CN110672544A - Glucose visualization sensor based on cyclic peptide recognition element and preparation method and application thereof - Google Patents

Abstract

The invention provides a glucose visualization sensor based on a cyclic peptide recognition element, a preparation method and application thereof. The sensor is mainly composed of gold nanoparticles with catalytic properties, a cyclic peptide for specific binding of glucose and a colorimetric probe. 4-nitrophenol composition. The advantage of the invention is that the cyclic peptide is used as the recognition element of glucose for the first time, and combined with the colorimetric sensing technology, a new fast, convenient and sensitive detection method is provided for the detection of small molecule glucose, which can be used in a short time. (10 minutes), without the aid of large precision instruments, the experimental results can be directly observed with the naked eye. The technical problems such as long detection time, expensive instrument, complicated operation and poor specificity in traditional detection methods are overcome. It can be used for the detection of glucose in many fields such as medicine, biology and food.

Description

A kind of glucose visualization sensor based on cyclic peptide recognition element and preparation method thereof and application

technical field

The invention belongs to the technical field of molecular detection, and in particular relates to a glucose visualization sensor based on a cyclic peptide identification element and its use in glucose detection.

Background technique

Glucose is the main energy source of the human body and participates in an important part of human metabolism. Disorders of glucose metabolism can cause a series of diseases such as diabetes. Chronic hyperglycemia of diabetes can damage the body in the long term, leading to failure of different organs, especially the heart, blood vessels, nerves, kidneys, eyes, etc. At present, there are many methods for measuring glucose concentration, which can be roughly divided into two categories: enzymatic methods (including spectrophotometry and blood glucose meters) and non-enzymatic instruments (such as high performance liquid chromatography systems and their related detectors). However, existing enzymatic methods need to overcome problems such as high cost, instability, purification and storage difficulties. Traditional instrument-based detection methods have problems such as long detection time, expensive equipment, complex preparation, and poor selection performance. Therefore, it is of great significance to develop fast, simple, highly reactive and highly selective sensors.

Colorimetric sensors are based on changes in color intensity caused by a specific chemical reaction between the target analyte and the sensing probe. The output of the colorimetric sensor can be qualitatively analyzed with the naked eye or quantitatively analyzed by simple spectrophotometry. It has the advantages of short response time, simple operation, and no need for complicated instruments. Peptides are designed to act as recognition elements by mimicking the interaction between proteins and receptors while maintaining a small size. They are less toxic and can be immobilized at high densities while maintaining their stability and specificity, greatly reducing the cost of technology and having great potential for development.

At present, the detection of cyclic peptides as recognition elements combined with colorimetric sensing for glucose visualization has not been reported yet.

SUMMARY OF THE INVENTION

The present invention utilizes AuNPs with catalytic properties, cyclic peptides that specifically bind to glucose, and 4-nitrophenol as a colorimetric probe to prepare a colorimetric biosensor and provide a colorimetric biosensor for use in practical samples A visual method for the detection of glucose. The sensor is mainly composed of gold nanoparticles with catalytic properties, a cyclic peptide sequence cyclo[-CNDNHCRDNDC-] for specific binding of glucose, and 4-nitrophenol as a colorimetric probe.

The principle is as follows: In the absence of glucose, 4-nitrophenol can easily reach the exposed surface of AuNPs, and due to the catalysis of AuNPs, 4-nitrophenol is reduced to 4-aminophenol, and the color of the solution changes from yellow to no color. In the presence of glucose, the binding site of cyclic peptide native glucose-binding protein with glucose, acting as a glucose receptor, specifically recognizes and binds glucose, thereby shielding part of the surface of AuNPs. It is difficult for 4-nitrophenol to access the surface of AuNPs, which leads to an increase in the reaction time of ₄ -nitrophenol and NaBH, and a slow color change of the solution. Therefore, the presence of glucose molecules can be directly observed with the naked eye through the color change phenomenon induced by the exposed surface of AuNPs.

