Construction method of electrochemical-glucometer dual-mode sensor based on copper phosphate hybrid nanoflowers

Abstract

The invention relates to a construction method of an electrochemical-glucometer dual-mode sensor based on streptavidin modified copper phosphate hybridized nanoflowers (SA-Cu3 (PO4) 2 HNFs), a dual-function signal probe based on SA-Cu3 (PO4) 2 HNFs is prepared for the first time, electrochemistry and a glucometer are combined to construct the dual-mode sensor, the experimental steps are simplified, the detection accuracy and reliability are improved, and the application prospect is wide. And field detection can be realized. Jointly incubating the signal probe, the magnetic bead and the aptamer to construct a signal probe / aptamer / magnetic bead system. Aflatoxin B1 is added into a composite system, the signal probe falls off, the free signal probe in supernate is dropwise added to the surface of a gold electrode after magnetic separation, and electrochemical signals are detected by an electrochemical workstation after incubation. Meanwhile, a sucrose solution is added into the precipitate for reaction, the glucose content is measured through a glucometer, and finally electrochemical-glucometer dual-mode detection of the aflatoxin B1 is achieved through response to an electrochemical signal and a glucometer signal.

Description

Technical Field

[0001] The invention belongs to the technical field of harmful substance detection, and in particular relates to a method for constructing an electrochemical-blood glucose meter dual-mode sensor based on copper phosphate hybrid nanoflowers. Background Art

[0002] Aflatoxin B1 (AFB1) is a toxic metabolite produced by molds. These molds are mainly Aspergillus, such as Aspergillus flavus and Aspergillus parasiticus. These molds are widely present in the natural environment and are prone to contaminating many foods and feeds. AFB1 is the most toxic aflatoxin, which has teratogenic and carcinogenic effects, can easily cause liver damage, and affect animal and human health. Many countries and regions have established strict standards to limit the residue of AFB1 in food and feed.

[0003] Traditional AFB1 detection methods mainly focus on chromatography-mass spectrometry techniques, such as high-performance liquid chromatography, high-performance liquid chromatography-tandem mass spectrometry, and thin-layer chromatography. Although these methods are accurate and reliable, they are complex, time-consuming, and expensive, and cannot meet the requirements of on-site detection and rapid detection. In recent years, aptamer-based methods have been widely used in the rapid detection of AFB1, mainly involving electrochemistry, fluorescence, colorimetry, and surface-enhanced Raman scattering. Among them, electrochemical sensors have been used in the detection of fungal toxins due to their advantages such as strong specificity, high sensitivity, and fast response speed. However, the relatively complex readout mode limits its application in on-site detection, and the single output mode is easily affected by the environment, resulting in false positive results. Therefore, it is very necessary to develop an AFB1 on-site detection method that is simple to operate and fast to detect.

[0004] Blood glucose meters are small in size, low in cost, simple to operate, and can achieve on-site detection, but can only detect glucose. Therefore, in the present invention, a dual-function signal probe is prepared based on copper phosphate hybrid nanoflowers, which realizes the quantitative detection of non-glucose targets, and an electrochemical-blood glucose meter dual-mode sensor is developed. The dual output mode improves the detection accuracy and reliability of the sensor, and can achieve on-site rapid detection of AFB1, which has broad application prospects. Summary of the invention

[0005] A method for constructing an electrochemical-glucose meter dual-mode sensor based on copper phosphate hybrid nanoflowers comprises the following steps: (1) Preparation of signal probe: Streptavidin SA and copper sulfate were added to phosphate buffer, reacted at room temperature for 12 h, and then the solution was centrifuged to obtain streptavidin-modified copper phosphate hybrid nanoflowers (SA-Cu3(PO4)2HNFs). Complementary chain cDNA was added and mixed and incubated for 2 h to form SA-Cu3(PO4)2HNFs / DNA complex. Biotinylated convertase was added to the SA-Cu3(PO4)2HNFs / DNA complex, mixed and incubated at room temperature for 2 h, and the reaction was continued at 4°C overnight to complete the preparation of signal probe. (2) Construction of signal probe / aptamer / magnetic bead system: The streptavidin magnetic beads SA-MBs and the aptamer Apt were reacted at 37°C for 1 h to complete the fixation of Apt and SA-MBs. The signal probe was added to the composite system of Apt and SA-MBs, and the system was shaken at 37°C for 1 h to complete the construction of the signal probe / aptamer / magnetic bead system. (3) Construction of electrochemical-blood glucose meter dual-mode aptamer sensor: Different concentrations of AFB1 were added to the signal probe / aptamer / magnetic bead system. Apt bound to AFB1 and the signal probe fell off. After magnetic separation, the free signal probe in the supernatant was dripped onto the surface of the gold electrode. After incubation, the electrochemical signal was detected using an electrochemical workstation. At the same time, sucrose solution was added to the precipitate for reaction. The invertase on the signal probe converted sucrose into glucose, which was measured using a blood glucose meter. Finally, the electrochemical-blood glucose meter dual-mode detection of AFB1 was achieved by responding to the electrochemical signal and the blood glucose meter signal.

