A glucose sensor and its preparation method and application

Abstract

The present invention discloses a glucose sensor and its preparation method and application. The glucose sensor comprises an electrode, the surface of which is connected with a triblock nucleic acid and a short-chain thiol molecule, the triblock nucleic acid comprises a first sequence, a PolyA sequence and a second sequence connected in sequence, the first sequence and the second sequence are not complementary, the first sequence is connected to glucose oxidase through a complementary pairing sequence of the first sequence, the second sequence is connected to horseradish peroxidase through a complementary pairing sequence of the second sequence, or the first sequence is connected to horseradish peroxidase through a complementary pairing sequence with the first sequence, and the second sequence is connected to glucose oxidase through a complementary pairing sequence of the second sequence. The present invention utilizes triblock nucleic acids to connect glucose oxidase and horseradish peroxidase, effectively regulates the density, distribution and configuration of the enzyme on the electrode surface, and provides a glucose sensor with high stability and sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors and relates to a glucose sensor and a preparation method and application thereof. Background Art

[0002] Glucose is an important substance in human metabolism. Simple and rapid glucose detection technology is essential for monitoring glucose levels in human body fluids such as blood, urine, and tears. Electrochemical sensors are simple, rapid, and sensitive detection technologies. Enzyme-based electrochemical sensors can obtain rapid and specific glucose detection results. They are not only suitable for glucose testing in hospital outpatient clinics, but also for glucose testing at home.

[0003] However, there are still some problems with enzyme-based electrochemical sensors. First, as a biologically active substance, enzymes are easily affected by external factors, resulting in reduced enzyme activity or even inactivation. Second, enzyme reactions often lead to high background signals, greatly reducing the detection limit of electrochemical sensors. In addition, there is randomness in the fixation of enzymes on the electrochemical interface, and the composition, structure and morphology of the enzymes are difficult to adjust, all of which may have a negative impact on the activity of the enzymes. For example, CN114778634A discloses a method for preparing a modified graphene enzyme electrode glucose sensor, comprising: modifying carboxyl groups on the surface of graphene to obtain carboxylated graphene; mixing carboxylated graphene with ferroferric oxide to obtain a carboxylated graphene / ferroferric oxide nanocomposite material; mixing and fixing the carboxylated graphene / ferroferric oxide nanocomposite material and glucose oxidase on the surface of a screen-printed carbon electrode to obtain a carboxylated graphene / ferroferric oxide / glucose oxidase screen-printed carbon electrode; coating a layer of polyurethane outer film on the carboxylated graphene / ferroferric oxide / glucose oxidase screen-printed carbon electrode to obtain a carboxylated graphene / ferroferric oxide / glucose oxidase / polyurethane screen-printed carbon electrode.

[0004] In summary, how to develop new and efficient glucose sensors is still one of the urgent problems to be solved in the field of biosensor technology. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a glucose sensor and a preparation method and application thereof. The present invention designs a sensor with a specific structure to effectively control the enzyme cascade reaction and significantly improve stability and sensitivity.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a glucose sensor, the glucose sensor comprising an electrode;

[0008] The surface of the electrode is connected with a triblock nucleic acid and a short-chain thiol molecule, the triblock nucleic acid comprises a first sequence, a PolyA sequence and a second sequence connected in sequence, the first sequence and the second sequence are not complementary, the first sequence is connected to glucose oxidase via a complementary pairing sequence of the first sequence, the second sequence is connected to horseradish peroxidase via a complementary pairing sequence of the second sequence, or the first sequence is connected to horseradish peroxidase via a complementary pairing sequence with the first sequence, and the second sequence is connected to glucose oxidase via a complementary pairing sequence of the second sequence.

[0009] In the present invention, the triblock nucleic acid is connected to the electrode by relying on the adsorption of the PolyA sequence in the middle region of the triblock nucleic acid and the electrode. Subsequently, two single-stranded DNA segments connected with glucose oxidase (GOx) and horseradish peroxide (HRP) are hybridized with the triblock nucleic acid on the electrode surface to prepare an electrochemical sensor for glucose detection. The schematic diagram is shown in FIG. Figure 1 As shown in the figure, the principle of the sensor is to use glucose oxidase to catalyze oxygen molecules to oxidize glucose to generate hydrogen peroxide and gluconic acid. Hydrogen peroxide reacts with tetramethylbenzidine (TMB) as a substrate, and produces obvious electrochemical signal changes under the oxidation of horseradish peroxidase (HRP). The enzyme connected to the single-stranded DNA can be placed in a 4°C refrigerator for half a year, and then added to the electrode surface for hybridization when used, which can well keep the enzyme activity from being affected by the external environment; secondly, the glucose detection on the electrode surface undergoes a cascade amplification reaction, which not only has a lower background, but also a higher signal (detection system containing glucose), which greatly improves the sensitivity of the sensor.

