CN114486870A - Nano enzyme functionalized paper-based sensor for rapidly detecting blood sugar and detection method thereof - Google Patents
Nano enzyme functionalized paper-based sensor for rapidly detecting blood sugar and detection method thereof Download PDFInfo
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Abstract
The invention provides a nano enzyme functionalized paper-based sensor for quickly detecting blood sugar. The invention uses AB-Hemin with POD activity to form a sensing interface on the surface of the paper base, and specifically detects the blood sugar of a patient. The carbon material is modified by Hemin, so that the POD (peroxidase) activity of the carbon material is greatly improved, AB without POD activity is found after modification of AB by Hemin, and 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 device can be applied to hospitals, families, and ports such as customs, airports and the like which need on-site rapid detection in food safety and inspection and quarantine.
Description
Technical Field
The invention belongs to the technical field of nano composite materials, and relates to a nano enzyme functionalized paper-based sensor for quickly detecting blood sugar and a detection method thereof.
Background
Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia due to multiple causes. Diabetes is usually accompanied by various complications, and the diabetes easily affects micro-vascular and macro-vascular diseases, causes complications of kidney, eye, nervous system, cardiovascular system and the like, and is a main cause of death and disability of diabetic patients. Detection of an excessively high blood glucose level early in a diabetic patient helps control the patient's blood glucose and subsequent symptom control.
Clinical blood sugar detection is mainly performed by biochemical analyzers, household blood sugar detection is mainly performed by blood sugar meters, and other methods include micro-needle methods and the like. Although the biochemical analyzer is called as the gold standard of blood sugar test, the application of the biochemical analyzer needs special instruments and professional operators, while the household blood sugar meter needs to be matched with corresponding test paper, and the micro-needle method is difficult to directly obtain the blood sugar value.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a nano enzyme functionalized paper-based sensor for quickly detecting blood sugar. The invention immobilizes AB-Hemin with excellent POD activity on the surface of a paper base, can effectively capture a color signal of glucose which is catalytically displayed, accurately determines the content of the glucose and provides a new method for clinically detecting the blood sugar.
Except for special description, the parts are parts by weight, and the percentages are mass percentages.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a nanometer enzyme functionalized paper-based sensor for rapidly detecting blood sugar is characterized in that: the paper-based sensor is constructed by acetylene black nanoenzyme modified by hemin.
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, placing in an ultrasonic cleaner for ultrasonic dispersion for 40-60min, stirring at normal temperature for 15-20h, centrifuging at the rotating speed of 10000 plus 15000rpm/min, and obtaining precipitate, namely Hemin-modified acetylene black nanoenzyme (AB-Hemin).
The construction method of the nanometer enzyme functionalized paper-based sensor for quickly detecting blood sugar comprises the following steps: cutting Whatman CHR 1 filter paper into round paper sheets by using a puncher, dripping AB-Hemin water solution onto the cut round paper sheets, and drying for 30-40min at 37 ℃; and (3) dropwise adding an ethanol solution of TMB (tetramethylbenzidine) to the surface of the dried paper base, and finishing the construction of the paper base sensor after the ethanol is volatilized until the paper base is dry at normal temperature.
The aqueous AB-Hemin solution was a 20 μ L dilution of AB-Hemin in 1mL of a pH 4 sodium acetate buffer.
The ethanol solution of TMB was 35 mM.
A nanometer enzyme functional paper-based sensor for rapidly detecting blood sugar is constructed by acetylene black nanometer enzyme modified by hemin; the preparation method is characterized by comprising the following steps:
(1) preparation of AB-Hemin:
weighing 4mg Hemin (Hemin) and adding 4mL of N, N-Dimethylformamide (DMF) solution; dissolving 4mg of Acetylene Black (AB) in 4mL of ultrapure water in an ultrasonic cleaner, carrying out ultrasonic treatment at normal temperature for 5min to fully dissolve the Acetylene Black (AB), then completely and slowly adding the acetylene black into the DMF solution of the Hemin under the condition of stirring at normal temperature, continuously stirring for 5min, then placing the solution in the ultrasonic cleaner for ultrasonic dispersion for 50min, then stirring the mixed solution after ultrasonic treatment at normal temperature for 16h, carrying out centrifugal cleaning at the rotating speed of 12000rpm/min until the supernatant is transparent and colorless, carrying out redispersion the solution with the ultrapure water, and carrying out constant volume to 4mL to obtain the AB-Hemin.
(2) Construction of paper-based sensor: cutting Whatman CHR 1 filter paper into 6mm circular paper sheets by using a 6 mm-diameter puncher, dripping 20 mu L of AB-Hemin water solution onto the cut circular paper sheets, and drying at 37 ℃ for 35 min; dripping an ethanol solution of TMB (tetramethylbenzidine) on the surface of the dried paper base, and finishing the construction of the paper base sensor when the ethanol is volatilized until the paper base is dry at normal temperature; the ethanol solution of TMB is 35mM, and the dosage is 5 mu L.
The invention also provides a method for detecting blood sugar by using the nano enzyme functionalized paper-based sensor, which is simple to operate and strong in practicability.
