novel electron transfer medium according to one experimental example of the present invention. Figure 6 shows (a) redox characteristics and (b) changes in oxidation according to glucose concentration of a blood glucose detection sensor fabricated using a novel electron transfer medium according to another embodiment of the present invention. FIG. 7 shows the performance evaluation of the sensor under optimized glucose detection conditions for a blood glucose detection sensor according to the present invention, where (a) is the change in the time-amplitude signal according to glucose concentration and (b) shows the corresponding calibration curve. FIG. 8 evaluates the (a, b) interference effect, (c) stability, and (d) reproducibility of a blood glucose detection sensor according to the present invention. Figure 9 shows the results of evaluation using actual samples. Specific details for implementing the invention

[0019] The present invention will be described in detail below.

[0021] In order to improve the aforementioned problems, the inventors synthesized a novel electron transporter 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 completed the present invention by confirming that a sensor strip using the synthesized novel electron transporter exhibits excellent glucose detection performance even in the presence of interfering substances.

[0023] The present invention provides a novel iron complex or a chloride compound thereof.

[0024] Preferably, the iron complex or its chloride compound can be represented by the following chemical formula 1:

[0025] [Chemical Formula 1]

[0026]

[0027] In the above chemical formula 1, R 1 , R 2 and R 3 Each may be the same or different and may be selected from hydrogen, alkali metal, ammonium or (C1 to C4) alkyl, preferably selected from H, Na, Li, K, Cs, NH4, -CH3, or -CH2CH3, but is not limited thereto.

[0028] The above iron complex or its salt compound may be in the form in which a ligand derived from 3-amino-4-hydroxybenzene sulfonic acid (AHBS) is bound to the central metal ion, iron (Fe(III)).

[0029] The above iron complex or its salt compound can be utilized as an electron transfer medium to facilitate 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 immobilized on the electrode of the enzyme-based biosensor.

[0030] The above biosensor may be a sensor for detecting blood glucose, but is not limited thereto.

[0032] The present invention provides a method for manufacturing the above-mentioned novel iron complex or its chloride compound.

[0033] A method for preparing an iron complex or a chloride compound thereof according to the present invention may include 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 obtaining a reaction product by adding the prepared ligand solution dropwise to the prepared iron precursor solution.

[0034] The step of preparing the above ligand solution can be performed by adding AHBS to a basic solution, wherein the basic solution may be one or more selected from the group consisting of NaOH, KOH, CsOH and NH4OH, preferably NaOH, but is not limited thereto.

[0035] 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 one or more selected from the group consisting of iron nitrate (III), iron chloride (III), iron citrate (III), iron acetylacetonate (III), iron pyrophosphate (III), iron tintate (III), iron bromide (III), and iron sulfate (III), but is not limited thereto, and may include all precursor materials capable of providing iron ions.

[0036] The step of obtaining the 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 for 2 to 24 hours at the same temperature range to terminate the reaction, and then recovering the reaction product by chromatography, centrifugation, freeze-drying, or vacuum evaporation.

[0038] The present invention provides a biosensor for blood glucose detection comprising the novel iron complex or a chloride compound thereof.

[0039] More specifically, the biosensor according to the present invention may comprise a substrate; an operating electrode and an auxiliary electrode disposed on the substrate; and an enzyme layer formed on the electrode and comprising an enzyme and an electron transfer medium, wherein the electron transfer medium may comprise the novel iron complex described above or a salt compound thereof.

[0040] The above electrode may be selected from the group consisting of carbon electrodes, gold electrodes, silver electrodes, and copper electrodes, but is not limited thereto, and electrodes commonly used in the art may be used.

[0041] The above enzyme may be changed depending on the target to be measured by the biosensor, and in the case of the blood glucose detection biosensor according to the present invention, the enzyme may be one or more 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 may be FAD / GDH, but is not limited thereto.

[0042] The above biosensor uses Ru as the electron transfer medium. 3+ / Ru 2+ , Os 3+ / Os 2+ and Fc + One or more selected from the group consisting of / Fc may be additionally included, and blood glucose detection performance may be improved by additionally including these.

[0044] The biosensor for blood glucose detection according to the present invention can suppress interference effects caused by various substances in the blood.

[0045] The various substances in the blood mentioned above may be 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), but are not limited thereto.

[0046] In addition, the above biosensor has high reproducibility and shows stable performance even after 6 months, so reproducibility and long-term stability may be improved.

[0048] In addition, the present invention provides a blood glucose detection method comprising the step of contacting the above-described blood glucose detection biosensor with a blood sample.

[0049] The above blood glucose detection method can evaluate whether or not blood glucose is detected or the extent thereof using cyclic voltammetry or chronoamperometry.

[0051] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0053] <Example 1> Synthesis of a Novel Electron Transfer Medium (FeAHBS)

[0054] A novel electron transfer medium (FeAHBS) was synthesized as shown in Fig. 1 through the synthesis process below.

