CN106053410A - Compounded liquid of quercetin and cyclodextrin and applications thereof - Google Patents

Abstract

The invention discloses a compound solution of quercetin and cyclodextrin and its application. The compound solution is a binary compound solution of quercetin and cyclodextrin, wherein the concentration of quercetin is 1×10 ^{‑ 5} mol•L ^‑1 , the cyclodextrin is 2‑hydroxypropyl‑β‑cyclodextrin, and the mass concentration ratio of cyclodextrin to quercetin is 100~900:1. The invention uses the quercetin-cyclodextrin binary system compound solution as a fluorescent probe to detect copper ions in the aqueous solution, which is simple and quick to operate, has strong ability to identify copper ions, is less interfered by other metal ions, and has high sensitivity. , large linear range and low detection limit. Compared with quercetin alone, cyclodextrin can significantly enhance the fluorescence intensity of quercetin in this compound solution, and the fluorescence intensity of quercetin solution gradually decreases with time, while quercetin-cyclodextrin two The fluorescence intensity of the metasystem does not change much with time, and the stability is greatly improved. The quercetin-cyclodextrin binary system can produce fluorescence in cells and is non-toxic to cells. It is expected to be used to detect copper ion content in cells.

Description

Compound solution of quercetin and cyclodextrin and its application

technical field

The invention belongs to the technical field of spectral analysis and detection, and specifically relates to a compound solution of quercetin and cyclodextrin and an application thereof.

Background technique

Copper widely exists in nature and the human body, and is an indispensable trace element for the human body. At the same time, copper is widely used in metal smelting, electronics, packaging and other industries closely related to human life. With the accelerated development of human society, mineral resources The phenomenon of indiscriminate mining is becoming more and more serious. In addition to the three industrial wastes, domestic garbage and electronic waste are also increasing, which eventually leads to the loss of heavy metal ions, resulting in excessive heavy metal ion content in soil and water. Although copper is an essential trace element for the human body, excessive copper content can cause gastrointestinal discomfort in mild cases, nerve damage, Parkinson’s syndrome, and even life-threatening in severe cases. Therefore, it is necessary to monitor the concentration of copper ions in the environment content appears to be important.

At present, there are many methods for detecting copper ions, such as inductively coupled plasma mass spectrometry, flame atomic absorption method, electrochemical method, etc. High cost and low efficiency. In order to overcome the above shortcomings, there are more and more reports on the detection of copper ions by fluorescence method. In addition to the advantages of high sensitivity and good selectivity, the fluorescence method also has the advantages of rapid detection, simplicity, and low cost. However, at present, most of the fluorescent probes used to detect copper ions are obtained through organic synthesis, and many toxic chemical reagents are used in the experimental process, which virtually causes secondary pollution to the environment.

Quercetin (C ₁₅ H ₁₀ O ₇ , 3,5,7-trihydroxy-2-(3,4-dihydroxyphenyl)benzopyran-4-one) is a flavonoid compound widely present in nature Plants, such as: Ginkgo biloba, onion, Sophora japonica rice, etc. Quercetin has many biological activities such as anti-oxidation, dilation of blood vessels, and anti-tumor, but there are few studies on its fluorescence properties.

Contents of the invention

Purpose of the invention: In view of the deficiencies in the prior art, the purpose of the invention is to provide a compound solution of quercetin and cyclodextrin, which can meet the needs of detecting copper ions. Another object of the present invention is to provide the application of the compound solution of quercetin and cyclodextrin, which has the characteristics of good selectivity, high sensitivity, wide linear range, simplicity, environmental protection and low toxicity.

Technical solution: In order to realize the above-mentioned purpose of the invention, the technical solution adopted in the present invention is:

Application of the compound solution of quercetin and cyclodextrin in the determination of copper ions.

The application is characterized in that it comprises the following steps:

(1) prepare the buffer solution of quercetin and cyclodextrin binary system;

(2) Add different volumes of copper ion solutions to the buffer solution of the quercetin-cyclodextrin binary system respectively, use a fluorescence spectrometer to record the fluorescence intensity of each solution, and draw the standard curve of the fluorescence intensity to the copper ion concentration;

(3) Add the copper ion test solution to the quercetin-cyclodextrin binary system solution, and use a fluorescence spectrometer to record the fluorescence intensity of the solution;

(4) Determine the concentration of copper ions in the solution to be tested according to the standard curve.

The buffer solution is CH ₃ OH-PBS buffer solution, the pH of the solution is 7.4, and the volume ratio of methanol to phosphate buffer saline solution is 1-5:99.