[0006] It is further defined that in step (1), the signal probe itself has an electrochemical signal, and the invertase on the signal probe converts sucrose into glucose.

[0007] It is further defined that in step (1), one end of the complementary chain cDNA is modified with a group that binds to SA-Cu3(PO4)2HNFs, and the other end is modified with a group that binds to a gold electrode, which is one or more of biotin, amino, carboxyl, and thiol.

[0008] It is further defined that in step (1), the biotinylated converting enzyme is obtained by reacting the converting enzyme with one or more of a biotin solution, biotin amide hexanoyl hydrazide, and biotin-N-succinimidyl ester.

[0009] It is further defined that in step (2), one end of the aptamer Apt is modified with a group that binds to SA-MBs, which is one or more of biotin, amino, carboxyl, and thiol.

[0010] It is further defined that in step (2), the SA-MB concentration is 10 mg / mL and the volume used is 5-10 μL.

[0011] It is further defined that in step (3), the amount of the signal probe added to the surface of the gold electrode is 5-10 μL, and the incubation time is 1-2 h.

[0012] It is further defined that in step (3), the concentration of the sucrose solution is 200 mg / mL, the amount added is 20-50 μL, and the reaction temperature is 40-70°C.

[0013] Compared with the prior art, the present invention has the following significant advantages: 1. The present invention first synthesized SA-Cu3(PO4)2HNFs with electrochemical signals, and combined complementary chain cDNA and biotinylated convertase as a bifunctional signal probe, which was simultaneously used for electrochemical and blood glucose meter detection.

[0014] 2. The present invention avoids the complex reaction of the DNA chain, simplifies the experimental steps, and improves the stability of the sensor.

[0015] 3. The present invention utilizes dual-mode signals to self-supervise the detection results, which is conducive to achieving more accurate detection performance.

[0016] 4. The blood glucose meter is small in size, low in cost, and has a fast signal response. It can be used for rapid on-site testing, which is conducive to the promotion and application of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the construction of electrochemical-blood glucose meter dual-mode sensor based on Cu3(PO4)2 hybrid nanoflowers.

[0018] Figure 2 This is a scanning electron microscope image of the SA-Cu3(PO4)2HNFs prepared in Example 1 of the present invention.

[0019] Figure 3 The standard curves of electrochemical detection (A) and blood glucose meter detection (B) of AFB1 in Example 1 of the present invention.

[0020] Figure 4 This is a comparative diagram of the selectivity of the electrochemical-blood glucose meter dual-mode sensor constructed in Example 2 of the present invention for AFB1 in the presence of other interfering toxins (Figure A is electrochemical detection, and Figure B is blood glucose meter detection). DETAILED DESCRIPTION

[0021] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1

[0022] A method for constructing an electrochemical-glucose meter dual-mode sensor based on Cu3(PO4)2 hybrid nanoflowers, the implementation method of which is as follows Figure 1 shown.

[0023] A method for constructing an electrochemical-glucose meter dual-mode sensor based on Cu3(PO4)2 hybrid nanoflowers, comprising the following steps: (1) Preparation of SA-Cu3(PO4)2HNFs: First, add 20 μL, 5 mg / mL streptavidin SA and 6.7 μL, 120 mM copper sulfate solution into phosphate buffer (1 mL, 30 mM, pH=7.4). After reacting at room temperature for 12 h, the solution was centrifuged to collect the SA-Cu3(PO4)2HNFs precipitate. Figure 2 Shown is the scanning electron microscopy image of SA-Cu3(PO4)2HNFs.