[0010] It is understood that any electrode commonly used in the art that can bind to the Poly A sequence is applicable to the present invention without any special limitation.

[0011] Preferably, the electrode comprises a gold electrode, a glassy carbon electrode deposited with gold nanoparticles, or the like.

[0012] Preferably, the length of the first sequence is 10-40 nt, including but not limited to 11 nt, 12nt, 13nt, 15nt, 18nt, 20nt, 25nt, 30nt, 32nt, 34nt, 36nt, 38nt or 39nt.

[0013] Preferably, the length of the PolyA sequence is 10-100 nt, including but not limited to 11 nt, 12 nt, 13 nt, 15 nt, 18 nt, 20 nt, 30 nt, 40 nt, 52 nt, 60 nt, 70 nt, 80 nt, 85 nt, 88 nt, 90 nt, 95 nt, 98 nt or 99 nt, preferably 10 to 60 nt.

[0014] Preferably, the length of the second sequence is 10-40 nt, including but not limited to 11 nt, 12 nt, 13 nt, 15 nt, 18 nt, 20 nt, 25 nt, 30 nt, 32 nt, 34 nt, 36 nt, 38 nt or 39 nt.

[0015] In the present invention, by controlling the density and length of the triblock nucleic acid connected to the electrode surface, the density, distribution and configuration of the enzyme on the electrode surface can be effectively regulated, and the efficiency of the enzyme cascade reaction on the electrode surface can be further improved.

[0016] It can be understood that the first sequence and the second sequence in the present invention are not limited to specific sequences, and as long as they are not complementary, they can be connected to the enzyme respectively through their complementary sequences.

[0017] Optionally, the first sequence includes the sequence shown in SEQ ID NO.1.

[0018] Optionally, the second sequence includes the sequence shown in SEQ ID NO.2.

[0019] Optionally, the complementary pairing sequence of the first sequence includes the sequence shown in SEQ ID NO.3.

[0020] Optionally, the complementary pairing sequence of the second sequence includes the sequence shown in SEQ ID NO.4.

[0021] SEQ ID NO. 1: TATCATCCTTACACCTCACT.

[0022] SEQ ID NO. 2: CCCTCTAACTTCCATCACA.

[0023] SEQ ID NO. 3: AGTGAGGTGTAAGGATGATATTTTTTTTTT-SH (thiol).

[0024] SEQ ID NO. 4: SH-TTTTTTTTTTTGTGATGGAAGTTAGAGGGT.

[0025] In a second aspect, the present invention provides a method for preparing the glucose sensor according to the first aspect, the preparation method comprising:

[0026] Mixing glucose oxidase with the complementary pairing sequence of the first sequence and a cross-linking agent, and mixing horseradish peroxidase with the complementary pairing sequence of the second sequence and a cross-linking agent to obtain an enzyme mixture, or,

[0027] Mixing horseradish peroxidase with the complementary pairing sequence of the first sequence and a cross-linking agent, and mixing glucose oxidase with the complementary pairing sequence of the second sequence and a cross-linking agent to obtain an enzyme mixture;

[0028] The enzyme mixture, triblock nucleic acid and mercaptohexanol are added to an electrode to obtain the glucose sensor.

[0029] Preferably, the cross-linking agent includes SPDP (3-[2-pyridyldithio]succinimidyl propionate) or SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxyl-(6-amidohexanoate)) and the like.

[0030] Preferably, the concentrations of the glucose oxidase and horseradish peroxidase are independently 0.02-1 μmol / L, including but not limited to 0.03 μmol / L, 0.04 μmol / L, 0.05 μmol / L, 0.06 μmol / L, 0.08 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.25 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.8 μmol / L or 0.9 μmol / L.

[0031] In the present invention, controlling the concentrations of glucose oxidase and horseradish peroxidase can further improve the cascade reaction efficiency.

