CN114486870B - Nanometer enzyme functionalized paper-based sensor for rapid blood glucose detection and detection method thereof - Google Patents
Nanometer enzyme functionalized paper-based sensor for rapid blood glucose detection and detection method thereof Download PDFInfo
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Abstract
The invention provides a nano enzyme functionalized paper-based sensor for rapid blood glucose detection. The invention uses AB-Hemin with POD activity to form a sensing interface on the paper-based surface, and specifically detects the blood sugar of a patient. The POD (peroxidase) activity of the carbon material can be greatly improved by modifying the carbon material with the Hemin, and the AB which does not have the POD activity is found after the AB is modified by using the Hemin, so that the modified AB has considerable enzyme activity and can be used for colorimetric detection of glucose. The detection method is simple to operate and easy to popularize and apply; the method can be applied to quick detection mechanisms in the field, such as hospitals, families, customs of food safety and inspection and quarantine, airports and the like.
Description
Technical Field
The invention belongs to the technical field of nanocomposite materials, and relates to a nano enzyme functionalized paper-based sensor for rapid detection of blood sugar and a detection method thereof.
Background
Diabetes is a group of metabolic diseases caused by multiple causes characterized by chronic hyperglycemia. Diabetes is usually accompanied by various complications, and is susceptible to microvascular and macrovascular lesions, which lead to complications of the kidney, eye, nervous system, cardiovascular system, etc., and are the main cause of fatal disability for diabetics. The early detection of excessive blood glucose levels in diabetics helps control the patient's blood glucose and subsequent symptom control.
The reliable way of clinical blood sugar detection is mainly a biochemical analyzer, and the domestic blood sugar detection way is mainly a blood sugar meter, and other blood sugar detection ways are also micro-needle methods and the like. Although the biochemical analyzer is called a gold standard for blood glucose test, the application of the biochemical analyzer requires a special instrument and a professional operator, and the household blood glucose analyzer is usually matched with a corresponding test paper, so that the blood glucose value is difficult to directly obtain by a micro-needle method.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a nano enzyme functionalized paper-based sensor for rapid blood glucose detection. The AB-Hemin with excellent POD activity is immobilized on the paper-based surface, so that a color signal of glucose which is catalyzed and displayed can be effectively captured, the content of glucose is accurately measured, and a novel method for clinically detecting the blood sugar is provided.
Except for special descriptions, the parts are parts by weight, and the percentages are mass percentages.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a nano enzyme functionalized paper-based sensor for rapid blood glucose detection, which is characterized in that: the paper-based sensor is constructed by using hemin modified acetylene black nano-enzyme.
The preparation method of the Hemin modified acetylene black nanoenzyme (AB-Hemin) comprises the following steps: dissolving Hemin (Hemin) with N, N-Dimethylformamide (DMF), adding Acetylene Black (AB) solution dissolved with ultrapure water, stirring for 3-8min, then placing in an ultrasonic cleaner for ultrasonic dispersion for 40-60min, stirring for 15-20h at normal temperature, centrifuging at a rotation speed of 10000-15000rpm/min, and obtaining precipitate, namely Hemin modified acetylene black nanoenzyme (AB-Hemin).
The construction method of the nano enzyme functionalized paper-based sensor for rapid blood glucose detection comprises the following steps: cutting Whatman CHR No. 1 filter paper into round paper sheets by using a puncher, dripping AB-Hemin water solution onto the cut round paper sheets, and drying at 37 ℃ for 30-40min; and (3) dropwise adding an ethanol solution of TMB to the surface of the dried paper base, and waiting for the volatilization of ethanol until the paper base is dry at normal temperature, thereby completing the construction of the paper base sensor.
The above-mentioned aqueous solution of AB-Hemin was a dilution of 20. Mu.L of AB-Hemin in 1mL of sodium acetate buffer of pH=4.
The ethanol solution of TMB was 35mM.
A nano enzyme functionalized paper-based sensor for rapid detection of blood sugar is constructed by a chloromethemoglobin modified acetylene black nano enzyme; the preparation method is characterized by comprising the following steps:
(1) Preparation of AB-Hemin:
4mg of Hemin (Hemin) was weighed and 4mL of a solution of N, N-Dimethylformamide (DMF) was added; dissolving 4mg of Acetylene Black (AB) in 4mL of ultrapure water in an ultrasonic cleaner at normal temperature for 5min to fully dissolve, then slowly adding the 4mg of Acetylene Black (AB) into the DMF solution of the Hemin under the condition of stirring at normal temperature, continuously stirring for 5min, then placing the mixture in the ultrasonic cleaner for ultrasonic dispersion for 50min, stirring the mixture after ultrasonic treatment for 16h at normal temperature, centrifugally cleaning the mixture to obtain clear and colorless supernatant at the rotating speed of 12000rpm/min, redispersing the supernatant with ultrapure water, and fixing the volume to 4mL to obtain AB-Hemin.