The conditions for measuring the fluorescence intensity are that the excitation slit is 5nm, the emission slit is 15nm, the scanning range is 400-700nm, the excitation wavelength is 390nm, and the emission wavelength is 553nm.

The concentration of quercetin is 1×10 ^-5 mol·L ^-1 , the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin, and the mass concentration ratio of cyclodextrin to quercetin is 100～ 900:1.

The linear concentration range of copper ions is 5.0×10 ^-8 -8.3×10 ^-6 mol·L ^-1 , and the detection limit is 2.3×10 ^{- 8} mol·L ^-1 .

Said application, the standard curve of fluorescence intensity versus copper ion concentration, y=-9.24x+844.51, y is fluorescence intensity, x is copper ion concentration, and the linear correlation coefficient is R ² =0.997.

Application of the compound solution of quercetin and cyclodextrin in the detection of copper ions in cells.

A compound solution for detecting copper ions is a binary compound solution of quercetin and cyclodextrin, wherein the concentration of quercetin is 1×10 ^-5 mol·L ^-1 , and the concentration of cyclodextrin is 2-hydroxypropyl-β-cyclodextrin, the mass concentration ratio of cyclodextrin and quercetin is 100-900:1.

In the compound solution for detecting copper ions, both quercetin and cyclodextrin are dissolved in CH ₃ OH-PBS buffer solution, the pH of the solution is 7.4, and the volume ratio of methanol to phosphate buffered saline solution is 1-5 :99.

Beneficial effects: Compared with the prior art, the present invention uses the quercetin-cyclodextrin binary system compound solution as a fluorescent probe to detect copper ions in the aqueous solution, and utilizes the quercetin-cyclodextrin before and after adding copper ions The changing law of the fluorescence intensity of the binary system, draw the standard curve of the fluorescence intensity to the concentration of copper ions, and calculate the concentration of copper ions in the unknown sample according to the standard curve. The operation is simple and fast, and the ability to identify copper ions is strong. Small interference, high sensitivity, large linear range, low detection limit. Compared with quercetin alone, cyclodextrin can significantly enhance the fluorescence intensity of quercetin in this compound solution, and the fluorescence intensity of quercetin solution gradually decreases with time, while quercetin-cyclodextrin two The fluorescence intensity of the metasystem does not change much with time, and the stability is greatly improved. The quercetin-cyclodextrin binary system can produce fluorescence in cells and is non-toxic to cells. It is expected to be used to detect the content of copper ions in cells.

Description of drawings

Figure 1 is a graph showing the enhancing effect of cyclodextrin on the fluorescence intensity of quercetin;

Figure 2 is a graph showing the change in fluorescence intensity of quercetin and quercetin-cyclodextrin binary system over time;

Fig. 3 is the fluorescence titration spectrogram of quercetin-cyclodextrin binary system to copper ion;

Fig. 4 is the fluorescence titration curve figure of quercetin-cyclodextrin binary system to copper ion;

Fig. 5 is the selectivity figure of quercetin-cyclodextrin binary system to different metal ions;

Figure 6 is the fluorescence imaging diagram of the quercetin-cyclodextrin binary system in HeLa cells; in the figure, a, b, and c are cultured with the culture medium containing the quercetin-cyclodextrin binary system, and a is the fluorescence Imaging, b is the bright field, c is the overlay of a and b; d, e, f are the blank control, cultured only with culture medium, d is the fluorescence imaging, e is the bright field, f is the overlay of d and e;

Figure 7 is the fluorescence imaging diagram of HeLa cells cultured with quercetin-cyclodextrin binary system first, and then with copper ions; in the figure, a is the fluorescence imaging without adding copper ions, and b is after adding copper ions fluorescence imaging.

detailed description

The present invention will be further described below in conjunction with specific embodiments.

Example 1

(1) Accurately weigh 0.0030g of quercetin, dilute it to a 10mL volumetric flask with methanol, and prepare a solution with a concentration of 1× ^10-3 mol·L ^{- 1} ; accurately weigh 0.7500g of 2-hydroxypropyl-β - Cyclodextrin, dilute to 25mL volumetric flask with PBS buffer solution (pH=7.40).

(2) Take 1.0 mL of quercetin solution, dilute it to a 100 mL volumetric flask with PBS buffer solution (pH=7.40), measure the fluorescence intensity with a fluorescence spectrometer, and measure the fluorescence intensity every 1 h.

(3) Take 9.0 mL of cyclodextrin solution, dilute it into a 100 mL volumetric flask with PBS buffer solution (pH=7.40), and measure the fluorescence intensity with a fluorescence spectrometer.

