阅读说明：本技术 基于金簇和金纳米颗粒的荧光阵列传感器及其制备方法及应用 (Fluorescent array sensor based on gold clusters and gold nanoparticles and preparation method and application thereof ) 是由 黄略略 李彬 江帆 范大明 梁勇 于 2020-06-05 设计创作，主要内容包括：本发明公开了一种基于金簇和金纳米颗粒的荧光阵列传感器,其包括表面带负电的金纳米簇、以及带正电的半胱氨酸修饰的金纳米颗粒和柠檬酸-三聚氰胺修饰的金纳米颗粒。本发明还提供了所述基于金簇和金纳米颗粒的荧光阵列传感器的制备方法。本发明还提供了所述基于金簇和金纳米颗粒的荧光阵列传感器用于分析检测维生素B6、B9、B12的用途。本发明还提供了一种用于分析检测维生素B6、B9、B12的方法,其包括将含有维生素B6、B9、B12的样品加入所述基于金簇和金纳米颗粒的荧光阵列传感器中,测定加入前后的荧光信号强度变化。本发明能够有效、简便、灵敏地检测并分析维生素B6、B9、B12。(The invention discloses a fluorescence array sensor based on gold clusters and gold nanoparticles, which comprises gold nanoclusters with negative electricity on the surface, gold nanoparticles modified by cysteine and gold nanoparticles modified by citric acid-melamine with positive electricity. The invention also provides a preparation method of the fluorescence array sensor based on the gold clusters and the gold nanoparticles. The invention also provides application of the fluorescent array sensor based on the gold clusters and the gold nanoparticles to analysis and detection of vitamins B6, B9 and B12. The invention also provides a method for analyzing and detecting the vitamins B6, B9 and B12, which comprises the steps of adding a sample containing the vitamins B6, B9 and B12 into the gold cluster and gold nanoparticle-based fluorescence array sensor, and measuring the change of the fluorescence signal intensity before and after the addition. The invention can effectively, simply and sensitively detect and analyze the vitamins B6, B9 and B12.)

1. A fluorescence array sensor based on gold clusters and gold nanoparticles comprises gold nanoclusters with negative surfaces, cysteine modified gold nanoparticles with positive surfaces and citric acid-melamine modified gold nanoparticles.

2. The gold cluster and gold nanoparticle-based fluorescence array sensor of claim 1, wherein the gold nanoclusters are synthesized from bovine serum albumin as a template.

3. The method for preparing a gold cluster and gold nanoparticle based fluorescence array sensor according to claim 1 or 2, comprising the steps of:

step 1: preparing gold nanoclusters;

step 2: preparing gold nanoparticles modified by cysteine;

and step 3: preparing the citric acid-melamine modified gold nanoparticles.

4. The method of claim 3, wherein the gold nanoclusters are prepared by the steps of: 5.0mL of HAuCl at a concentration of 10mmol/L₄Adding the solution into 5.0mL of bovine serum albumin solution with the concentration of 50mg/mL, vigorously stirring at 37 ℃ for 2 minutes, quickly adding 0.5mL of NaOH solution with the concentration of 1mol/L into the reaction solution, and vigorously stirring at 37 ℃ for 12 hours to obtain the gold nanocluster solution.

5. The method of claim 3, wherein the cysteine-modified gold nanoparticles are prepared by the steps of: 500. mu.L of 215mmol/L cysteine solution and 2.23mL of 25.5mmol/L HAuCl₄The solution was mixed well, 37.5mL of distilled water was added, stirred at room temperature for 20 minutes, and then 10. mu.L of NaBH at a concentration of 10mmol/L was added₄And (3) rapidly stirring the solution at room temperature for 40 minutes, and gradually changing the solution into deep red to obtain the cysteine modified gold nanoparticles.

6. The method according to claim 3, wherein the citric acid-melamine modified gold nanoparticles are prepared by the following steps: 100mL of 0.01% HAuCl₄Condensing and refluxing the solution, heating to a boiling state, adding 5mL of a citric acid solution with the mass concentration of 1%, continuing boiling for 15 minutes after the solution turns to be wine red to obtain the citric acid modified gold nanoparticles, mixing the citric acid modified gold nanoparticles and melamine with the mass concentration of 20:1, and incubating for 10 minutes to obtain the citric acid-melamine modified gold nanoparticles.