(2) Construction of paper-based sensor: cutting Whatman CHR No. 1 filter paper into circular paper sheets with the diameter of 6mm by using a puncher with the diameter of 6mm, dripping 20 mu L of AB-Hemin water solution onto the cut circular paper sheets, and drying at 37 ℃ for 35min; dropwise adding an ethanol solution of TMB (TMB) to the surface of the dried paper base, and waiting for the volatilization of ethanol until the paper base is dry at normal temperature, thereby completing the construction of the paper base sensor; the ethanol solution of TMB was 35mM, in an amount of 5. Mu.L.
The invention also provides a method for detecting blood sugar by using the nano enzyme functionalized paper-based sensor, and the method is simple to operate and has strong practicability.
A method for detecting blood glucose by a nano enzyme functionalized paper-based sensor, which is characterized by comprising the following steps:
(1) Preparing standard solutions with different concentrations by using glucose standard substances, respectively mixing the standard solutions with PBS (phosphate buffer solution) dissolved with GOD, and reacting in a 37 ℃ oven for 30min to obtain a plurality of standard mixed solutions;
(2) Dripping each standard mixed liquid on the surface of the paper-based sensor, and after reacting for 18min, reading RGB color values of color signals by using a smart phone to establish a linear relation between glucose concentration and the color signals;
(4) Mixing PBS (pH=6) solution dissolved with GOD with serum, and mixing at 37deg.C for 30min; and (2) dripping the mixed solution on the surface of the paper-based sensor, reading RGB values of color signals by using a smart phone after waiting for 18min of reaction, and substituting the RGB values into the linear relation curve obtained in the step (2) after signal processing, so as to obtain the blood glucose concentration of the serum to be detected.
The beneficial effects are that:
the invention uses AB-Hemin with POD activity to form a sensing interface on the paper-based surface, and specifically detects the blood sugar of a patient. The POD (peroxidase) activity of the carbon material can be greatly improved by modifying the carbon material with the Hemin, and the AB which does not have the POD activity is found after the AB is modified by using the Hemin, so that the modified AB has considerable enzyme activity and can be used for colorimetric detection of glucose.
Compared with the prior art, the paper-based sensor for detecting glucose in blood has the outstanding characteristics that:
(1) According to the invention, the Hemin is used for functionalizing the AB for the first time, so that the AB POD activity is remarkably endowed, and a very obvious color signal can be generated in colorimetric application.
(2) The invention combines paper base with acetylene black for paper base colorimetric detection of blood sugar with low cost and no pollution, has high sensitivity, good stability, wide application range, simple operation and use method and easy popularization and application; the method can be applied to quick detection mechanisms in the field, such as hospitals, families, customs of food safety and inspection and quarantine, airports and the like.
(3) The invention can specifically identify glucose, can realize quantitative detection of blood sugar by utilizing the smart phone, does not need complex detection equipment, and can obtain results on site.
Drawings
FIG. 1 shows the results of the sensor of the present invention for detecting different concentrations of glucose and a linear calibration line.
FIG. 2 shows the results of glucose specific detection by the sensor of the present invention.
FIG. 3 is a graph showing the results of the batch-to-batch variation of the sensors prepared according to the present invention.
FIG. 4 is a graph showing the stability test results of the sensor of the present invention.
Detailed Description
The present invention is described in detail below by way of specific examples, which are given herein for the purpose of further illustration only and are not to be construed as limiting the scope of the present invention, as many insubstantial modifications and variations of the present invention will become apparent to those skilled in the art in light of the foregoing disclosure. The raw materials and the reagents used in the invention are all commercial products.
Example 1
The main chemical reagents used in the examples of the present invention are as follows:
hemin was purchased from Sigma-Aldrich (USA). AB was purchased from lean (Shanghai) investment management company, inc. (Shanghai, china). N, N-Dimethylformamide (DMF) was purchased from Michael Biochemical technology Co., ltd (Shanghai, china).
Example 1 nanomaterial AB-Hemin was prepared.
The operation is as follows (the construction schematic is shown in fig. 1):
(1) 4mg of Hemin (Hemin) was weighed and added to 4mL of N, N-Dimethylformamide (DMF) solution; dissolving 4mg of Acetylene Black (AB) in 4mL of ultrapure water, performing ultrasonic treatment at normal temperature for 5min to fully dissolve the Acetylene Black (AB), then adding a DMF solution dissolved with Hemin slowly under the condition of stirring at normal temperature, continuously stirring for 5min, performing ultrasonic dispersion for 50min in the ultrasonic cleaner, stirring for 16h at normal temperature, washing with ultrapure water three times at a speed of 12000rpm/min for 5min each time, re-dispersing with ultrapure water, and fixing the volume to form an AB-Hemin solution of 1 mg/mL.