[0024] (2) Preparation of signal probe: 200 μL, 2 μM biotin-modified complementary chain cDNA was mixed with SA-Cu3(PO4)2HNFs and incubated for 2 h to form a SA-Cu3(PO4)2HNFs / DNA complex. Biotinylation convertase was added to the SA-Cu3(PO4)2HNFs / DNA complex, mixed and incubated at room temperature for 2 h, and the reaction was continued at 4°C overnight to complete the preparation of the signal probe.

[0025] (3) Construction of signal probe / aptamer / magnetic bead system: 5 μL, 10 mg / mL SA-MBs and 10 μL, 2 μM biotin-modified aptamer bio-Apt were reacted at 37°C for 1 h to complete the fixation of bio-Apt and SA-MBs. Unbound bio-Apt was removed from the supernatant by magnetic separation to obtain the bio-Apt / SA-MBs complex. 20 μL of signal probe was added to the substrate and the signal probe / aptamer / magnetic bead system was constructed by constant temperature shaking at 37°C for 1 h.

[0026] (4) Construction of electrochemical-glucose meter dual-mode sensor: Different concentrations of AFB1 were added to the signal probe / aptamer / magnetic bead system and kept at 37°C for 30 min. Bio-Apt bound to AFB1 and caused the signal probe to fall off. After magnetic separation, the free signal probe was dispersed in the supernatant. 5 μL was added to the surface of the gold electrode and incubated at 37°C for 1 h. At the same time, 200 mg / mL sucrose solution was added to the precipitate and reacted at 50°C for 1 h.

[0027] (5) Electrochemical-glucose meter dual-mode detection of AFB1: In the electrochemical mode, a saturated silver chloride electrode is used as the reference electrode and a platinum wire electrode is used as the counter electrode. The detection of AFB1 is completed by the response of SA-Cu3(PO4)2HNFs in a phosphate buffer solution. The corresponding linear relationship curve is drawn based on the relationship between the measured current value and the concentration of AFB1 added. After adding sucrose solution to the precipitate, sucrose is converted into glucose under the action of invertase and measured with a blood glucose meter. The corresponding linear relationship curve is drawn based on the relationship between the measured blood glucose meter reading and the concentration of AFB1 added.

[0028] like Figure 3 As shown in A, the electrochemical signal increases with the increase of AFB1 concentration, and the linear regression equation is I=1.121LgC AFB1 +6.98(R 2 =0.994), and the minimum detection limit (LOD) was 0.49 pg / mL. Figure 3 As shown in B, the blood glucose meter reading decreases with the increase of AFB1 concentration, and the linear regression equation is Y=-5.293 LgC AFB1 +11.36(R 2 =0.996), LOD was 5.4 pg / mL. Where I represents the current value, Y represents the reading of the blood glucose meter, and C represents the concentration of AFB1 (ng / mL). Example 2

[0029] A method for constructing an electrochemical-glucose meter dual-mode sensor based on copper phosphate hybrid nanoflowers comprises the following steps: In order to verify that the prepared electrochemical-glucose meter dual-mode sensor has specific recognition of AFB1, AFB1 standard was added to the buffer solution to make its concentration 10 ng / mL; other interfering toxins (AFB2, AFM1, AFG1, FB1, OTA, OTB) standard solutions with concentrations of 100 ng / mL were prepared with the buffer solution. The above-mentioned different interfering toxin standards were tested according to the detection system constructed in Example 1. The test results are shown in the figure. Figure 4 As shown, it is shown that the method of the present invention has a higher selectivity for AFB1. Example 3

[0030] A method for constructing an electrochemical-glucose meter dual-mode sensor based on copper phosphate hybrid nanoflowers comprises the following steps: (1) Actual sample processing: Take 0.5 g of buckwheat flour spiked sample and add it to 10 mL of methanol / water solution (ratio 7:3), place it in a water bath constant temperature oscillator and shake for 30 min. After shaking, take it out and place it in a centrifuge for centrifugal separation. After centrifugation, filter it through a disposable syringe filter (0.22 μm) to obtain the supernatant.

[0031] (2) Sample detection: According to steps (1) to (5) of Example 1, the current value and the blood glucose meter reading were obtained, and the concentration of AFB1 in the sample was obtained by substituting the standard curve into the standard curve. Each sample was measured three times and the average value was taken, and the relative standard deviation (RSD) and the average recovery rate were calculated.