7. Use of a gold cluster and gold nanoparticle based fluorescent array sensor according to claim 1 or 2 for the analytical detection of vitamins B6, B9, B12.

8. A method for the analytical detection of vitamins B6, B9, B12, characterized in that it comprises the following steps: adding a sample containing vitamins B6, B9, B12 to the gold cluster and gold nanoparticle-based fluorescence array sensor according to claim 1 or 2, and measuring the change in fluorescence signal intensity before and after the addition.

Technical Field

The invention belongs to the field of organic chemistry, and particularly relates to a fluorescence array sensor based on gold clusters and gold nanoparticles, and a preparation method and application thereof.

Background

Vitamin B, also known as vitamin B, the B vitamin family or the B vitamin complex, is a generic term for B vitamins and is usually derived from the same food source, such as yeast. Vitamin B was mistaken for a single structural organic compound, and later studies showed that vitamin B is a group of compounds having different structures, such as vitamin B1, B2, B6, and the like. Vitamin B is water-soluble vitamins, and has synergistic effect in regulating metabolism, maintaining skin and muscle health, improving immune system and nervous system function, and promoting cell growth and division. Wherein, the three vitamins B of B6, B9 and B12 have important effect on pregnant women.

Vitamin B6(Vitamin B6) is a water-soluble Vitamin, also called pyridoxine, including pyridoxine, pyridoxamine and pyridoxal, which exists in the body in the form of phosphate esters. The vitamin B6 is colorless crystal, is stable in acid solution, and is easily destroyed in alkali solution; pyridoxine is thermostable, but pyridoxamine and pyridoxal are not. Vitamin B6 is present in yeast, liver, fish, egg, meat, beans and grain in high amounts. Vitamin B6 is a component of some coenzymes in human bodies and is involved in various metabolic reactions, particularly in close relation with amino acid metabolism. The vitamin B6 preparation is usually used clinically to prevent and treat vomiting of pregnancy and vomiting of radiation sickness in women. Vitamin B6 is an essential substance for metabolism of sugar and fat in human body, and vitamin B6 is more required for female estrogen metabolism, so that B6 has great effect on preventing and treating some gynecological diseases. Many women may have feelings of low mood, splenic irritability, feelings of hypodynamia, etc. due to taking contraceptive, and only need to supplement about 60mg per day to relieve symptoms. Still some women suffer from premenstrual tension syndrome, which is often manifested as edema of the eyelid and hands and feet before menstruation, or insomnia and amnesia, and can be completely relieved by eating 50-100mg of vitamin B6 every day.

Folic Acid (FA) is a water-soluble vitamin (B9), and has the main functions of participating in the synthesis and metabolism of ribonucleic Acid and deoxyribonucleic Acid, also participating in amino Acid metabolism, promoting the maturation of juvenile cells in bone marrow, and participating in the production of red blood cells and white blood cells, thereby enhancing the autoimmune capability of human body. If the human body is lack of folic acid or the intake of folic acid is insufficient, various diseases can occur, such as gradual reduction of leukocytes in the body, depression, senile dementia, cardiovascular diseases and Down syndrome, and even body canceration. In addition, folic acid is important for the normal development of the fetus in the mother, and proper folic acid supplementation can reduce the probability of the occurrence of spinal fissure, neural tube development defects, and brain and skull deformity of the fetus before and during pregnancy. Since the human body cannot synthesize folic acid by itself, the folic acid must be obtained from food, and the U.S. Food and Drug Administration (FDA) has made a mandatory regulation, requiring that the folic acid content of some food products be above 1400 mg/kg-1. Therefore, the assay of folate is important for human health and in particular for the normal development of the fetal nervous system in the mother.