(4) Take 1.0mL of quercetin solution and 9.0mL of cyclodextrin solution, and use PBS buffer solution (pH=7.40) to dilute to 100mL volumetric flask to make quercetin-cyclodextrin binary system, and The fluorescence intensity was detected by a fluorescence spectrometer, and the fluorescence intensity was measured every 1 h.

The effect of cyclodextrin on fluorescence enhancement of quercetin is shown in Figure 1, and cyclodextrin can significantly enhance the fluorescence intensity of quercetin. The fluorescence intensity of quercetin and quercetin-cyclodextrin binary system changed with time as shown in Figure 2. The fluorescence intensity of quercetin solution gradually decreased with time, while the quercetin-cyclodextrin The fluorescence intensity of the binary system does not change much with time, and the stability is greatly improved.

Example 2

The application of the quercetin-cyclodextrin binary system in the determination of copper ions specifically comprises the following steps:

(1) Accurately weigh 0.0030g of quercetin, dilute it to a 10mL volumetric flask with methanol, and prepare a solution with a concentration of 1× ^10-3 mol·L ^{- 1} ; accurately weigh 0.7500g of 2-hydroxypropyl-β - Cyclodextrin, dilute to 25mL volumetric flask with PBS buffer solution (pH=7.40). Take 1.0mL of quercetin solution and 9.0mL of cyclodextrin solution, and dilute to 100mL volumetric flask with PBS buffer solution (pH=7.40) to prepare a quercetin-cyclodextrin binary system for use.

(2) Accurately weigh 0.0200g Cu(AC) ₂ ·H ₂ O into a 10mL volumetric flask with water to prepare a solution containing copper ions with a concentration of 1×10 ^-2 mol·L ^-1 , and dilute in turn to form 1×10 ^-3 , 1×10 ^-4 , 1×10 ^-5 mol·L ^-1 solution, ready for use.

(3) Prepare a number of dry and clean sample tubes, add quercetin-cyclodextrin binary system solution and copper ion solution with different volumes and concentrations respectively, so that the concentration of quercetin is 1×10 ^-5 mol· L ^-1 , copper ion concentrations are 0×10 ^-7 mol·L ^-1 , 0.5×10 ^-7 mol·L ^-1 , 1.0×10 ^-7 mol·L ^-1 , 3.0×10 ^-7 mol·L ^-1 , 5.0×10 ^-7 mol·L ^-1 , 7.0×10 ^-7 mol·L ^-1 , 10.0×10 ^-7 mol·L ^-1 , 13.0×10 ^-7 mol·L ^-1 , 15.0×10 ^-7 mol·L ^-1 , 17.0×10 ^-7 mol·L ^-1 , 20.0×10 ^-7 mol·L ^-1 , 23.0×10 ^-7 mol·L ^{-1 ,} 25.0×10 ^-7 mol·L -1 ¹ , 27.0×10 ^-7 mol·L ^-1 , 30.0×10 ^-7 mol·L ^-1 , 33.0×10 ^-7 mol·L ^-1 , 35.0×10 ^-7 mol·L ^{-1 ,} 37.0×10 ^{- 7} mol·L ^-1 , 40.0×10 ^-7 mol·L ^-1 , 43.0×10 ^-7 mol·L ^-1 , 45.0×10 ^-7 mol·L ^-1 , 47.0×10 ^-7 mol·L ^-1 , 50.0×10 ^-7 mol·L ^-1 , 53.0×10 ^-7 mol·L ^-1 , 55.0×10 ^-7 mol·L ^-1 , 57.0×10 ^-7 mol·L ^-1 , 60.0×10 ^-7 mol·L ^-1 , 63.0×10 ^-7 mol·L ^-1 , 65.0×10 ^-7 mol·L ^-1 , 67.0×10 ^-7 mol·L ^-1 , 70.0× ^{10 -7} mol·L ^-1 , 73.0×10 ^-7 mol·L ^-1 , 75.0×10 ^-7 mol·L ^-1 , 77.0×10 ^-7 mol·L ^-1 , 80.0×10 ^-7 mol·L ^-1 , 81.0×10 ^-7 mol ·L ^-1 , 82.0×10 ^-7 mol·L ^-1 , 83.0×10 ^-7 mol·L ^-1 solution.