The specific scheme of the present invention is: a glucose visualization sensor based on a cyclic peptide recognition element, characterized in that the sensor is mainly composed of gold nanoparticles and cyclic peptides

Preferably, the cyclic peptide sequence is cyclo[-CNDNHCRDNDC-].

Preferably, the amount of the gold nanoparticles is 90-110ul, the amount of the cyclic peptide is 100-200ul, and the concentration is 0.6mM. The amount of peptide should not be too much, too much will shield the colloidal gold in large quantities, and there will be no difference before and after adding glucose. The amount of peptide should not be too small, too little, the binding of glucose will be less, and the detection range will be too narrow.

A preparation method of a glucose visualization sensor based on a cyclic peptide recognition element, comprising the following steps:

(1) Preparation of gold nanoparticles

All glassware for synthesizing gold nanoparticles was thoroughly cleaned with chromic acid solution before use, and AuNPs were synthesized by reaction with chloroauric acid and sodium citrate as raw materials; first, 250 mL, 1 mM chloroauric acid solution was added into a round-bottomed flask and stirred Heat to boiling; then, quickly add 25 mL of 38.8 mM sodium citrate to the above boiling solution, observe that the color of the solution changes from light yellow to wine red and start timing, stir vigorously for 10 minutes, stop heating, and cool the mixed solution to room temperature; The obtained gold nanoparticles were uniformly dispersed, with a particle size of 13 nm, and were stored in a 4°C refrigerator for later use;

(2) Preparation of cyclic peptide-modified AuNPs

The sequence of the cyclic peptide is: cyclo[-CNDNHCRDNDC-], 150 μL of the prepared cyclic peptide solution with a concentration of 0.6 mM was added to 100 μL of the gold nanoparticle solution, and incubated overnight at 4 °C to obtain CP-AuNPs; the self-assembled CP-AuNPs were obtained; - The AuNPs solution was centrifuged at 8000 rpm for 20 min in order to remove free cyclic peptides from the surface of AuNPs, thereby avoiding non-specific adsorption. The self-assembled monolayers of AuNPs bound to cyclic peptides were formed by Au-S covalent interactions between the -SH groups in the cysteine residues of the peptides and the Au surface.

Preferably, the incubation time of the cyclic peptide is 9 hours

A cyclic peptide recognition element-based glucose visualization sensor for glucose detection,

The detection steps are as follows:

100 μL of glucose with different concentrations was added to 250 μL of CP-AuNPs solution and incubated at room temperature for 60 min; the mixed solution was centrifuged at 8000 rpm for about 20 min to remove the free glucose present in the supernatant to avoid non-specific adsorption; then , 200 μL of 0.01M 4-nitrophenol solution and newly prepared 200 μL of 0.6M, NaBH ₄ solution were added to 100 μL of the above mixed solution, and reacted for 10 minutes. After diluting the reaction solution by 50 times, it was measured in a UV-Vis spectrophotometer. , the wavelength range of the detection absorption spectrum is 250nm-500nm, and the absorption peak of 4-nitrophenol at 400nm is recorded; the qualitative detection process is realized by observing the color change of the solution with naked eyes and taking pictures.

Preferably, the incubation time with glucose is 1 hour.

Determination of glucose content

According to the specific recognition of the target by the cyclic peptide, the catalytic performance of AuNPs was inhibited. By changing the concentration of the target, different inhibitory effects are produced, resulting in different colorimetric signals and optical signals, and the qualitative and quantitative detection of the target is realized. With the increase of target concentration, the shielding effect on the surface of AuNPs is stronger, the fading reaction catalyzed by AuNPs is weakened, and the colorimetric and optical signals generated will increase.

The quantitative detection process is realized by UV-Vis spectrophotometer. The response value of relative absorbance has a good linear relationship with glucose between 0.1mM and 20mM. The standard curve of glucose: (AA ₀ )/A ₀ =7.50C(mM)+33.50, the slope is 7.50, and the correlation coefficient R ² is 0.997. The minimum detection limit was 0.04mM.