Vitamin B12, also known as cyanocobalamin or cobalamin, is a cobalt-containing B-complex vitamin consisting of porphyrins. After 20 years of research, scientists finally separated a red substance named vitamin B12 from the liver in 1948, wherein the red substance has the effect of treating pernicious anemia. The vitamin B12 is the latest vitamin found in all B vitamins, is easily soluble in water and ethanol, is most stable in a weak acid condition with the pH value of about 4.5-5.0, is decomposed under a strong acid or alkaline condition, is damaged to a certain extent by heat, but is not greatly sterilized at high temperature in a short time, and is easily damaged by ultraviolet rays or strong light. Vitamin B12 has the following main effects: the coenzyme exists in human body in the form of coenzyme, can improve the utilization rate of folic acid and promote the metabolism of fat, carbohydrate and protein; promoting the growth and maturation of erythrocytes to maintain the hematopoietic function of the human body in a normal state, thereby preventing pernicious anemia and maintaining the health of the nervous system; has the functions of promoting the synthesis of nucleic acid and activating amino acid, promoting the synthesis of protein, and has important effects on the growth and development of infants and juveniles; is a vitamin necessary for the functional health of the nervous system because it is involved in the formation of a lipoprotein in nervous tissue; eliminate anxiety, help to concentrate attention, and enhance memory and sense of balance.

At present, the detection methods of vitamin B group molecules are various, such as electrochemical method, enzyme-linked immunosorbent assay, chromatography and the like, but the methods are easily interfered by other components of a sample during actual detection, and besides, some methods have the defects of poor stability, low sensitivity, high detection cost, long consumed time or various steps.

The fluorescent array sensor is one of optical sensors, and has the advantages of rich output signals, high sensitivity, imaging and the like. In the last decade, researchers have developed many fluorescent array sensors based on the principle of fluorescence sensing for detecting metal ions, biological macromolecules and some organic compounds.

The metal nanoclusters contain several to hundreds of different metal atoms, generally have particle sizes smaller than 3 nanometers, have different electronic energy levels and discontinuous energy band structures, and have molecular characteristics such as catalytic activity, electricity, magnetism, light, heat and the like. Compared with the traditional organic molecules, the metal nanocluster not only has quite ideal biocompatibility, but also has low cytotoxicity and strong photobleaching resistance, and particularly, the gold nanoclusters (AuNCs) show the excellent characteristics, so that the metal nanocluster has important application significance in medical diagnosis, biological detection and environmental analysis and monitoring.

Gold nanoparticles (AuNPs) are a nano material in early research, are generally called colloidal gold in biological research, have the diameter of 1-100 nm, have good stability and show different colors along with the change of particle size. The gold nanoparticles have high electron density and good contrast under an electron microscope, have the advantages of sensitive optical property, easy surface modification, good biocompatibility and the like, become ideal functional materials in the fields of analytical chemistry, environmental monitoring, medical diagnosis and the like, can be used as probes for accurately positioning biomacromolecules such as cell surface and intracellular polysaccharide, protein, polypeptide, antigen, hormone, nucleic acid and the like, can also be used for conventional immunodiagnosis and immunohistochemical positioning, and can be widely applied to the aspects of clinical diagnosis, drug detection and the like.

Disclosure of Invention

An object of the present invention is to provide an optical sensor for analyzing and detecting vitamin B group substances with good stability, high sensitivity, and simplicity and convenience.

Another object of the present invention is to provide a method for manufacturing the above optical sensor.

It is yet another object of the present invention to provide a method for analytically detecting a vitamin B group substance.

In order to achieve the above object, the present invention provides a gold cluster and gold nanoparticle-based fluorescence array sensor, which comprises gold nanoclusters with a negatively charged surface, and cysteine-modified gold nanoparticles and citric acid-melamine-modified gold nanoparticles with a positively charged surface.

According to the fluorescence array sensor based on the gold clusters and the gold nanoparticles, the gold nanoclusters are synthesized by taking bovine serum albumin as a template.

On the other hand, the invention also provides a preparation method of the fluorescence array sensor based on the gold clusters and the gold nanoparticles, which comprises the following steps:

step 1: preparing gold nanoclusters;

step 2: preparing gold nanoparticles modified by cysteine;

and step 3: preparing the citric acid-melamine modified gold nanoparticles.

Preferably, the gold nanoclusters are prepared by the following steps:

5.0mL of HAuCl at a concentration of 10mmol/L₄Adding the solution into 5.0mL of bovine serum albumin solution with the concentration of 50mg/mL, vigorously stirring at 37 ℃ for 2 minutes, quickly adding 0.5mL of NaOH solution with the concentration of 1mol/L into the reaction solution, and vigorously stirring at 37 ℃ for 12 hours to obtain the gold nanocluster solution.