[0032] The electrochemical-glucose meter dual-mode sensor has been verified to have high accuracy, good sensitivity and good stability in the detection of AFB1. At the same time, the test results of actual samples (such as buckwheat flour) show that the dual-mode sensor has very good practical application value.

[0033] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0034] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for constructing an electrochemical-glucose meter dual-mode sensor based on copper phosphate hybrid nanoflowers, characterized in that: The following steps are included (1) Preparation of signal probe: Streptavidin SA and copper sulfate were added to phosphate buffer, reacted at room temperature for 12 h, and then the solution was centrifuged to obtain streptavidin-modified copper phosphate hybrid nanoflowers (SA-Cu3(PO4)2HNFs). Complementary chain cDNA was added and mixed and incubated for 2 h to form SA-Cu3(PO4)2HNFs / DNA complex. Biotinylated convertase was added to the SA-Cu3(PO4)2HNFs / DNA complex, mixed and incubated at room temperature for 2 h, and the reaction was continued at 4°C overnight to complete the preparation of signal probe. (2) Construction of signal probe / aptamer / magnetic bead system: The streptavidin magnetic beads SA-MBs and the aptamer Apt were reacted at 37°C for 1 h to complete the fixation of Apt and SA-MBs. The signal probe was added to the composite system of Apt and SA-MBs, and the system was shaken at 37°C for 1 h to complete the construction of the signal probe / aptamer / magnetic bead system. (3) Construction of electrochemical-blood glucose meter dual-mode aptamer sensor: Different concentrations of aflatoxin B1 were added to the signal probe / aptamer / magnetic bead system. Apt bound to aflatoxin B1 and the signal probe fell off. After magnetic separation, the free signal probe in the supernatant was dripped onto the surface of the gold electrode. After incubation, the electrochemical signal was detected using an electrochemical workstation. At the same time, sucrose solution was added to the precipitate for reaction. The invertase on the signal probe converted sucrose into glucose, which was measured using a blood glucose meter. Finally, the electrochemical-blood glucose meter dual-mode detection of aflatoxin B1 was achieved by responding to the electrochemical signal and the blood glucose meter signal.

2. The method for constructing an electrochemical-blood glucose meter dual-mode aptasensor based on SA-Cu3(PO4)2HNFs according to claim 1, characterized in that: In step (1), the signal probe itself has an electrochemical signal, and the invertase on the signal probe converts sucrose into glucose.

3. The method for constructing an electrochemical-glucose meter dual-mode aptasensor based on SA-Cu3(PO4)2HNFs according to claim 1, characterized in that: In step (1), one end of the complementary chain cDNA is modified with a group that binds to SA-Cu3(PO4)2HNFs, and the other end is modified with a group that binds to a gold electrode, which is one or more of biotin, amino, carboxyl, and thiol.

4. The method for constructing an electrochemical-blood glucose meter dual-mode aptasensor based on SA-Cu3(PO4)2HNFs according to claim 1, characterized in that: In step (1), the biotinylated converting enzyme is obtained by reacting the converting enzyme with one or more of a biotin solution, biotin amide hexanoyl hydrazide, and biotin-N-succinimidyl ester.

5. The method for constructing an electrochemical-blood glucose meter dual-mode aptasensor based on SA-Cu3(PO4)2HNFs according to claim 1, characterized in that: In step (2), one end of the aptamer Apt is modified with a group that binds to SA-MBs, which is one or more of biotin, amino, carboxyl, and thiol.

6. The method for constructing an electrochemical-glucose meter dual-mode aptasensor based on SA-Cu3(PO4)2HNFs according to claim 1, characterized in that: In step (2), the SA-MB concentration is 10 mg / mL and the volume used is 5-10 μL.

7. The method for constructing an electrochemical-blood glucose meter dual-mode aptasensor based on SA-Cu3(PO4)2HNFs according to claim 1, characterized in that: In step (3), the signal probe is added to the surface of the gold electrode in an amount of 5 to 10 μL, and the incubation time is 1 to 2 h.

8. The method for constructing an electrochemical-blood glucose meter dual-mode aptasensor based on SA-Cu3(PO4)2HNFs according to claim 1, characterized in that: In step (3), the concentration of the sucrose solution is 200 mg / mL, the amount added is 20~50 μL, and the reaction temperature is 40~70°C.

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