[0055] 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) (containing 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, Iron(III) sulfate) was prepared in 50 to 200 mL of distilled water sufficiently purged with nitrogen. A reaction vessel was prepared by attaching a dropping funnel to a Schlenk flask, and the Fe(III) solution was injected into the Schlenk flask and the ligand into the dropping funnel. The ligand solution was then slowly added dropwise at a reaction temperature of 0 to 100 °C. After adding the solution dropwise, the reaction was terminated by stirring at the same temperature for 2 to 24 hours, and the mediator was recovered by methods such as chromatography, centrifugation, freeze-drying, and vacuum evaporation.

[0057] The novel electron transfer medium synthesized by the above method was confirmed using FT-IR and XPS.

[0058] Referring to Fig. 2(a), as can be seen from the black line, the ligand itself is 3260 cm⁻¹ -1 and 2940 cm -1 It exhibited sharp -NH2 and -OH stretching peaks. On the other hand, for the novel medium complexed with Fe, the -NH and -OH peaks broadened, and the overall FT-IR band positions shifted due to coordination between the metal and the ligand. Additionally, for both chemical species, the C=C stretching and S=O stretching bands were at 1638 and 1357 cm⁻¹, respectively.-1 It appeared in. Referring to Fig. 2(b), XPS analysis of the synthesized medium showed Fe, C, N, O, S, and Na, which matched the types of elements of the synthesized medium.

[0059] Referring to Fig. 3, in the C 1s diagram of (a), the CC sp of the ligand 2 (284.46 eV), CC sp 3 (285.11 eV), CN / CS (285.96 eV), and CO (286.77 eV) bonds were observed. In the N 1s diagram of (b), it was confirmed that the -NH2 peak (398.40 eV) and CN peak (400.01 eV), which were present in the ligand itself, shifted to -NH2 peak (399.01 eV) and CN peak (400.13 eV) after complex formation. In the case of the O 1s diagram of (c), the COH group observed in the ligand disappeared after complex formation. Based on this, it can be inferred that Fe in (d) is a compound coordinated to the 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.

[0061] <Example 2> Fabrication of a blood glucose detection sensor using a novel electron transfer medium

[0062] A blood glucose detection sensor was fabricated using the novel electron transfer medium synthesized according to Example 1 above (Fig. 4). The fabrication process is as follows:

[0063] First, prepare the synthesized novel electron transfer media to a concentration of 1–40 mM in 0.1–5 mL of PBS solution, add 1–30 mg of fungal-derived FAD (flavin adenine dinucleotide)-dependent GDH (glucose dehydrogenase) (FAD / GDH) enzyme to the prepared solution, and then add 1–100 mmol of Ru 3+ / Ru 2+A final enzyme solution was prepared by adding a couple. A blood glucose sensor was fabricated by dropping 0.1 to 5 μL of the prepared final enzyme solution onto a screen-printed electrode and drying it for 1 to 36 hours at 0 to 40 ℃. The electrode of the blood glucose sensor used a screen-printed electrode consisting of an operating electrode and an auxiliary electrode, and carbon ink was used for both electrodes.

[0065] <Comparative Example 1> Ru 3+ / Ru 2+ Fabrication of a blood glucose detection sensor utilizing a couple, excluding couples

[0066] In the above Example 2, Ru, which is a secondary electron transfer medium when manufacturing the blood glucose sensor 3+ / Ru 2+ A blood glucose sensor was manufactured using the same method without introducing a couple.

[0068] <Experimental Example 1> Measurement of Oxidation / Reduction Current of Novel Electron Transfer Medium According to Electrochemical Scan Rate

[0069] To confirm the electron transfer characteristics of the novel electron transfer medium (hereinafter referred to as FeAHBS) synthesized according to Example 1 of the present invention, the novel electron transfer medium was dissolved in 1M KCl to a concentration of 1 mM, and then evaluated by cyclic voltammetry using a glassy oxide electrode as the 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 was varied from 20 mV / s to 200 mV / s, resulting in the findings shown in Fig. 5. Based on the results of this experiment, it was confirmed that FeAHBS is a chemical species with high electrochemical reversibility, and the electron transfer rate was calculated using Equation 1 below:

[0070] [Equation 1]

[0071]

[0072] In the above equation, ψ is the movement parameter, D ox and D redε 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 value of ΔEp, Epa, and Epc was determined to be 66 mV at 100 mV / s. The Randles-Sevcik equation is D ox and D red The value of each is 1.5×10 -5 and 1.53×10 -5 cm 2 It was calculated as / s. 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.

[0074] <Experimental Example 2> Performance Evaluation of Modified Sensor for Glucose Oxidation

[0075] The oxidation / reduction results of the electron transporter were confirmed by measuring the potential range of -0.4 to 0.3 V and the scan rate of 50 mV / s of a solution of 10 mM glucose dissolved in 0.1 M PBS (pH 7.4) on the blood glucose sensor prepared according to Example 2 and Comparative Example 1 above using cyclic voltammetry.