A method for detecting blood sugar by a nano enzyme functionalized paper-based sensor is characterized by comprising the following steps:
(1) preparing standard solutions with different concentrations by using glucose standards, mixing the standard solutions with a PBS solution dissolved with GOD, and reacting in a 37 ℃ oven for 30min to prepare a plurality of standard mixed solutions;
(2) dripping each standard mixed liquid on the surface of the paper-based sensor, reading RGB color values of color signals by using a smart phone after reacting for 18min, and establishing a linear relation between the glucose concentration and the color signals;
(4) mixing GOD dissolved in PBS (pH 6) with serum, and reacting at 37 deg.C for 30 min; and (3) then, dripping the mixed solution on the surface of the paper-based sensor, waiting for reaction for 18min, reading RGB values of the color signals by using a smart phone, carrying out signal processing on the RGB values, and substituting the RGB values into the linear relation curve obtained in the step (2) to obtain the blood glucose concentration of the serum to be measured.
Has the advantages that:
the invention uses AB-Hemin with POD activity to form a sensing interface on the surface of the paper base, and specifically detects the blood sugar of a patient. The carbon material is modified by Hemin, so that the POD (peroxidase) activity of the carbon material is greatly improved, AB without POD activity is found after modification of AB by Hemin, and 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) the invention firstly applies Hemin to functionalize AB, obviously endows AB POD activity, and can generate very obvious color signals in colorimetric application.
(2) The method combines the low-cost and pollution-free paper base and the acetylene black for the paper base colorimetric detection of the blood sugar, and has the advantages of high sensitivity, good stability, wide application range, simple operation and use method and easy popularization and application; the device can be applied to hospitals, families, and ports such as customs, airports and the like which need on-site rapid detection in food safety and inspection and quarantine.
(3) The invention can specifically identify glucose, can realize quantitative detection of blood glucose by using a smart phone, does not need complex detection equipment, and can obtain results on site.
Drawings
FIG. 1 shows the results of the detection of glucose with different concentrations by the sensor of the present invention and the linear calibration curve.
FIG. 2 shows the result of specific detection of glucose by the sensor of the present invention.
FIG. 3 is the results of lot-to-lot variation of sensors prepared according to the present invention.
Fig. 4 is a stability test result of the sensor of the present invention.
Detailed Description
The present invention is described in detail in the following examples, which are given for the purpose of illustration only and are not to be construed as limiting the scope of the invention, which is defined in the claims. The raw materials and 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 purchased from yirui stone (shanghai) investment management limited (shanghai, china). N, N-Dimethylformamide (DMF) was purchased from Michelin Biochemical technology Ltd (Shanghai, China).
Example 1 nanomaterial AB-Hemin was prepared.
The method comprises the following steps (constructing a schematic diagram as shown in figure 1):
(1) weighing 4mg Hemin (Hemin) and adding into 4mL of N, N-Dimethylformamide (DMF) solution; dissolving 4mg of Acetylene Black (AB) in 4mL of ultrapure water, carrying out ultrasonic treatment in an ultrasonic cleaner at normal temperature for 5min to fully dissolve the Acetylene Black (AB), then completely and slowly adding a DMF solution dissolved with 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 for 16h at normal temperature, washing the mixture with the ultrapure water at the rotating speed of 12000rpm/min for three times, each time for 5min, redispersing the mixture with the ultrapure water, and fixing the volume to 1mg/mL of the AB-Hemin solution.
Example 2 construction of a paper-based sensor for Rapid detection of blood glucose
(1) 20 μ L of the AB-Hemin solution prepared in example 1 was dispersed in 1mL of NaAc-HAc buffer solution at pH 4, and vortexed to disperse the solution uniformly.
(2) The filter paper was cut into small 6mm diameter circular sheets with a 6mm diameter punch and was ready for use.
(3) Dripping 20 mu L of the buffer solution of AB-Hemin prepared in the step (1) to the surface of the paper sheet prepared in the step (2), and drying at 37 ℃ for 35min to obtain a dry state.
(4) 8.5mg of TMB was dissolved in 1mL of absolute ethanol to obtain 1mL of a 35mM TMB solution.
(5) Dripping 5 mu L of the TMB solution prepared in the step (4) on the surface of the paper base dried in the step (3), and airing in the air to obtain the nano enzyme functionalized paper base sensor for quickly detecting blood sugar.
Example 3 blood glucose detection Using paper-based sensor
The paper-based sensor constructed in example 2 was used for detecting blood glucose, and the following procedure was followed:
(1) drawing a standard curve:
glucose standard solutions with the concentrations of 0.2, 0.5, 0.6, 0.8, 0.9, 1.0, 2.0, 5.0, 10, 20 and 30mM are prepared respectively, 20 mu L of the glucose standard solution is mixed with 0.5mg/mL of PBS (pH 6) solution of GOD respectively, the mixture is reacted for 30min at 37 ℃, 10 mu L of the reaction solution is dripped on the surface of the paper-based sensor constructed in example 2, and after 18min, the color signals are read by a smart phone respectively for RGB values. And (3) drawing a standard curve, wherein the detection result shows that the two are in a good linear relation in a concentration range, the linear correlation coefficient is 0.9973, the detection limit is 0.1mM, and the result is shown in figure 1 in detail.