[0032] Preferably, the molar concentration ratio of the glucose oxidase to the cross-linking agent is 1:(2-20), including but not limited to 1:2, 1:3, 1:4, 1:5, 1:10, 1:12, 1:14, 1:16, 1:18 or 1:19.

[0033] Preferably, the molar concentration ratio of the horseradish peroxidase to the cross-linking agent is 1:(2-20), including but not limited to 1:2, 1:3, 1:4, 1:5, 1:10, 1:12, 1:14, 1:16, 1:18 or 1:19.

[0034] In the present invention, controlling the ratio of glucose oxidase, horseradish peroxidase and cross-linking agent can further improve the efficiency of the cascade reaction.

[0035] Preferably, the molar concentration ratio of the glucose oxidase to the horseradish peroxidase is (0.2-20):1, 0.2:1, 0.3:1, 0.5:1, 0.8:1, 1:1, 51, 8:1, 10:1, 15:1, 16:1, 18:1 or 19:1.

[0036] In the present invention, controlling the ratio of glucose oxidase to horseradish peroxidase can further improve the cascade reaction efficiency.

[0037] Preferably, the concentration of the triblock nucleic acid is 0.1 to 5 μmol / L (μM), including but not limited to 0.2 μmol / L, 0.3 μmol / L, 0.5 μmol / L, 0.8 μmol / L, 1 μmol / L, 1.2 μmol / L, 2 μmol / L, 3 μmol / L, 3.5 μmol / L, 3.8 μmol / L, 4 μmol / L, 4.2 μmol / L, 4.5 μmol / L, 4.8 μmol / L or 4.9 μmol / L.

[0038] Preferably, the concentration of the mercaptohexanol is 0.1-1 mmol / L (mM), including but not limited to 0.2 mmol / L, 0.3 mmol / L, 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L or 0.9 mmol / L.

[0039] In the present invention, controlling the concentration of mercaptohexanol can further improve the cascade reaction efficiency.

[0040] In a third aspect, the present invention provides use of the glucose sensor described in the first aspect in preparing a glucose detection product.

[0041] In a fourth aspect, the present invention provides a glucose detection device, wherein the glucose detection device comprises the glucose sensor described in the first aspect.

[0042] Preferably, the glucose detection device further comprises:

[0043] Transmitter: used to receive the current signal generated by the glucose sensor and send the current signal to the receiver; Receiver: used to process data and display the glucose concentration value in real time.

[0044] In a fifth aspect, the present invention provides use of the glucose sensor described in the first aspect in glucose detection.

[0045] In a sixth aspect, the present invention provides a method for detecting glucose concentration, the method comprising:

[0046] The glucose sensor described in the first aspect is brought into contact with a sample to be tested to detect the glucose concentration therein.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention designs a new glucose sensor structure, uses triblock nucleic acid to connect glucose oxidase (GOx) and horseradish peroxide (HRP), detects glucose through an enzyme cascade reaction, and effectively regulates the density, distribution and configuration of the enzyme on the electrode surface by controlling the density and length of the triblock nucleic acid, which can further improve the efficiency of the enzyme cascade reaction on the electrode surface, providing a glucose sensor with high stability and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a schematic diagram of the principle of the glucose sensor of the present invention;

[0050] Figure 2A IT curve diagram of glucose sensor of the present invention detecting glucose of different concentrations;

[0051] Figure 2B This is a standard curve diagram of the glucose sensor of the present invention detecting different concentrations of glucose;

[0052] Figure 3 This is a graph showing the results of the glucose sensor of the present invention detecting different sugars;

[0053] Figure 4 This is a diagram of the test results of different glucose sensors in Example 2;

[0054] Figure 5A This is a diagram of the test results of different glucose sensors in Example 3;

[0055] Figure 5B This is a diagram of the test results of different glucose sensors in Example 4;

[0056] Figure 5C This is a diagram of the test results of different glucose sensors in Example 5;

[0057] Figure 5D This is a diagram of the test results of different glucose sensors in Example 6;

[0058] Figure 5E This is a diagram of the test results of different glucose sensors in Example 7;

[0059] Fig. 5F This is a diagram of the test results of different glucose sensors in Example 7;

[0060] Figure 6 The glucose sensor detection results in Example 1 and Comparative Example 1 are shown;

[0061] Figure 7 The graphs are the test results of the glucose sensor in Example 1 and Comparative Examples 2-4. DETAILED DESCRIPTION

[0062] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0063] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0064] Example 1

