CN112098382A

CN112098382A - Ratiometric fluorescent probes and their use in penicillamine detection

Info

Publication number: CN112098382A
Application number: CN202010991714.9A
Authority: CN
Inventors: 姚永杰; 张奎
Original assignee: Anhui University of Technology AHUT
Current assignee: Anhui University of Technology AHUT
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2020-12-18
Anticipated expiration: 2040-09-16
Also published as: CN112098382B

Abstract

The invention discloses a ratiometric fluorescent probe and application thereof in penicillamine detection. The ratio-type fluorescence sensor of the present invention has two emission wavelengths, and the amount of the target substance is quantified based on the relationship between the ratio of the fluorescence intensity at the two wavelengths and the concentration of the analyte to be detected. By establishing the internal standard, the method has a self-regulation function, and greatly weakens the interference of variable or difficult-to-quantify factors on experiments and results. Moreover, the color of the ratiometric probe changes gradually with the change of the concentration of the analyte, so that the analyte can be visually detected, and semi-quantitative and qualitative analysis of the analyte can be realized according to the color of the probe.

Description

Ratiometric fluorescent probes and their use in penicillamine detection

Technical Field

The invention relates to a ratiometric fluorescent nanoprobe phenolic resin-Au cluster-Cu²⁺A detection system and construction thereof, and also relates to application of the ratiometric fluorescent nano probe in detecting penicillamine.

Background

Penicilliamine (D-Penicilliamine, D-PA) chemical name 3, 3-dimethyl- (D) -cysteine, is a sulfhydryl-containing amino acid with the ability to chelate metal ions, in which Cu is chelated²⁺The effect of (2) is particularly outstanding. Therefore, the compound has outstanding capacity in treating hepatolenticular degeneration (HLD), a copper metabolism disorder disease, heavy metal poisoning and the like. On the other hand, the mercapto group of D-penicillamine can decompose the rheumatoid factor belonging to macroglobulin by the cleavage of disulfide bondThis serves to reduce the level of serum rheumatoid factors. Based on the above, penicillamine has been widely used in autoimmune diseases such as scleroderma and rheumatoid arthritis. In addition, penicillamine can interfere the cross-linking of collagen (tropicolen) into insoluble collagenous tissues, and can prevent the maturation of soluble collagen, so that the penicillamine can be used for promoting the proliferation of connective tissues. Penicillamine also has an anti-inflammatory effect, and is mainly expressed in inhibiting the release of lysosomal enzymes to stabilize lysosomal membranes. In addition, the study of Xuhong et al on mice shows that high dosage of penicillamine achieves the ideal effect of reducing the copper content in vivo, but causes some side effects; low doses of penicillamine are not as effective; the penicillamine with medium dosage can not only obviously remove the copper element in serum and liver, but also has no adverse effect on experimental rats. Therefore, the simple and rapid detection of the concentration of the penicillamine in the medicine and the body has important significance for the research of the biomedicine and pharmacology.

Fluorescent metal nanoclusters composed of several metal atoms are a promising fluorescent probe due to their small size, good photostability, customizable surface properties and high photoluminescence depending on size, as well as a wide range of applications. Compared with the silver and copper nanoclusters which are easy to oxidize, the luminescent gold nanoclusters are stable in chemical property and easy to control in size. Therefore, gold nanoclusters have recently been considered as a satisfactory candidate material for imaging, detecting metal ions, and for fabricating fluorescent sensors.