(2) And (3) specific detection of a sensor:
in order to detect the specificity of the sensor of the present invention, the following interfering substances are detected: maltose (maltose, 5mM), sucrose (sucrose, 5mM), Fructose (Fructose,5mM), lactose (lactose, 5 mM). The RGB signals of the 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) And (3) detecting the stability of the nano material:
the prepared material is respectively placed at-20 ℃, 4 ℃ and normal temperature for 10 days, and then a sensor is constructed, and the result shows that the response value of the sensor to glucose measurement is still greater than 80% of the initial value after the sensor is stored for 10 days; the data (fig. 3) show that the material produces a sensor with acceptable stability.
(4) Sensor reproducibility detection:
glucose (10mM) was measured under the same conditions using 5 different sensors of the same lot prepared according to the present invention, and the results are shown in detail in FIG. 4, where the Relative Standard Deviation (RSD) of the current response values was 1.37%, indicating that the sensor was less in-lot variation and more reproducible.
(5) Practical sample analysis application:
in order to evaluate the practical applicability and accuracy of the proposed paper-based sensor, 20 μ L of human serum sample was mixed with 0.5mg/mL of a PBS (pH 6) solution of GOD, and RGB value reading calculation was performed using the prepared sensor assay, 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 glucometer 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 practical analysis.
Claims (6)
1. A nanometer enzyme functionalized paper-based sensor for rapidly detecting blood sugar is characterized in that: the paper-based sensor is constructed by acetylene black nanoenzyme modified by hemin; 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, placing in an ultrasonic cleaner for ultrasonic dispersion for 40-60min, stirring at normal temperature for 15-20h, centrifuging at the rotating speed of 10000 plus 15000rpm/min, and obtaining precipitate, namely Hemin-modified acetylene black nanoenzyme (AB-Hemin).
2. The method of claim 1, wherein the nanoenzyme functionalized paper-based sensor for rapid detection of blood glucose is constructed by: cutting Whatman CHR 1 filter paper into round paper sheets by using a puncher, dripping AB-Hemin water solution onto the cut round paper sheets, and drying for 30-40min at 37 ℃; and (3) dropwise adding an ethanol solution of TMB (tetramethylbenzidine) to the surface of the dried paper base, and finishing the construction of the paper base sensor after the ethanol is volatilized until the paper base is dry at normal temperature.
3. The method of claim 1, wherein: the aqueous AB-Hemin solution was a 20 μ L dilution of AB-Hemin in 1mL of pH 4 sodium acetate buffer.
4. The method of claim 1, wherein: the ethanol solution of TMB was 35 mM.
5. A nanometer enzyme functional paper-based sensor for rapidly detecting blood sugar is constructed by acetylene black nanometer enzyme modified by hemin; the preparation method is characterized by comprising the following steps:
(1) preparation of AB-Hemin:
weighing 4mg Hemin (Hemin) and adding 4mL of N, N-Dimethylformamide (DMF) solution; dissolving 4mg of Acetylene Black (AB) in 4mL of ultrapure water in an ultrasonic cleaner, performing ultrasonic treatment for 5min at normal temperature to fully dissolve the Acetylene Black (AB), then completely and slowly adding the acetylene black into the Hemin DMF solution 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, then stirring the ultrasonically treated mixed solution for 16h at normal temperature, performing centrifugal cleaning at the rotating speed of 12000rpm/min until the supernatant is transparent and colorless, re-dispersing the supernatant with the ultrapure water, and performing constant volume treatment to 4mL to obtain AB-Hemin;
(2) construction of paper-based sensor: cutting Whatman CHR 1 filter paper into 6mm circular paper sheets by using a puncher with the diameter of 6mm, dripping 20 mu LAB-Hemin water solution onto the cut circular paper sheets, and drying for 35min at 37 ℃; dripping an ethanol solution of TMB (tetramethylbenzidine) on the surface of the dried paper base, and finishing the construction of the paper base sensor when the ethanol is volatilized until the paper base is dry at normal temperature; the ethanol solution of TMB is 35mM, and the dosage is 5 mu L.
6. A method for detecting blood glucose using a nanoenzyme functionalized paper-based sensor according to any one of claims 1 to 5, comprising the steps of:
(1) preparing standard solutions with different concentrations by using glucose standards, mixing the standard solutions with a PBS solution dissolved with GOD, and reacting in a 37 ℃ oven for 30min to prepare a plurality of standard mixed solutions;
(2) dripping each standard mixed liquid on the surface of the paper-based sensor, reading RGB color values of color signals by using a smart phone after reacting for 18min, and establishing a linear relation between the glucose concentration and the color signals;
(4) mixing GOD dissolved in PBS (pH 6) with serum, and reacting at 37 deg.C for 30 min; and (3) then, dripping the mixed solution on the surface of the paper-based sensor, waiting for reaction for 18min, reading RGB values of the color signals by using a smart phone, carrying out signal processing on the RGB values, and substituting the RGB values into the linear relation curve obtained in the step (2) to obtain the blood glucose concentration of the serum to be measured.
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