[0065] This embodiment provides a glucose sensor, and the preparation method of the glucose sensor comprises the following steps:

[0066] 1. First, the enzyme-DNA complex was prepared. In short, 100 μL of 40 μM GOx or HRP in 50 mM HEPES (pH 7.5) was reacted with 10-fold excess SPDP at 25°C in the dark for 2 hours, and then the excess SPDP was removed using a 30 kD ultrafiltration membrane. At the same time, the thiol-modified DNA (L1 or L2, the sequence is shown in Table 2) was incubated with 20-fold TCEP at 25°C in the dark for 2 hours to prepare fresh DNA, and the excess TCEP was removed using a 3 kD ultrafiltration membrane. Next, the SPDP-modified enzyme was connected to 10-fold excess SH-DNA at 25°C in the dark for 2 hours, GOx was coupled to L1 DNA, and HRP was coupled to L2 DNA. Finally, the excess SH-DNA was removed by washing and filtered using a 30 kD ultrafiltration membrane.

[0067] 2. Preparation of electrochemical sensor. First, a gold electrode with a diameter of 2 mm was polished with 0.05 μm alumina particles on a microcloth, and then ultrasonicated in ethanol and Milli-Q water for 3 minutes respectively. 2 SO 4 The solution was electrochemically treated through a series of oxidation and reduction cycles. Finally, the electrode was rinsed with water and dried with nitrogen. The washed electrode was incubated overnight at 25°C in a fixation buffer containing 1 μM triblock polyA capture probe (CP50). The probe-modified electrode was then blocked with 0.1 μM mercaptohexanol (MCH) at 25°C for 30 min and rinsed thoroughly with washing buffer. Subsequently, the electrode was incubated at 37°C for 2 h in a hybridization buffer containing 0.5 μM GOx-L1 and 0.05 μM HRP-L2. Finally, the electrode was rinsed with washing buffer and electrochemical measurements were performed.

[0068] The prepared glucose sensor was used to detect different concentrations of glucose. Electrochemical measurements were performed in TMB substrate using an electrochemical workstation and a conventional three-electrode system consisting of a silver / silver chloride reference electrode, a platinum counter electrode, and a gold working electrode. After the HRP-catalyzed reaction reached a steady state, electrochemical reduction cyclic voltammetry (CV) was performed at a scan rate of 50 mV / s, amperometric detection was performed at 100 mV, and the electrochemical reduction current was recorded for 100 s. A standard curve was constructed, and the results are shown in Figure 2. Figure 2A and Figure 2B In addition, the glucose sensor was further used to detect serum samples with known glucose concentrations. The results are shown in Table 1, indicating that the glucose sensor of the present invention can accurately and effectively detect glucose.

[0069] The prepared sensor was used to detect galactose, fructose, sucrose, xylose and mannose samples. The results are shown in Figure 3 As shown, it is demonstrated that the glucose sensor of the present invention has high specificity.

[0070] Table 1

[0071] sample Actual concentration (mM) Detection concentration (mM) Recovery rate (%) 1 5 5.09±0.25 101.8 2 20 20.36±0.74 101.8

[0072] Example 2

[0073] This embodiment provides a glucose sensor, which is prepared by using triblock nucleic acids with different polyA sequence lengths (as shown in Table 2), and the preparation method includes the following steps:

[0074] A 2 mm diameter gold electrode was first polished with 0.05 μm alumina particles on a micro-cloth for 2 min and then sonicated in ethanol and Milli-Q water for 3 min, respectively. 2 SO 4 The solution was electrochemically treated through a series of oxidation and reduction cycles. Finally, the electrode was rinsed with water and dried with nitrogen. The washed electrode was incubated overnight at 25°C in a fixation buffer containing 1 μM triblock polyA capture probe (CP10, CP20, CP30, CP40, CP50 or CP60). The probe-modified electrode was then blocked with 0.1 μM MCH at 25°C for 30 minutes and rinsed thoroughly with wash buffer. Subsequently, the electrode was incubated at 37°C for 2 hours in a hybridization buffer containing 0.5 μM GOx-L1 and 0.05 μM HRP-L2. Finally, the electrode was rinsed with wash buffer and electrochemical measurements were performed.

[0075] Table 2

[0076]

[0077] The prepared sensor was used to detect 10 mM glucose, and the electrochemical signals were compared and analyzed. The results are as follows: Figure 4 As shown, it is shown that controlling the length of the polyA sequence in the triblock nucleic acid can further improve the efficiency of the sensor cascade reaction.