(4) Set the excitation wavelength of the fluorescence spectrometer to 390nm, the excitation and emission slit widths are 5nm and 15nm respectively, and the scanning range is set to 400nm to 700nm; the above solution is tested, and the fluorescence intensity at the emission wavelength of 553nm is recorded respectively. , the results are shown in Figure 3, from 1 to 38, the copper ion concentration is 0×10 ^-7 mol·L ^-1 , 0.5×10 ^-7 mol·L ^-1 , 1.0×10 ^{-7 mol·L -1} , 3.0×10 ^-7 mol·L ^-1 , 5.0×10 ^-7 mol·L ^-1 , 7.0×10 ^-7 mol·L ^-1 , 10.0×10 ^-7 mol·L ^-1 , 13.0×10 ^-7 mol·L ^-1 , 15.0×10 ^-7 mol·L ^-1 , 17.0×10 ^-7 mol·L ^-1 , 20.0×10 ^-7 mol·L ^-1 , 23.0×10 ^-7 mol·L ^-1 , 25.0×10 ^-7 mol·L ^-1 , 27.0×10 ^-7 mol·L ^-1 , 30.0×10 ^{- 7} mol·L ^-1 , 33.0×10 ^-7 mol·L ^-1 , 35.0×10 ^-7 mol ·L ^-1 , 37.0×10 ^-7 mol·L ^-1 , 40.0×10 ^-7 mol·L ^-1 , 43.0×10 ^-7 mol·L ^-1 , 45.0×10 ^-7 mol·L ^-1 , 47.0 ×10 ^-7 mol·L ^-1 , 50.0×10 ^-7 mol·L ^-1 , 53.0×10 ^-7 mol·L ^-1 , 55.0×10 ^-7 mol·L ^-1 , 57.0×10 ^-7 mol· L ^-1 , 60.0×10 ^-7 mol·L ^-1 , 63.0×10 ^-7 mol·L ^-1 , 65.0×10 ^-7 mol·L ^-1 , 67.0×10 ^-7 mol·L ^-1 , 70.0× 10 ^-7 mol·L ^-1 , 73.0×10 ^-7 mol·L ^-1 , 75.0×10 ^-7 mol·L ^-1 , 77.0×10 ^-7 mol·L ^-1 , 80.0×10 ^{-7 mol·L -1} , 81.0×10 ^-7 mol·L ^-1 , 82.0×10 ^-7 mol·L ^-1 , 83.0×10 ^-7 mol·L ^-1 .

(5) Draw the standard curve of fluorescence intensity to copper ion concentration, as shown in Figure 4. The obtained linear equation is: y=-9.24x+844.51, y is the fluorescence intensity, x is the copper ion concentration, the linear correlation coefficient is R ² =0.997, and the linear range is 5.0×10 ^-8 -8.3×10 ^-6 mol·L ^-1 , the detection limit is 2.3×10 ^-8 mol·L ^-1 , which can complete the detection of trace copper ions.

(6) Determination of samples: prepare the standard sample and the sample to be tested (unknown concentration) according to the above method, the concentration of quercetin in the solution is 1×10 ^-5 mol·L ^-1 , and the concentration of copper ion in the standard sample is 22.0× 10 ^-7 mol·L ^-1 (standard sample 1), 42.0×10 ^-7 mol·L ^-1 (standard sample 2), 52.0×10 ^-7 mol·L ^-1 (standard sample 3); Above-mentioned solution carries out fluorescence test, record above-mentioned solution at emission wavelength and be the fluorescence intensity of 553nm place, measure repeatedly three times, according to the standard curve y=-9.24x+844.51 of fluorescence intensity to copper ion concentration, calculate the concentration of copper ion, concrete result As shown in Table 1.

Table 1 Determination result table.

In the table, the concentration unit is 10 ^-7 mol·L ^-1 .

Example 3

In the quercetin-cyclodextrin binary system, add Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ , ^{Ca 2+} , Al ³⁺ , Cr ³⁺ , Mn ²⁺ , Fe ²⁺ , Fe ³⁺ , Co ²⁺ , Ni ²⁺ , Cu ²⁺ , Zn ²⁺ , Pb ²⁺ , Cd ²⁺ , La ³⁺ , Ce ³⁺ , Nd ³⁺ , Eu ³⁺ , Gd ³⁺ , Dy ^{3 +} salt solution, in which the concentration of Li ⁺ , Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ is 5 times the concentration of copper ions, Al ³⁺ , Cr ³⁺ , Mn ²⁺ , Fe ²⁺ , Fe ³⁺ , Co ²⁺ , Ni ²⁺ , Zn ²⁺ , Pb ²⁺ , Cd ²⁺ , La ³⁺ , Ce ³⁺ , Eu ³⁺ , Gd ³⁺ , and Dy ³⁺ are twice the concentration of copper ions. The fluorescence intensity measured under the same conditions and method as in Example 2 is shown in FIG. 5 . It can be seen from Figure 5 that only the addition of copper ions can cause obvious fluorescence quenching of the quercetin-cyclodextrin binary system, and other metal ions have a great influence on the fluorescence intensity of the quercetin-cyclodextrin binary system. Small and negligible, it shows that the quercetin-cyclodextrin binary system has a good selectivity for copper ions. In addition, it can be seen from the fluorescence titration spectrum and titration curve that the quercetin-cyclodextrin binary system has a large linear range and a low detection limit for the detection of copper ions, which shows that the quercetin-cyclodextrin binary system is sensitive to copper ions. ions have better sensitivity.