At present, various methods for measuring the penicillamine content exist. Such as high performance liquid chromatography, infrared spectroscopy, ultraviolet spectrophotometry, and the like. However, the above methods, such as high performance liquid chromatography, have disadvantages of high price, small detection amount, need of using different types of packed columns, difficulty in analyzing inorganic ions and bio-macromolecules, large consumption of mobile phase, much toxicity, and extra-column effect. The infrared spectroscopy is not suitable for analyzing a water-containing sample because hydroxyl peaks in water have large interference on measurement, has large error and low sensitivity when quantitative analysis is carried out, and mainly depends on experience when pattern analysis is carried out. The ultraviolet spectrophotometry is expensive, the ultraviolet absorption of different substances is different, the measurement result has certain error, and the experimental result is influenced and limited by the cuvette material, the pH value of the solution, the buffer medium solution, the light source and other factors. Although the sensitivity of fluorescence detection has become very high with the continuous progress of fluorescein and fluorescence detection technology in recent years, the detection level of isotope labeling is reached, and compared with the isotope labeling method, the fluorescence labeling method has the advantages of safety, convenience, no radioactive pollution and the like, so that the fluorescence probe method can be selected to detect the concentration of penicillamine. However, the penicillamine molecule itself is not fluorescent, so that the measurement of penicillamine by derivatization fluorescence spectroscopy becomes an option. However, the derivatization method is difficult to be popularized in practical application due to the reasons of complicated operation, low sensitivity, poor reproducibility and the like, and a brand new method is urgently needed to make up for various defects of the method so as to achieve the purpose of rapidly and accurately detecting penicillamine. Single emission fluorescence sensors have considerable advantages and make up for the deficiencies of some conventional approaches. However, the single-emission signal fluorescence sensor has some defects, such as the quantum dot nanoprobe is easily interfered by a plurality of variable or hard-to-quantify factors, such as probe concentration, polarity, temperature, environmental pH value, stability and the like, thereby leading to the uncertainty of the signal.

Disclosure of Invention

The invention provides a ratiometric fluorescent probe phenolic resin-Au cluster-Cu for overcoming the defects of the existing penicillamine detection technology²⁺The invention is used for detecting penicillamine, and has the advantages of high detection speed, high accuracy and sensitivity and easy operation.

The invention researches and constructs the phenolic resin-Au quantum dot of the ratiometric fluorescence sensor with dual-emission fluorescence, and the sensor passes through Cu²⁺Has quenching effect on the fluorescence of Au quantum dots, and penicillamine can react with Cu²⁺Interaction to release Cu² ⁺The Au quantum dots gradually recover fluorescence to realize high sensitivity and high fluorescence of the D-penicillamineAnd (4) detecting the accuracy. And fluorescent substance Phenol Formaldehyde Resin (PFR) as internal standard to Cu²⁺And does not respond to penicillamine. The fluorescence intensity of the Au quantum dots was thus observed with PFR as an internal standard to make a linear relationship between the ratiometric fluorescence and the penicillamine concentration.

The ratio-type fluorescence sensor of the present invention has two emission wavelengths, and the amount of the target substance is quantified based on the relationship between the ratio of the fluorescence intensity at the two wavelengths and the concentration of the analyte to be detected. By establishing the internal standard, the self-regulation function is realized, and the interference of variable or difficult-to-quantify factors on the experimental result is greatly weakened. Moreover, along with the change of the concentration of the analyte, the color of the ratiometric probe is changed step by step, so that the visualized detection of the analyte is realized, the detection easiness in identification is improved, the detection speed is improved, and the semi-quantitative and qualitative analysis of the analyte can be realized according to the color of the probe. The established sensor mode improves the range of dynamic response by means of the change of the fluorescence intensity ratio.

If only a single fluorescent probe of an Au cluster is adopted for detecting the penicillamine, the orange-red fluorescence with the change of single fluorescence intensity is difficult to be directly distinguished by naked eyes (the orange-red fluorescence is faded down along with the reduction of the penicillamine concentration, and the faded-down of the single color is difficult to be distinguished by the naked eyes); in contrast, in the present invention, the phenol resin-Au cluster ratiometric fluorescent probe is used, and the color change that is easily distinguished appears as the blue-green light of the fluorescent phenol resin gradually changes from red to blue with the decrease of the penicillamine concentration, as shown in fig. 4. Therefore, compared with a detection method for single fluorescence intensity change, the ratiometric fluorescence detection method is more sensitive, and the visual detection is more reliable and easier to distinguish.

Drawings

FIG. 1 is a fluorescence emission curve (610nm emission peak) of the prepared Au cluster under 270nm excitation light and an ultraviolet-visible absorption curve thereof.

