CN110672544A - Glucose visualization sensor based on cyclic peptide recognition element and preparation method and application thereof - Google Patents
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Abstract
The invention provides a glucose visualization sensor based on a cyclic peptide recognition element, and a preparation method and application thereof. The invention has the advantages that the cyclic peptide is firstly used as a glucose recognition element and is combined with a colorimetric sensing technology, a new rapid, convenient and sensitive detection method is provided for the detection of the glucose of a small molecular substance, and the experimental result can be directly observed by naked eyes within a short time (10 minutes) without the help of a large-scale precise instrument. The technical problems of long detection time, expensive instrument, complex operation, poor specificity and the like in the traditional detection method are solved. Can be used for detecting glucose in the fields of medicine, biology, food and the like.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a glucose visualization sensor based on a cyclic peptide recognition element and application of the glucose visualization sensor to glucose detection.
Background
Glucose is the main energy source of human body and is an important part participating in human metabolism. Sugar metabolism disorder can cause a series of diseases such as diabetes. Chronic hyperglycemia in diabetes can cause long-term damage to the body, leading to failure of various organs, particularly the heart, blood vessels, nerves, kidneys, eyes, etc. Currently, there are many methods for measuring glucose concentration, and the methods can be roughly classified into two types: enzymatic (including spectrophotometry and glucometers) and non-enzymatic instruments (such as high performance liquid chromatography systems and their associated detectors). However, the existing enzyme methods need to overcome the problems of high cost, instability, difficulty in purification and storage, and the like. The traditional detection method based on the instrument has the problems of long detection time, expensive equipment, complex preparation, poor selectivity and the like. Therefore, the development of a sensor which is rapid, simple, high in reactivity and high in selectivity is of great significance.
Colorimetric sensors are based on a change in color intensity caused by a specific chemical reaction between a target analyte and a sensing probe. The output of the colorimetric sensor may be qualitatively analyzed by the naked eye or quantitatively analyzed by a simple spectrophotometric method. The method has the advantages of short response time, simple operation, no need of complex instruments and the like. Peptides are designed to be recognition elements by mimicking the interaction between proteins and receptors, and maintaining a small size. They have low toxicity, can be fixed at high density, simultaneously maintain the stability and specificity, greatly reduce the technical cost and have great development potential.
At present, the cyclic peptide is used as an identification element and is combined with colorimetric sensing, and the detection research for glucose visualization is not reported yet.
Disclosure of Invention
The invention utilizes AuNPs with catalytic property, cyclic peptide specifically combined with glucose and 4-nitrophenol serving as a colorimetric probe to prepare a colorimetric biosensor and provide a visual detection method for glucose in an actual sample. The sensor mainly comprises gold nanoparticles with catalytic performance, cyclic peptide sequence for specifically binding glucose, cyclo [ -CNDNHCRDNDC- ] and 4-nitrophenol serving as a colorimetric probe.
The principle is as follows: in the absence of glucose, 4-nitrophenol easily reaches the exposed surface of AuNPs, and due to the catalytic action of AuNPs, 4-nitrophenol is reduced to 4-aminophenol, and the color of the solution changes from yellow to colorless. When glucose is present, the binding site of the cyclic peptide native glucose binding protein to glucose, acting as a glucose receptor, specifically recognizes and binds glucose, thereby masking a portion of the surface of AuNPs. The 4-nitrophenol is difficult to access the AuNPs surface, resulting in 4-nitrophenol and NaBH4The reaction time of (a) increases and the color of the solution changes slowly. Therefore, the presence of glucose molecules can be directly observed with the naked eye by the phenomenon of color change induced by the naked surface of AuNPs.
The specific scheme of the invention is as follows: a glucose visualization sensor based on cyclic peptide recognition element is characterized in that the sensor mainly comprises gold nanoparticles and cyclic peptide
Preferably, the cyclic peptide sequence is cyclo [ -CNDNHCRDNDC- ].
Preferably, the dosage of the gold nanoparticles is 90-110ul, the dosage of the cyclic peptide is 100-200ul, and the concentration is 0.6 mM. The amount of peptide should not be excessive, which would result in a large amount of shielding of the colloidal gold, and there was no difference between before and after addition of glucose. The amount of peptide should not be too small, too little binding to glucose is small, and the detection range is too narrow.
A preparation method of a glucose visualization sensor based on a cyclic peptide recognition element comprises 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 in order to remove free cyclic peptides from the AuNPs surface and thereby avoid non-specific adsorption. Self-assembled monolayers of AuNPs bound to cyclic peptides are formed by the Au-S covalent bond interaction between-SH groups in cysteine residues of the peptides and the Au surface.
Preferably, the cyclic peptide is incubated for 9 hours
A glucose visualization sensor based on cyclic peptide recognition element is used for detecting glucose,
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 20 min to remove free glucose in the supernatant to avoid non-specific adsorption; then, 200. mu.L of a 0.01M solution of 4-nitrophenol and 200. mu.L of a freshly prepared 0.6M solution of 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.
Preferably, the incubation time for glucose is 1 hour.
Determination of glucose content
According to the specific recognition of the cyclic peptide to the target object, the catalytic performance of AuNPs is inhibited. Different inhibition effects are generated by changing the concentration of the target substance, so that the generated colorimetric signal and optical signal are different, and qualitative and quantitative detection of the target substance is realized. With the increase of the concentration of the target object, the shading effect on the surfaces of AuNPs is stronger, the change of fading reaction catalyzed by the AuNPs is weakened, and the generated specific color and optical signal are increased.