[0078] Example 3

[0079] This embodiment provides a glucose sensor. Compared with Embodiment 1, the glucose sensor is prepared using triblock nucleic acids of different concentrations. The preparation method includes the following steps:

[0080] A gold electrode with a diameter of 2 mm was first polished with 0.05 μm alumina particles on a micro-cloth and then sonicated in ethanol and Milli-Q water for 3 min, respectively. 2 SO 4 The solution was electrochemically treated through a series of oxidation and reduction cycles. Finally, the electrode was rinsed with water and dried with nitrogen. The washed electrode was incubated overnight at 25°C in a fixed buffer containing (0.1μM, 0.5μM, 1μM or 5μM) triblock polyA capture probe (CP30). The probe-modified electrode was then blocked with 0.1μM MCH at 25°C for 30 minutes and rinsed thoroughly with washing buffer. Subsequently, the electrode was incubated at 37°C for 2 hours in a hybridization buffer containing 0.5μM GOx-L1 and 0.5μM HRP-L2. Finally, the electrode was rinsed with washing buffer and electrochemical measurements were performed.

[0081] The prepared sensor was used to detect 10 mM glucose, and the electrochemical signals were compared and analyzed. The results are as follows: Figure 5A As shown, it is shown that controlling the concentration of triblock nucleic acid can further improve the efficiency of the sensor cascade reaction.

[0082] Example 4

[0083] This embodiment provides a glucose sensor. Compared with Embodiment 1, the glucose sensor is prepared by using different concentrations of mercaptohexanol (0 μM, 0.1 μM, 0.5 μM, 1 μM), and the rest is the same as Embodiment 1.

[0084] The prepared sensor was used to detect 10 mM glucose, and the electrochemical signals were compared and analyzed. The results are as follows: Figure 5B As shown, it is shown that controlling the concentration of mercaptohexanol can further improve the efficiency of the sensor cascade reaction.

[0085] Example 5

[0086] This embodiment provides a glucose sensor. Compared with Example 1, the glucose sensor is prepared using different concentrations of glucose oxidase and horseradish peroxidase (0.02 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, and the concentrations of glucose oxidase and horseradish peroxidase are the same), and the rest is the same as Example 1.

[0087] The prepared sensor was used to detect 10 mM glucose, and the electrochemical signals were compared and analyzed. The results are as follows: Figure 5C As shown, it indicates that controlling the concentrations of glucose oxidase and horseradish peroxidase can further improve the efficiency of the sensor cascade reaction.

[0088] Example 6

[0089] This embodiment provides a glucose sensor. Compared with Embodiment 4, the only difference is that the molar concentration ratios of glucose oxidase and horseradish peroxidase are controlled to be 0.2:1, 1:1, 5:1, 10:1 and 20:1 respectively, and the concentration of horseradish peroxidase is 0.05 μM.

[0090] The prepared sensor was used to detect 10 mM glucose, and the electrochemical signals were compared and analyzed. The results are as follows: Figure 5D As shown, it indicates that controlling the concentration ratio of glucose oxidase and horseradish peroxidase can further improve the efficiency of the sensor cascade reaction.

[0091] Example 7

[0092] This embodiment provides a glucose sensor. Compared with Embodiment 4, the only difference is that the molar concentration ratios of glucose oxidase and horseradish peroxidase to SPDP are controlled to be 1:2, 1:5, 1:10 and 1:20 respectively, and the concentration of horseradish peroxidase is 0.05 μM.

[0093] The prepared sensor was used to detect 10 mM glucose, and the electrochemical signals were compared and analyzed. The results are as follows: Figure 5E and Fig. 5F As shown, it is shown that controlling the concentration ratio of glucose oxidase and horseradish peroxidase to cross-linking agent can further improve the efficiency of the sensor cascade reaction.