FIG. 2 is a fluorescence emission curve (410nm emission peak) of the prepared phenolic resin under 312nm excitation light and an ultraviolet-visible absorption curve thereof.

FIG. 3 shows the construction of a system for detecting Cu in penicillamine solution²⁺Fluorescence emission curve for quenching Au clusterA wire.

FIG. 4 shows a phenol resin-Au cluster-Cu²⁺And (3) when the solution system detects penicillamine, the penicillamine enables the Au cluster to recover fluorescence.

FIG. 5 shows the ratio of penicillamine concentration to Au cluster fluorescence intensity (I) of phenolic resin₆₁₀/I₄₁₀) The relationship of (1).

FIG. 6 shows a phenol resin-Au cluster-Cu cluster²⁺The detection system tests the selectivity of the penicillamine.

Fig. 7 is an SEM image of fluorescent phenolic resin.

Detailed Description

Example 1

The preparation method of the Au cluster comprises the following steps:

accurately preparing a 0.01M chloroauric acid solution (1g chloroauric acid is dissolved in deionized water, and then the volume is determined by a 250mL volumetric flask); then 200mL of 1M NaOH solution is prepared for standby. A50 mL round-bottom flask was charged with 10mL deionized water, 0.0544g of MUA (11-mercaptoundecanoic acid) was weighed into the round-bottom flask, and after sonication for 5min, 1M NaOH solution was added while sonication to completely dissolve the MUA, and the addition of NaOH was stopped. Then 6250. mu.L of the HAuCl previously prepared was slowly added with stirring₄The solution, if floc appears, is stirred for half an hour, the precipitate disappears, NaOH solution (1M) is added dropwise until the solution becomes clear, and the solution is stirred for 24 hours at room temperature. Dialyzing the reacted solution for 24h, wherein the obtained solution is colorless under sunlight and strong orange-red light under 312nm ultraviolet light, namely Au cluster solution, centrifuging at 10000rpm for 10min before use, discarding white precipitate, and taking supernatant for use.

Example 2

The preparation method of the phenolic resin comprises the following steps:

0.05mmol of phenol and 0.025mmol of Hexamethylenetetramine (HMT) are dispersed in 22ml of ultrapure water, are fully dissolved and then are transferred into a hydrothermal reaction kettle to react for 4 hours at the constant temperature of 160 ℃. And (4) storing the product obtained by the reaction at low temperature. The obtained phenolic resin aqueous solution is blue-green under 312nm ultraviolet light. Before the experiment, the reaction kettle inner container is made of concentrated HNO with the volume ratio of 10:1₃And H₂Soaking the mixture of O for 4h, adding ultrapure water, washing for 4h at the constant temperature of 140 ℃,and finally drying. From FIG. 7, the phenolic resin of the present invention is uniformly distributed and has a uniform size.

Example 3

The construction method of the solution system for detecting penicillamine by ratio fluorescence comprises the following steps:

the penicillamine with the concentration of 10mM is accurately prepared, and 1000 mu L of the penicillamine with the concentration of 10mM and 9.0ml of deionized water are absorbed to prepare the penicillamine with the concentration of 1.0mM for use.

② accurately preparing 10mM Cu (NO)₃)₂The solution was pipetted 1000. mu.L of 10mM Cu (NO)₃)₂The solution was made up to 1.0mM Cu (NO) with 9.0mL of deionized water₃)₂The solution was left for use.

③ adding 2mL of water, 1mL of Au cluster solution and 1mL of phenolic resin aqueous solution into a test tube for use.

Adding 2mL of water and 100 mu L of phenolic resin-Au cluster mixed solution into a cuvette, sequentially adding 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 mu L of 1mM Cu (NO3)2 solution to enable the concentration of Cu (NO3)2 in a detection system to reach 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 mu M in sequence, and recording a fluorescence spectrum chart in sequence, wherein after the 28 mu L of 1mM Cu (NO3)2 solution is added, the fluorescence intensity of the Au cluster is not reduced any more, namely the phenolic resin-Au cluster-Cu cluster²⁺Detection system (ratiometric fluorescent probes), as in FIG. 3. The copper ions are added to quench the fluorescence of the Au clusters in the fluorescent probe.