Example 2 construction of paper-based sensor for rapid blood glucose detection
(1) 20 μl of the AB-Hemin solution prepared in example 1 was dispersed in 1mL of NaAc-HAc buffer at pH=4, and vortexed to disperse uniformly.
(2) The filter paper is cut into small round paper sheets with the diameter of 6mm by a puncher with the diameter of 6mm for standby.
(3) And (3) dripping 20 mu L of the AB-Hemin buffer solution prepared in the step (1) to the surface of the paper sheet prepared in the step (2), and drying at 37 ℃ for 35min to form a dry state.
(4) TMB 8.5mg was weighed out in 1mL of absolute ethanol to give 1mL of TMB solution having a concentration of 35mM.
(5) And (3) dripping 5 mu L of the TMB solution prepared in the step (4) to the dried paper-based surface in the step (3), and airing in air to obtain the nano enzyme functionalized paper-based sensor for rapidly detecting blood sugar.
Example 3 detection of blood glucose Using paper-based sensor
Blood glucose was measured using the paper-based sensor constructed in example 2, and the procedure was as follows:
(1) Drawing a standard curve:
glucose standard solutions with concentrations of 0.2,0.5,0.6,0.8,0.9,1.0,2.0,5.0, 10, 20 and 30mM are respectively prepared, 20 mu L of the glucose standard solution is respectively mixed with 0.5mg/mL of PBS (pH=6) solution of GOD, the mixture is reacted at 37 ℃ for 30min, 10 mu L of the reaction solution is respectively dripped on the surface of the paper-based sensor constructed in the embodiment 2, and after waiting for 18min, a smart phone is used for respectively reading RGB values of color signals. A standard curve is drawn, the detection result shows that the two are in good linear relation in the concentration range, the linear correlation coefficient is 0.9973, the detection limit is 0.1mM, and the result is shown in figure 1.
(2) Sensor-specific detection:
in order to detect the specificity of the sensor of the invention, the following interfering substances were detected: maltose (5 mM), sucrose (5 mM), fructose (5 mM), lactose (5 mM). The RGB signals of different interfering substances on the paper substrate were measured under the same conditions. The results show (FIG. 2) that the biosensor of the present invention has satisfactory specificity.
(3) Stability detection of nanomaterial:
the prepared materials are respectively placed at the temperature of minus 20 ℃ and the temperature of 4 ℃ and the normal temperature for 10 days, and then the sensor is constructed, and the result shows that the response value to the measurement of glucose after 10 days of storage is still more than 80% of the initial value; the data (fig. 3) indicate that the sensor prepared from this material has acceptable stability.
(4) Sensor reproducibility detection:
glucose (10 mM) was measured under the same conditions using 5 different sensors of the same batch prepared according to the present invention, and the results are shown in FIG. 4, wherein the Relative Standard Deviation (RSD) of the current response values is 1.37%, indicating smaller variation in sensor batch and better reproducibility.
(5) Practical sample analysis application:
to evaluate the practical applicability and accuracy of the proposed paper-based sensor, 20 μl of human serum samples were mixed with 0.5mg/mL of GOD in PBS (ph=6), and RGB value reading calculation was performed using the prepared sensor test, and the blood glucose value was calculated by substituting into the standard curve equation obtained in step (1) of example 3, and compared with the commercial blood glucose meter measurement value. The results are shown below (Table 1).
Table 1 paper-based sensor prepared according to the present invention for detecting blood glucose
The results showed RSD values of 2.32% to 6.96%. Therefore, the paper-based sensor prepared by the invention is feasible for detecting blood sugar, and can meet the requirement of actual analysis.
Claims (5)
1. A nano enzyme functionalized paper-based sensor for rapid blood glucose detection, which is characterized in that: the paper-based sensor is formed by dripping aqueous solution of hemin modified acetylene black nano-enzyme on paper sheets, drying, dripping ethanol solution of TMB on the surface of the paper base, and drying; the preparation method of the hemin modified acetylene black nanoenzyme comprises the following steps: dissolving hemin with N, N-Dimethylformamide (DMF), adding acetylene black solution dissolved with ultrapure water, stirring for 3-8min, then placing in an ultrasonic cleaner for ultrasonic dispersion for 40-60min, stirring for 15-20h at normal temperature, and centrifuging at a rotating speed of 10000-15000rpm/min to obtain precipitate, namely the hemin modified acetylene black nanoenzyme.