[0076] As a result, referring to Fig. 6(a), FeAHBS showed Epa = 62.25 mV and Epc = -51.62 mV, and low reversibility was confirmed at the electrode coated with the glucose oxidase FAD / GDH due to reasons such as a decrease in electrical conductivity. Subsequently, Ru, a secondary electron transport mediator 3+ / Ru 2+ When introduced, the conductivity of the electrode surface increases threefold, and Ru 3+ / Ru 2+ The redox peaks of the couple were identified at Epa = 46.24 mV and Epc = -43.15 mV, respectively. When observed by cyclic voltammetry while varying the glucose solution from 0 to 50 mM using the final blood glucose sensor, it was confirmed that the signal magnitude changed linearly, as shown in Figure 6(b).

[0078] <Experimental Example 3> Time-based amperometric current evaluation of blood glucose sensor according to glucose concentration

[0079] The performance of the blood glucose sensor prepared according to Example 2 and Comparative Example 1 was evaluated under glucose detection conditions optimized for the sensor. The glucose solution was prepared in a 0.1 M PBS (pH 7.4) buffer solution, and the detection voltage was fixed at one potential between 100 and 300 mV, and the current value was measured at one time within the range of 1 to 10 seconds.

[0080] As a result, as shown in Fig. 7(a), the response current of the time-amplitude method increased as the glucose concentration increased. Referring to the calibration curve in Fig. 7(b), 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 0.05–50 mM. (S / N = 3)

[0082] <Experimental Example 4> Evaluation of Interference Effect, Stability, and Reproducibility of Blood Glucose Sensor

[0083] Interfering molecules in the blood that interfere with glucose detection include ascorbic acid (AA), uric acid (UA), dopamine (DA), acetaminophen (AP), mannose, lactose, xylose, and monosaccharides including fructose. Since their blood concentrations are very low compared to glucose, cyclic voltammetry was used to evaluate the selectivity of the sensor prepared according to the above example in the presence of 0.2 mM of AA, UA, DA, and AP relative to 5 mM glucose. As a result, as shown in Figures 8(a) and (b), no oxidation / reduction current was observed in the final sensor in the presence of interfering substances.

[0084] To evaluate the reproducibility of the sensor manufactured according to the above example, five sensors were measured with a glucose solution at a concentration of 10 mM. As shown in Fig. 8(c), the relative standard deviation (RSD) of the oxidation current was approximately 1.5%, confirming that the sensor has very high reproducibility.

[0085] In addition, to evaluate long-term stability, the electrode was stored at room temperature after manufacturing the sensor, and the signal was examined for 180 days with a glucose solution at a concentration of 10 mM. As shown in Fig. 8(d), the performance of the sensor remained very stable at 99.09% even after 6 months.

[0087] <Experimental Example 5> Evaluation of Actual Samples

[0088] After purchasing human whole blood (Na Heparin) samples from a single donor for actual sample analysis, an evaluation was performed following 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 measured using the same method as in Experimental Example 3 above.

[0089] As a result, time-amplitude amperometric results as shown in Fig. 9(a) were obtained, and plotting the current response according to concentration yielded Fig. 8(b). The correlation coefficient of the calibration curve for glucose measurement 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)

[0091] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 Iron complex represented by the following chemical formula 1 or its chloride compound: [Chemical Formula 1] In the above chemical formula 1, R 1 , R 2 and R 3 Each may be the same or different and is selected from H, Na, Li, K, Cs, NH4, or (C1 to C2) alkyl. Claim 2 The iron complex or its salt compound according to claim 1, characterized in that the iron complex or its salt compound is an electron transfer medium of an enzyme-based biosensor. Claim 3 A method for preparing an iron complex or a chloride compound thereof according to claim 1, 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 obtaining a reaction product by adding the prepared ligand solution dropwise to the prepared iron precursor solution. Claim 4 A method of preparation according to claim 3, wherein the basic solution is one or more selected from the group consisting of NaOH, KOH, CsOH, and NH4OH. Claim 5 A method of manufacturing according to claim 3, wherein the iron precursor is one or more selected from the group consisting of iron nitrate (III), iron chloride (III), iron citrate (III), iron acetylacetonate (III), iron pyrophosphate (III), iron tartrate (III), iron bromide (III), and iron sulfate (III). Claim 6 A biosensor for blood glucose detection, comprising: a substrate; an operating electrode and an auxiliary electrode disposed on the substrate; and an enzyme layer formed on the operating electrode and comprising an enzyme and an electron transfer medium, wherein the electron transfer medium is an iron complex according to claim 1 or a salt compound thereof. Claim 7 A biosensor for blood glucose detection according to claim 6, wherein the enzyme is one or more 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. Claim 8 In claim 6, the biosensor is Ru as the electron transfer medium. 3+ / Ru 2+ , Os 3+ / Os 2+ A biosensor for blood glucose detection, characterized by further comprising one or more selected from the group consisting of , and Fc+ / Fc. Claim 9 A biosensor for blood glucose detection according to claim 6, wherein the biosensor inhibits 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). Claim 10 In claim 6, the biosensor is a biosensor for blood glucose detection characterized by improved reproducibility and long-term stability. Claim 11 A blood glucose detection method comprising the step of contacting a blood glucose detection biosensor according to claim 6 with a blood sample. Claim 12 A blood glucose detection method according to claim 11, characterized in that the method evaluates blood glucose detection using cyclic voltammetry or chronoamperometry.

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