[0094] Comparative Example 1

[0095] This comparative example provides a glucose sensor. Compared with Example 1, glucose oxidase and horseradish peroxidase are respectively connected to the gold electrode surface through thiol DNA capture probes (CP1-SH and CP2-SH, the sequences are shown in Table 2). The specific preparation method includes the following steps:

[0096] A gold electrode with a diameter of 2 mm was first polished with 0.05 μm alumina particles on a micro-cloth and then sonicated in ethanol and Milli-Q water for 3 min, respectively. 2 SO 4 The solution was electrochemically treated through a series of oxidation and reduction cycles. Finally, the electrode was rinsed with water and dried with nitrogen. The washed electrode was incubated overnight at 25°C in a fixation buffer containing 1 μM each of CP1-SH and CP2-SH. The probe-modified electrode was then blocked with 0.1 μM MCH at 25°C for 30 minutes and rinsed thoroughly with washing buffer. Subsequently, the electrode was incubated at 37°C for 2 hours in a hybridization buffer containing 0.5 μM GOx-L1 and 0.05 μM HRP-L2. Finally, the electrode was rinsed with washing buffer and electrochemical measurements were performed.

[0097] Comparative Example 2

[0098] This comparative example provides a glucose sensor, which, compared with Example 1, is not connected with glucose oxidase and horseradish peroxidase, and the specific preparation method includes the following steps:

[0099] A gold electrode with a diameter of 2 mm was first polished with 0.05 μm alumina particles on a micro-cloth and then sonicated in ethanol and Milli-Q water for 3 min, respectively. 2 SO 4 The solution was electrochemically treated through a series of oxidation and reduction cycles. Finally, the electrode was rinsed with water and dried with nitrogen. The washed electrode was incubated overnight at 25°C in a fixation buffer containing 1 μM triblock polyA capture probe (CP50). The probe-modified electrode was then blocked with 0.1 μM MCH at 25°C for 30 minutes and rinsed thoroughly with wash buffer. Subsequently, the electrode was incubated in hybridization buffer at 37°C for 2 hours. Finally, the electrode was rinsed with wash buffer and electrochemical measurements were performed.

[0100] Comparative Example 3

[0101] This comparative example provides a glucose sensor, which, compared with Example 1, is only connected to glucose oxidase, and the specific preparation method includes the following steps:

[0102] A gold electrode with a diameter of 2 mm was first polished with 0.05 μm alumina particles on a micro-cloth and then sonicated in ethanol and Milli-Q water for 3 min, respectively. 2 SO 4The solution was electrochemically treated through a series of oxidation and reduction cycles. Finally, the electrode was rinsed with water and dried with nitrogen. The washed electrode was incubated overnight at 25°C in a fixation buffer containing 1 μM triblock polyA capture probe (CP50). The probe-modified electrode was then blocked with 0.1 μM MCH at 25°C for 30 minutes and rinsed thoroughly with washing buffer. Subsequently, the electrode was incubated at 37°C for 2 hours in a hybridization buffer containing 0.5 μM GOx-L1. Finally, the electrode was rinsed with washing buffer and electrochemical measurements were performed.

[0103] Comparative Example 4

[0104] This comparative example provides a glucose sensor, which, compared with Example 1, is only connected to horseradish peroxidase, and the specific preparation method includes the following steps:

[0105] A gold electrode with a diameter of 2 mm was first polished with 0.05 μm alumina particles on a micro-cloth and then sonicated in ethanol and Milli-Q water for 3 min, respectively. 2 SO 4 The solution is electrochemically treated through a series of oxidation and reduction cycles. Finally, the electrode is rinsed with water and blown dry with nitrogen. The cleaned electrode is incubated overnight at 25°C in a fixed buffer containing 1μM triblock polyA capture probe (CP50). The probe-modified electrode is then blocked with 0.1μM MCH at 25°C for 30 minutes and rinsed thoroughly with washing buffer. Subsequently, the electrode is incubated at 37°C for 2 hours in a hybridization buffer containing and 0.05μM HRP-L2. Finally, the electrode is rinsed with washing buffer and electrochemical measurements are performed. 5mM glucose was detected using the glucose sensors prepared in Example 1 and Comparative Examples 1-4, respectively, and the detected electrochemical signals were compared and analyzed. The results are shown as follows. Figure 6 and Figure 7 As shown, it is shown that only the glucose sensor with a specific structure of the present invention can perform efficient detection.

[0106] In summary, the present invention designs a new glucose sensor structure, uses triblock nucleic acid to connect glucose oxidase (GOx) and horseradish peroxide (HRP), detects glucose through an enzyme cascade reaction, and effectively regulates the density, distribution and configuration of the enzyme on the electrode surface by controlling the density and length of the triblock nucleic acid, which can further improve the efficiency of the enzyme cascade reaction on the electrode surface, and provides a glucose sensor with high stability and sensitivity.