The advantages of the invention are that: the cyclic peptide is used as the recognition element of glucose for the first time, and combined with the colorimetric sensing technology, a new fast, convenient and sensitive detection method is provided for the detection of small molecular substance glucose, which can be used in a short period of time. Within the time (10 minutes), the experimental results can be directly observed with the naked eye without the aid of large-scale precision instruments. Colloidal gold plays a catalytic role, and 4-nitrophenol is used for color development in the detection process. The technical problems such as long detection time, expensive instrument, complicated operation and poor specificity in traditional detection methods are overcome. It can be used for the detection of glucose in many fields such as medicine, biology and food.

Description of drawings

Fig. 1 is a schematic diagram of the visual sensing principle of glucose based on the cyclic peptide recognition element of the present invention;

Figures 2a and 2b are standard curves of glucose of the present invention;

Fig. 3 glucose visualization solution photo of the present invention and filter paper colorimetric card photo;

Figures 4a, 4b and 4c the selectivity of the invention;

Figure 5. Reproducibility analysis of the present invention.

Detailed ways

The exemplary embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

The cyclic peptide used in the present invention is designed by this research group and synthesized by Shanghai Botai Biotechnology Co., Ltd.

Example 1

(1) Preparation of gold nanoparticles (AuNPs)

All glassware for the synthesis of AuNPs was thoroughly cleaned with chromic acid solution before use. AuNPs were synthesized by chemical reduction reaction between chloroauric acid and sodium citrate. First, a solution of chloroauric acid (1 mM, 250 mL) was added to a round-bottomed flask and heated to boiling with stirring. Subsequently, sodium citrate (38.8 mM, 25 mL) was rapidly added to the above boiling solution. When the color of the solution changed from light yellow to wine red, the time was started. After vigorous stirring for 10 minutes, the heating was stopped, and the mixed solution was cooled to room temperature. The obtained AuNPs were uniformly dispersed, with a particle size of about 13 nm, and were stored in a refrigerator at 4 °C for later use;

(2) Preparation of cyclic peptide-modified AuNPs

The sequence of the cyclic peptide is: cyclo[-CNDNHCRDNDC-]. The prepared cyclic peptide solution (0.6 mM, 150 μL) was added to the AuNPs solution (100 μL) and incubated at 4°C for 9 hours. The self-assembled CP-AuNPs solution was centrifuged at 8,000 rpm for 20 min, in order to remove free cyclic peptides from the surface of AuNPs, thereby avoiding nonspecific adsorption. The self-assembled monolayer of AuNPs bound to the cyclic peptide is formed by the Au-S covalent interaction between the -SH group in the cysteine residue of the peptide and the Au surface;

Example 2

(1) Detection method:

In the detection state, first, different concentrations of glucose (100 μL) were added to 250 μL CP-AuNPs solution and incubated at room temperature for 1 hour. The mixed solution was centrifuged at 8000 rpm for about 20 minutes to remove free glucose present in the supernatant to avoid non-specific adsorption. Then, 4-nitrophenol solution (0.01M, 200μL) and newly prepared NaBH4 solution (0.6M, 200μL) were added to 100μL of the above mixed solution, and reacted for 10 minutes. After diluting the reaction solution by 50 times, it was subjected to UV-Vis spectroscopy. Measured in a photometer, the wavelength range of the detection absorption spectrum is 250nm-500nm, and the absorption peak of 4-nitrophenol at 400nm is recorded;

(2) Determination of glucose content

According to the specific recognition of the target by the cyclic peptide, the catalytic performance of AuNPs was inhibited. By changing the concentration of the target, different inhibitory effects are produced, resulting in different colorimetric signals and optical signals, and the qualitative and quantitative detection of the target is realized. With the increase of target concentration, the shielding effect on the surface of AuNPs is stronger, the fading reaction catalyzed by AuNPs is weakened, and the colorimetric and optical signals generated will increase.