Example 4

Phenolic resin-Au cluster-Cu²⁺The detection system detects penicillamine by the following method:

the phenolic resin-Au cluster-Cu prepared in the example 3 is taken for detection by adopting a fluorescence spectroscopy method²⁺A solution detection system, wherein 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μ L of 1mM penicillamine are sequentially added to ensure that the concentration of the penicillamine in the detection system sequentially

reaches

1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 μ M, and the fluorescence spectrum of the solution system is sequentially recorded, as shown in figure 4; the relationship between the ratio of the fluorescence intensity of the Au cluster to the fluorescence intensity of the phenol resin and the penicillamine concentration was obtained from the fluorescence spectrum, as shown in fig. 5.

Example 5

Phenolic resin-Au cluster-Cu²⁺The selective detection experiment of the detection system on the penicillamine comprises the following steps;

respectively preparing 1mM of L-lysine, L-valine, D-alanine, L-leucine, L-glutamic acid, glucose, NaCl, KCl and CaCl₂And penicillamine solutions, 5. mu.L of each solution was separately added to the phenolic resin-Au cluster-Cu prepared in example 3²⁺In the solution detection system, the fluorescence spectroscopy is adopted to respectively record corresponding fluorescence spectrograms, and the change delta I of the fluorescence intensity of the Au cluster before and after the solution is added is recorded, and the result is shown in figure 6, compared with other results, the phenolic resin-Au cluster-Cu composite material provided by the invention²⁺The detection system (ratiometric fluorescent probe) has the most sensitive detection response to penicillamine.

Example 6

Phenolic resin-Au cluster-Cu²⁺The detection system detects the concentration of penicillamine in urine by the following method:

a2 mL urine sample was taken and placed in a cuvette, and 100. mu.L of the mixed solution of phenol resin and Au clusters (prepared in example 3) and 28. mu.L of 1mM Cu (NO) were added₃)₂The method comprises the steps of adding 4, 10 and 16 mu L of 1mM penicillamine solution into a solution independently respectively to enable the concentration of penicillamine in a system to reach 2, 5 and 8 mu M, respectively recording corresponding spectrograms by adopting a fluorescence spectroscopy, calculating the fluorescence intensity ratio of Au clusters to phenolic resin according to the spectrograms, calculating the fluorescence intensity ratio of corresponding Au clusters to phenolic resin when the concentration of penicillamine reaches 2, 5 and 8 mu M according to the relation between the obtained penicillamine concentration and the fluorescence intensity ratio of Au clusters to phenolic resin, and comparing the fluorescence intensity ratio corresponding to the concentration of penicillamine in urine with the ratio. Add: adding penicillamine concentration, Found: detecting the concentration of penicillamine, Recovery: and (4) recovering rate.

TABLE 1 detection test for penicillamine in urine

Sample	Add(μM)	Found(μM)	Recovery(％)
				1	2.0	2.27	113.52
2	5.0	5.23	104.63
				3	8.0	8.12	101.54

It should be noted that the technical contents described above are only explained and illustrated to enable those skilled in the art to know the technical spirit of the present invention, and therefore, the technical contents are not to limit the scope of the present invention. The scope of the invention is defined by the appended claims. It should be understood by those skilled in the art that any modification, equivalent replacement, and improvement made based on the spirit of the present invention should be considered to be within the spirit and scope of the present invention.

Claims

1. A ratiometric fluorescent probe is mainly composed of phenolic resin, Au cluster and Cu²⁺And compounding to form a detection system.

2. The method for preparing ratiometric fluorescent probe of claim 1, comprising the steps of synthesizing fluorescent phenolic resin, preparing Au clusters, and preparing phenolic resin-Au clusters-Cu²⁺And (5) constructing a detection system.

3. The method for preparing the ratiometric fluorescent probe according to claim 2, wherein the fluorescent phenolic resin is synthesized by performing a hydrothermal reaction of phenol and hexamethylenetetramine at 150-180 ℃ for 3-6h, preferably at 160 ℃ for 4 h.