[0107] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A glucose sensor, characterized in that: The glucose sensor comprises electrodes; The surface of the electrode is connected with triblock nucleic acid and short-chain thiol molecules; The triblock nucleic acid comprises a first sequence, a PolyA sequence and a second sequence connected in sequence, wherein the first sequence and the second sequence are not complementary; the length of the PolyA sequence is 10-100 nt; The triblock nucleic acid is connected to the electrode by relying on the adsorption of the PolyA sequence in the middle region of the triblock nucleic acid to the electrode; The first sequence is connected to glucose oxidase via a complementary pairing sequence of the first sequence, and the second sequence is connected to horseradish peroxidase via a complementary pairing sequence of the second sequence, or, The first sequence is linked to horseradish peroxidase via a complementary pairing sequence to the first sequence, and the second sequence is linked to glucose oxidase via a complementary pairing sequence to the second sequence.

2. The glucose sensor according to claim 1, characterized in that The electrode comprises a gold electrode or a glassy carbon electrode on which gold nanoparticles are deposited.

3. The glucose sensor according to claim 1, characterized in that The short-chain thiol molecule includes any one of mercaptohexanol, dithiothreitol or mercaptoethanol, or a combination of at least two of them.

4. The glucose sensor according to claim 1, characterized in that The length of the first sequence is 10-40 nt.

5. The glucose sensor according to claim 1, characterized in that The length of the second sequence is 10-40 nt.

6. The glucose sensor according to claim 1 or 4, characterized in that: The first sequence includes the sequence shown in SEQ ID NO.

1.

7. The glucose sensor according to claim 1 or 5, characterized in that: The second sequence includes the sequence shown in SEQ ID NO.

2.

8. The glucose sensor according to claim 1, characterized in that The complementary pairing sequence of the first sequence includes the sequence shown in SEQ ID NO.

3.

9. The glucose sensor according to claim 1, characterized in that The complementary pairing sequence of the second sequence includes the sequence shown in SEQ ID NO.

4.

10. A method for preparing a glucose sensor according to any one of claims 1 to 9, characterized in that: The preparation method comprises: Mixing glucose oxidase with the complementary pairing sequence of the first sequence and a cross-linking agent, and mixing horseradish peroxidase with the complementary pairing sequence of the second sequence and a cross-linking agent to obtain an enzyme mixture, or, Mixing horseradish peroxidase with the complementary pairing sequence of the first sequence and a cross-linking agent, and mixing glucose oxidase with the complementary pairing sequence of the second sequence and a cross-linking agent to obtain an enzyme mixture; The enzyme mixture, triblock nucleic acid and mercaptohexanol are added to an electrode to obtain the glucose sensor.

11. The method for preparing a glucose sensor according to claim 10, characterized in that: The concentrations of the glucose oxidase and horseradish peroxidase are independently 0.02-1 μmol / L.

12. The method for preparing a glucose sensor according to claim 10, characterized in that: The molar concentration ratio of the glucose oxidase to the cross-linking agent is 1:(2-20).

13. The method for preparing a glucose sensor according to claim 10, characterized in that: The molar concentration ratio of the horseradish peroxidase to the cross-linking agent is 1:(2-20).

14. The method for preparing a glucose sensor according to claim 10, characterized in that: The molar concentration ratio of the glucose oxidase to the horseradish peroxidase is (0.2-20):

1.

15. The method for preparing a glucose sensor according to claim 10, characterized in that: The concentration of the triblock nucleic acid is 0.1-5 μmol / L.

16. The method for preparing a glucose sensor according to claim 10, characterized in that: The concentration of the mercaptohexanol is 0.1-1 mmol / L.

17. Use of the glucose sensor according to any one of claims 1 to 9 in the preparation of glucose detection products.

18. A glucose detection device, characterized in that: The glucose detection device comprises the glucose sensor according to any one of claims 1-9.

19. The glucose detection device according to claim 18, characterized in that: The glucose detection device also includes: Transmitter: used to receive the current signal generated by the glucose sensor and send the current signal to the receiver; Receiver: used for data processing and real-time display of glucose concentration values.

20. Use of the glucose sensor according to any one of claims 1 to 9 in glucose detection.

21. A method for detecting glucose concentration, characterized in that: The method comprises: The glucose sensor according to any one of claims 1 to 9 is contacted with a sample to be tested to detect the glucose concentration therein.

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