Preferably, the cysteine modified gold nanoparticles are prepared by the following steps:

500. mu.L of 215mmol/L cysteine solution and 2.23mL of 25.5mmol/L HAuCl₄The solution was mixed well, 37.5mL of distilled water was added, stirred at room temperature for 20 minutes, and then 10. mu.L of NaBH at a concentration of 10mmol/L was added₄And (3) rapidly stirring the solution at room temperature for 40 minutes, and gradually changing the solution into deep red to obtain the cysteine modified gold nanoparticles.

Preferably, the preparation steps of the citric acid-melamine modified gold nanoparticles are as follows:

100mL of 0.01% HAuCl₄Condensing and refluxing the solution, heating to a boiling state, adding 5mL of a citric acid solution with the mass concentration of 1%, continuing boiling for 15 minutes after the solution turns to be wine red to obtain the citric acid modified gold nanoparticles, mixing the citric acid modified gold nanoparticles and melamine with the mass concentration of 20:1, and incubating for 10 minutes to obtain the citric acid-melamine modified gold nanoparticles.

The invention also provides application of the fluorescent array sensor based on the gold clusters and the gold nanoparticles to analysis and detection of vitamins B6, B9 and B12.

The invention also provides a method for analyzing and detecting the vitamins B6, B9 and B12, which comprises the following steps: adding a sample containing vitamins B6, B9 and B12 into the fluorescent array sensor based on the gold clusters and the gold nanoparticles, and measuring the change of the fluorescence signal intensity before and after the addition.

The principle of the fluorescence array sensor of the invention is as follows: the gold clusters are negatively charged, while the two differently modified gold nanoparticles are both positively charged, and due to the electrostatic effect, the gold clusters are adsorbed to the surface of the gold nanoparticles, whereby fluorescence is quenched, and when a vitamin B (e.g., B6, B9, B12) analyte is added, the negatively charged vitamin B molecules cause the gold nanoparticles to aggregate, and the interaction between the gold clusters and the gold nanoparticles is destroyed, thereby restoring the fluorescence of the gold clusters, but to a different extent, that is, different vitamin B analytes can generate different fluorescence response spectra on the fluorescence array sensor due to different structural characteristics, and then the identification and discrimination results of the vitamin B analytes are obtained by utilizing Linear Discriminant Analysis (LDA) to draw fingerprint spectra, and classifying the sample on the fingerprint spectrum in the actual sample detection to realize the identification and detection of the three vitamin B molecules.

Drawings

Fig. 1 is a fourier infrared spectrum of gold nanoclusters.

Fig. 2 is a graph of the uv-vis absorption spectra of gold nanoclusters.

FIG. 3 is a fluorescence emission spectrum of gold nanoclusters.

FIG. 4 is a graph of fluorescence excitation spectra of gold nanoclusters.

FIG. 5 is a Fourier infrared spectrum of cysteine-modified gold nanoparticles cys-AuNPs.

FIG. 6 is a Fourier infrared spectrum of sodium citrate-melamine modified gold nanoparticles Mel-citr-AuNPs.

FIG. 7 is a UV-VIS absorption spectrum of cysteine modified gold nanoparticles cys-AuNPs.

FIG. 8 is a diagram of the ultraviolet-visible absorption spectrum of sodium citrate-melamine modified gold nanoparticles Mel-citr-AuNPs.

FIG. 9 is the effect of different concentrations of B6 solution on the fluorescence intensity of gold clusters.

FIG. 10 is a graph showing the effect of different concentrations of B9 solution on the fluorescence intensity of gold clusters.

FIG. 11 is a graph showing the effect of different concentrations of B12 solution on the fluorescence intensity of gold clusters.

Fig. 12 shows the results of the fluorescence response of three vitamin B group molecules on the sensing unit 1.

Fig. 13 shows the results of the fluorescence response of three vitamin B group molecules on the sensing unit 2.

FIG. 14 shows the results of the fluorescence response of three aqueous vitamin B group molecules (13.65. mu.M) on a fluorescence array sensor.

FIG. 15 is the LDA analysis of three solution samples (13.65. mu.M).

FIG. 16 is the result of HCA analysis of three solution samples (13.65. mu.M).

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only, and are not intended to limit the scope of the present invention.