The quantitative detection process is realized by an ultraviolet-visible spectrophotometer. The response value of the relative absorbance and the glucose are in a good linear relationship between 0.1mM and 20mM, and the glucose standard curve: (A-A)0)/A07.50c (mm) +33.50, slope 7.50, correlation coefficient R20.997 with a minimum detection limit of 0.04 mM.
The invention has the advantages that: the cyclic peptide is used as a glucose recognition element for the first time, is combined with a colorimetric sensing technology, provides a new rapid, convenient and sensitive detection method for detecting the glucose of a small molecular substance, and can directly observe an experimental result by naked eyes in a short time (10 minutes) without the help of a large-scale precision instrument. The colloidal gold has a catalytic effect, and the 4-nitrophenol is used for developing in the detection process. The technical problems of long detection time, expensive instrument, complex operation, poor specificity and the like in the traditional detection method are solved. Can be used for detecting glucose in the fields of medicine, biology, food and the like.
Drawings
FIG. 1 is a schematic diagram of the glucose visual sensing principle of the present invention based on cyclic peptide recognition element;
FIGS. 2a and 2b standard curves for glucose according to the invention;
FIG. 3 is a photograph of a glucose visualization solution and a filter paper colorimetric card of the present invention;
FIGS. 4a, 4b and 4c illustrate the selectivity of the present invention;
FIG. 5 reproducibility analysis of the present invention.
Detailed Description
The illustrative embodiments and descriptions of the present invention are provided to explain the present invention and not to limit the present invention unduly. The invention is described in further detail below with reference to the figures and the specific embodiments.
The cyclic peptide used in the present invention was designed by this group of subjects and synthesized by Shanghai Boratae Biotech Co.
Example 1
(1) Preparation of gold nanoparticles (AuNPs)
All glassware for the synthesis of AuNPs was thoroughly cleaned with chromic acid solution prior to use. The AuNPs is synthesized by taking chloroauric acid and sodium citrate as raw materials through a chemical reduction reaction between the chloroauric acid and the sodium citrate. First, a chloroauric acid solution (1mM,250mL) was added to a round bottom flask and heated to boiling with stirring. Subsequently, sodium citrate (38.8mM,25mL) was added rapidly to the above boiling solution. And (4) observing the color of the solution to be changed from light yellow to wine red, starting timing, stirring vigorously for 10 minutes, stopping heating, and cooling the mixed solution to room temperature. The obtained AuNPs are uniformly dispersed, have the particle size of about 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- ]. The prepared cyclic peptide solution (0.6mM, 150. mu.L) was added to AuNPs solution (100. mu.L) and incubated at 4 ℃ for 9 hours. The self-assembled CP-AuNPs solution was centrifuged at 8000rpm for 20 minutes in order to remove free cyclic peptides from the AuNPs surface and thereby avoid non-specific adsorption. The self-assembled monolayer combined by the AuNPs and the cyclic peptide is formed by the interaction of an Au-S covalent bond between-SH group in cysteine residue of the peptide and the Au surface;
example 2
(1) The detection method comprises the following steps:
in the test state, first, glucose (100. mu.L) was added at different concentrations to a 250. mu.L solution of LCP-AuNPs, and incubated at room temperature for 1 hour. The mixed solution was centrifuged at 8000rpm for about 20 minutes to remove free glucose present in the supernatant to avoid non-specific adsorption. Then, adding a 4-nitrophenol solution (0.01M,200 mu L) and a newly prepared NaBH4 solution (0.6M,200 mu L) into 100 mu L of the mixed solution, reacting for 10 minutes, diluting the reaction solution by 50 times, measuring in an ultraviolet-visible spectrophotometer, detecting that the wavelength range of an absorption spectrum is 250nm-500nm, and recording the absorption peak value of the 4-nitrophenol at 400 nm;
(2) determination of glucose content
According to the specific recognition of the cyclic peptide to the target object, the catalytic performance of AuNPs is inhibited. Different inhibition effects are generated by changing the concentration of the target substance, so that the generated colorimetric signal and optical signal are different, and qualitative and quantitative detection of the target substance is realized. With the increase of the concentration of the target object, the shading effect on the surfaces of AuNPs is stronger, the change of fading reaction catalyzed by the AuNPs is weakened, and the generated specific color and optical signal are increased.
The quantitative detection process is realized by an ultraviolet-visible spectrophotometer. The response value of the relative absorbance and the glucose are in a good linear relationship between 0.1mM and 20mM, and the glucose standard curve:
(a-a0)/a0 ═ 7.50c (mM) +33.50, slope 7.50, correlation coefficient R2 0.997, and minimum detection limit 0.04 mM.
Example 3
Determination of the glucose content in the actual sample:
the sensor and the glucose kit are respectively utilized to analyze and measure glucose in actual samples (rabbit serum, cabbage, pear and wheat flour), a standard addition method is adopted to carry out a standard addition recovery experiment, and three times of parallel measurement are respectively carried out under three concentrations. The recovery rates are 86.96% -101.66% respectively, which shows that the glucose visualization sensor constructed by the preparation method has higher accuracy in sample detection.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope 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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| CN109991217A (en) * | 2019-03-14 | 2019-07-09 | 厦门大学 | A kind of detection A β1-42The colorimetric bio sensor of oligomer |
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