4. The method for preparing ratiometric fluorescent probes of claim 3, wherein the molar ratio of phenol to hexamethylenetetramine is 1.8-2.2: 1, preferably in a molar ratio of 2: 1.

5. the method for preparing a ratiometric fluorescent probe according to claim 4, wherein concentrated HNO with a volume ratio of 10:1 is adopted in advance in an inner container of a reaction kettle for the hydrothermal reaction₃And H₂Soaking the mixture of O for 4-5h, adding ultrapure water, washing for 4-5h at constant temperature of 140-150 ℃, drying the inner container, and then carrying out hydrothermal reaction.

6. The method of claim 2, wherein the Au cluster is prepared by reacting 11-mercaptoundecanoic acid with a chloroauric acid solution.

7. The method of claim 2, wherein the phenolic resin-Au cluster-Cu cluster is used to form a ratiometric fluorescent probe² ⁺The construction of the detection system adopts the following steps: uniformly mixing the Au cluster solution and the phenolic resin solution to obtain an Au cluster-phenolic resin mixed solution for later use; respectively taking equal amount of Au cluster-phenolic resin mixed solution, and sequentially adding Cu with the same concentration and different dosage²⁺The solution is dissolved, and the fluorescence spectrum spectrograms are recorded in sequence until the fluorescence intensity of the Au cluster is not reduced any more, namely the phenolic resin-Au cluster-Cu²⁺And (3) a detection system.

8. The method for preparing a ratiometric fluorescent probe of claim 2,

(1) synthesis of fluorescent phenolic resin: 0.05mmol of phenol and 0.025mmol of hexamethylenetetramine are dispersed in 22ml of ultrapure water, are fully dissolved, are transferred into a hydrothermal reaction kettle, react for 4 hours at the constant temperature of 160 ℃, and the product obtained by the reaction is stored at low temperature;

(2) preparation of Au clusters: preparing a 0.01M chloroauric acid solution, dissolving 1g chloroauric acid in deionized water, and fixing the volume in a 250mL volumetric flask; preparing 200mL of 1MNaOH solution for later use; adding 10mL of deionized water into a 50mL round bottom flask, weighing 0.0544-0.0546g of 11-mercaptoundecanoic acid, adding into the round bottom flask, performing ultrasonic treatment for 5min, adding 1M NaOH solution while performing ultrasonic treatment to completely dissolve MUA, stopping adding NaOH, and slowly adding 6250 μ L of HAuCl prepared before under stirring₄Stirring the solution for half an hour if floccules appear, dripping 1MNaOH solution until the solution becomes clear if precipitates do not disappear, stirring the solution at room temperature for 24 hours, dialyzing the reacted solution for 24 hours, wherein the obtained solution is colorless under sunlight and is strong orange-red under 312nm ultraviolet light, namely the Au clusters;

(3) phenolic resin-Au cluster-Cu²⁺Construction of a detection system:

uniformly mixing the Au cluster solution obtained in the step (2) and the phenolic resin solution obtained in the step (1) to obtain an Au cluster-phenolic resin mixed solution for later use; respectively taking equal amount of Au cluster-phenolic resin mixed solution, and sequentially adding 1.0mM of Cu with different dosages²⁺The solution is dissolved, and the fluorescence spectrum spectrograms are recorded in sequence until the fluorescence intensity of the Au cluster is not reduced any more, namely the phenolic resin-Au cluster-Cu²⁺And (3) a detection system.

9. Use of the ratiometric fluorescent probe of claim 1 or obtained by the preparation process of any one of claims 2 to 8 for the detection of penicillamine.

10. The use of claim 9, wherein the equivalent amount of phenol-formaldehyde resin-Au cluster-Cu is measured by fluorescence spectroscopy²⁺Solution testingAnd (3) sequentially adding 1mM of penicillamine with different dosages into the system, sequentially recording the fluorescence spectrum of the solution system, and obtaining the relation between the ratio of the fluorescence intensity of the Au cluster to the fluorescence intensity of the phenolic resin and the concentration of the penicillamine according to the fluorescence spectrum.