2. The paper-based sensor according to claim 1, wherein the construction method of the nano enzyme functionalized paper-based sensor for rapid blood glucose detection comprises the following steps: cutting Whatman CHR No. 1 filter paper into round paper sheets by using a puncher, dripping the water solution of acetylene black nano enzyme on the cut round paper sheets, and drying at 37 ℃ for 30-40min; and (3) dropwise adding an ethanol solution of TMB to the surface of the dried paper base, and waiting for the volatilization of ethanol until the paper base is dry at normal temperature, thereby completing the construction of the paper base sensor.
3. The paper-based sensor of claim 1, wherein: the ethanol solution of TMB was 35mM.
4. The paper-based sensor of claim 1, prepared by the steps of:
(1) Preparation of acetylene black nano enzyme:
4mg of hemin was weighed and 4mL of N, N-Dimethylformamide (DMF) solution was added; dissolving 4mg of acetylene black in 4mL of ultrapure water in an ultrasonic cleaner at normal temperature for 5min to fully dissolve the acetylene black, then adding the 4mL of ultrapure water into DMF solution of hemin slowly under the condition of stirring at normal temperature, continuously stirring for 5min, then placing the mixture in the ultrasonic cleaner for ultrasonic dispersion for 50min, stirring the mixture after ultrasonic treatment at normal temperature for 16h, centrifugally cleaning the mixture at a rotating speed of 12000rpm/min until the supernatant is transparent and colorless, redispersing the mixture with ultrapure water, and fixing the volume to 4mL to obtain the aqueous solution of acetylene black nano-enzyme;
(2) Construction of paper-based sensor: cutting Whatman CHR No. 1 filter paper into circular paper sheets with the diameter of 6mm by using a puncher with the diameter of 6mm, dripping 20 mu L of aqueous solution of acetylene black nano-enzyme onto the cut circular paper sheets, and drying at 37 ℃ for 35min; dropwise adding an ethanol solution of TMB (TMB) to the surface of the dried paper base, and waiting for the volatilization of ethanol until the paper base is dry at normal temperature, thereby completing the construction of the paper base sensor; the ethanol solution of TMB was 35mM, in an amount of 5. Mu.L.
5. A method for detecting blood glucose using a nanoenzyme functionalized paper-based sensor according to any one of claims 1-4, comprising the steps of:
(1) Preparing standard solutions with different concentrations by using glucose standard substances, respectively mixing the standard solutions with PBS (phosphate buffer solution) dissolved with GOD, and reacting in a 37 ℃ oven for 30min to obtain a plurality of standard mixed solutions;
(2) Dripping each standard mixed liquid on the surface of the paper-based sensor, and after reacting for 18min, reading RGB color values of color signals by using a smart phone to establish a linear relation between glucose concentration and the color signals;
(3) Mixing PBS solution with dissolved GOD and pH=6 with serum, and mixing at 37deg.C for 30min; and (2) dripping the mixed solution on the surface of the paper-based sensor, reading RGB values of color signals by using a smart phone after waiting for 18min of reaction, and substituting the RGB values into the linear relation curve obtained in the step (2) after signal processing, so as to obtain the blood glucose concentration of the serum to be detected.
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| CN1751743A (en) * | 2005-09-12 | 2006-03-29 | 重庆医科大学附属第二医院 | Treat leukemic 131The proteinic preparation method of I marker GM-CSF |
| JP2008045938A (en) * | 2006-08-11 | 2008-02-28 | Toshiba Medical Systems Corp | Photoacoustic analysis and photoacoustic analyzer for concentration measurement of analysis object in specimen tissue |
| CN106442667A (en) * | 2016-09-23 | 2017-02-22 | 桂林电子科技大学 | Method for detecting blood glucose by virtue of persimmon tannin@graphene@Pt-Pd non-enzymatic sensor |
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| CN110694069B (en) * | 2019-11-08 | 2022-02-25 | 重庆医科大学附属第二医院 | Medicine for preventing and treating fulminant hepatitis |
| CN112316984B (en) * | 2020-11-20 | 2022-05-13 | 华中科技大学 | Supported metalloporphyrin/phthalocyanine catalyst, and preparation method and application thereof |
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| CN1751743A (en) * | 2005-09-12 | 2006-03-29 | 重庆医科大学附属第二医院 | Treat leukemic 131The proteinic preparation method of I marker GM-CSF |
| JP2008045938A (en) * | 2006-08-11 | 2008-02-28 | Toshiba Medical Systems Corp | Photoacoustic analysis and photoacoustic analyzer for concentration measurement of analysis object in specimen tissue |
| CN106442667A (en) * | 2016-09-23 | 2017-02-22 | 桂林电子科技大学 | Method for detecting blood glucose by virtue of persimmon tannin@graphene@Pt-Pd non-enzymatic sensor |
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