The quantitative detection process is realized by UV-Vis spectrophotometer. The response value of relative absorbance has a good linear relationship with glucose between 0.1mM and 20mM. The standard curve of glucose:

(A-A0)/A0=7.50C(mM)+33.50, the slope is 7.50, the correlation coefficient R2 is 0.997, and the minimum detection limit is 0.04mM.

Example 3

Determination of glucose content in actual samples:

The sensor and the glucose kit described in the present invention were respectively used to analyze and measure the glucose in the actual samples (rabbit serum, cabbage, pear, wheat flour), and the standard addition method was used to carry out the standard addition recovery experiment. Measured in parallel three times. The recoveries are respectively 86.96%-101.66%, indicating that the glucose visual sensor constructed by the preparation method of the present invention has high accuracy in sample detection.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the within the scope of protection of the present invention.

Claims (8)

1. A glucose visualization sensor based on a cyclic peptide recognition element is characterized by comprising gold nanoparticles and cyclic peptide phenol.

2. The glucose visualization sensor based on the cyclic peptide recognition element as claimed in claim 1, wherein the cyclic peptide sequence is cyclo [ -CNDNHCRDNDC- ].

3. The glucose visualization sensor based on the cyclic peptide identification element as claimed in claim 1, wherein the amount of the gold nanoparticles is 90-110ul, the amount of the cyclic peptide is 100-200ul, and the concentration is 0.6 mM.

4. A preparation method of a glucose visualization sensor based on a cyclic peptide recognition element is characterized by comprising the following steps:

(1) preparation of gold nanoparticles

Thoroughly cleaning all glassware for synthesizing gold nanoparticles by using a chromic acid solution before use, and reacting and synthesizing AuNPs by using chloroauric acid and sodium citrate as raw materials; firstly, adding 250mL of 1mM chloroauric acid solution into a round-bottom flask, stirring and heating to boil; then, 25mL38.8mM sodium citrate is rapidly added into the boiling solution, timing is started after the color of the solution is observed to be changed from light yellow to wine red, heating is stopped after the solution is vigorously stirred for 10 minutes, and the mixed solution is cooled to the room temperature; the obtained gold nanoparticles are uniformly dispersed, have the particle size of 13nm and are stored in a refrigerator at 4 ℃ for later use;

(2) preparation of cyclopeptide modified AuNPs

The sequence of the cyclic peptide is: cyclo [ -CNDNHCRDNDC- ], adding the prepared 150 μ L cyclic peptide solution with the concentration of 0.6mM into 100 μ L gold nanoparticle solution, and incubating at 4 ℃ overnight to obtain CP-AuNPs; the self-assembled CP-AuNPs solution was centrifuged at 8000rpm for 20 minutes.

5. The method for preparing a cyclic peptide recognition element-based glucose visualization sensor as claimed in claim 4, wherein the cyclic peptide is incubated for 9 hours.

6. A glucose visualization sensor based on cyclic peptide recognition element is used for detecting glucose.

7. The glucose visualization sensor based on the cyclic peptide recognition element of claim 6 is used for detecting glucose, and is characterized in that the detection steps are as follows:

adding 100 mu L of glucose with different concentrations into 250 mu of LCP-AuNPs solution, and incubating for 60min at room temperature; centrifuging the mixed solution at 8000rpm for about 20 min to remove free glucose in the supernatant; then, 200. mu. L0.01M 4 solution of 4-nitrophenol and 200. mu.L of freshly prepared 0.6M, NaBH₄Adding the solution into 100 mu L of the mixed solution, reacting for 10 minutes, diluting the reaction solution by 50 times, determining in an ultraviolet visible spectrophotometer, detecting that the wavelength range of an absorption spectrum is 250nm-500nm, and recording the absorption peak value of 4-nitrophenol at 400 nm; the qualitative detection process is realized by observing the color change of the solution with naked eyes and photographing.

8. The glucose visualization sensor based on the cyclic peptide recognition element of claim 7, which is used for detecting glucose, wherein the incubation time of glucose is 1 hour.

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