Example 4

HeLa cells were cultured at 37°C in an incubator containing 5% CO ₂ for 24 hours to ensure a cell density of 10 ⁵ cells/mL, washed with PBS (pH=7.40) to remove dead cells, and added quercetin-cyclodextrin The culture solution of the binary system (the mass concentration of quercetin is 12 μg/mL) was continued for 2-6 hours, the culture solution was discarded, washed three times with PBS, and the fluorescence imaging was observed on a confocal laser imager. Copper ions were added after the observation, and the fluorescence imaging was observed again after incubation for 1 hour, using an excitation wavelength of 405 nm. The results are shown in Figure 6 and Figure 7, the cells cultured with the culture medium containing the quercetin-cyclodextrin binary system, the fluorescence phenomenon in the cells can be observed on the laser confocal microscope; then add copper ions to culture 1 After ~4h, the fluorescence in the cells disappeared, indicating that the copper ions entered the cells and interacted with the quercetin-cyclodextrin binary system to produce fluorescence quenching.

It can be seen that, by adopting the method of the present invention, the quercetin-cyclodextrin binary system can be used as a fluorescent probe to detect trace copper ions, which not only has high selectivity and sensitivity, but also is fast, environmentally friendly and low-toxic. The result is ideal, and it is expected to be used to detect copper ions in cells.

Claims (10)

1. the compositional liquor of Quercetin and cyclodextrin application in measuring copper ion.

Application the most according to claim 1, it is characterised in that comprise the following steps:

(1) preparation Quercetin and the buffer solution of cyclodextrin binary system；

(2) respectively the copper ion solution of different volumes is joined in the buffer solution of Quercetin-cyclodextrin binary system, use The fluorescence intensity of each solution of fluorescence spectrophotometer record, draws the fluorescence intensity standard curve to copper ion concentration；

(3) liquid to be measured for copper ion is joined in Quercetin-cyclodextrin binary system solution, use fluorescence spectrophotometer recording solution Fluorescence intensity；

(4) concentration of copper ion in solution to be measured is determined according to standard curve.

Application the most according to claim 2, it is characterised in that: described buffer solution is CH₃OH-PBS buffer solution, molten The pH of liquid is 7.4, and methanol is 1 ~ 5:99 with the volume ratio of phosphate buffered saline(PBS).

Application the most according to claim 2, it is characterised in that: described fluorescent strength determining condition is to excite the slit to be 5nm, transmitting slit is 15nm, and sweep limits is 400-700nm, and excitation wavelength is 390nm, a length of 553nm of transmitted wave.

Application the most according to claim 1 and 2, it is characterised in that: the concentration of described Quercetin is 1 × 10^-5mol•L^-1, Cyclodextrin is 2-HP-BETA-CD, and cyclodextrin is 100 ~ 900:1 with the mass concentration ratio of Quercetin.

Application the most according to claim 2, it is characterised in that: described copper ion linear concentration scope is 5.0 × 10^-8- 8.3×10^-6 mol•L^-1, detection is limited to 2.3 × 10^-8 mol•L^-1。

Application the most according to claim 2, it is characterised in that: the fluorescence intensity standard curve to copper ion concentration,y = - 9.24x+ 844.51,yFor fluorescence intensity,xFor copper ion concentration, linearly dependent coefficient is R²=0.997。

8. the application in the compositional liquor of Quercetin and cyclodextrin copper ion in detection cell.

9. one kind for detecting the compositional liquor of copper ion, it is characterised in that: for the binary built liquid of Quercetin Yu cyclodextrin, its In, the concentration of Quercetin is 1 × 10^-5mol•L^-1, cyclodextrin is 2-HP-BETA-CD, cyclodextrin and the quality of Quercetin Concentration ratio is 100 ~ 900:1.

Compositional liquor for detecting copper ion the most according to claim 9, it is characterised in that: Quercetin is equal with cyclodextrin It is dissolved in CH₃OH-PBS buffer solution, the pH of solution is 7.4, and methanol is 1 ~ 5:99 with the volume ratio of phosphate buffered saline(PBS).