Method for detecting pantocuronium bromide based on supramolecular gold nano fluorescent probe

Abstract

The invention discloses a method for detecting pantocuronium bromide based on a supramolecular gold nano fluorescent probe, and belongs to the field of fluorescent nano probe drug detection methods. The method comprises the following steps: (1) preparing a supramolecular gold nano fluorescent probe solution; and (2) adding the supramolecular gold nano fluorescent probe solution into solutions containing different concentrations of pantocuronium bromide, uniformly mixing, determining the fluorescence intensity, and drawing a standard curve by taking a pantocuronium bromide concentration log value as a horizontal coordinate and a fluorescence intensity change value as a vertical coordinate, thereby realizing the detection of the supramolecular gold nano fluorescent probe on pantocuronium bromide. The supramolecular gold nano fluorescent probe is constructed based on self-assembly of a supramolecular host material and a polypeptide chain modified gold nano guest containing phenylalanine, pantocuronium bromide can compete with guest molecules to be combined with a supramolecular host to cause change of a fluorescence signal, detection of pantocuronium bromide is achieved, and the method has the advantages of being easy and convenient to operate, high in sensitivity, high in sensitivity and the like. The sensitivity is high; and the specificity is good.

Description

Technical Field

[0001] The present invention belongs to the field of methods for detecting drugs using fluorescent nanoprobes, and particularly relates to a method for detecting pancuronium bromide based on a supramolecular gold nanoparticle fluorescent probe. Background Art

[0002] Pancuronium bromide is an artificially synthesized bisquaternary ammonium steroid medium- and long-acting non-depolarizing muscle relaxant that blocks all M cholinergic receptors in the body. It is mainly used for tracheal intubation during general anesthesia and muscle relaxation during surgery. In addition, it has the characteristics of no histamine release, no ganglion block, and no hypotension. However, the widespread use of pancuronium bromide is not without controversy. Currently, the detection of pancuronium bromide in pharmaceutical preparations, illegal preparations, and biological samples mainly relies on LC-MS / MS technology, which has high detection costs and requires professional technical personnel. The lack of chromophores and functional functional groups in the structure of pancuronium bromide makes it difficult to develop a sensor probe for direct reaction.

[0003] Therefore, it is an urgent problem to be solved to develop a method for detecting pancuronium bromide with simple operation and rapid reaction. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to develop a method for detecting pancuronium bromide based on a supramolecular gold nanoparticle fluorescent probe with good detection effect, fast detection speed, and accurate results.

[0005] Technical Solution: The method for detecting pancuronium bromide based on a supramolecular gold nanoparticle fluorescent probe according to the present invention includes the following steps:

[0006] (1) Mix an aqueous solution of chloroauric acid and a polypeptide chain containing phenylalanine in an aqueous solution, adjust the pH to alkaline, add a reducing agent, and then adjust the pH to acidic to quench the excess reducing agent. React at room temperature. After the reaction is completed, concentrate with a microporous centrifugal filter tube and freeze-dry to obtain a gold nanomaterial powder; Mix the obtained gold nanomaterial powder with a supramolecular host material in a PBS buffer system for self-assembly to prepare a supramolecular gold nanoparticle fluorescent probe solution

[0007] (2) Add the supramolecular gold nanoparticle fluorescent probe solution to a solution containing different concentrations of pancuronium bromide, mix well and measure the fluorescence intensity. Then, take the logarithm value of the pancuronium bromide concentration as the abscissa and the change value of the fluorescence intensity as the ordinate to plot a standard curve, thereby realizing the detection of pancuronium bromide by the supramolecular gold nanoparticle fluorescent probe.

[0008] Further, in step (1), the polypeptide chain containing phenylalanine has a length of 4-7 amino acids. The length of the peptide chain is related to the aggregation of the gold nanomaterials caused by the supramolecular cucurbituril, thereby affecting the detection effect of pancuronium bromide. Preferably, it is FGGC, the full Chinese name of which is phenylalanine-glycine-glycine-cysteine, and the solvent is ultrapure water. The dosage ratio of the chloroauric acid aqueous solution, the polypeptide chain containing phenylalanine, and the solvent is 9 μL: 5 mg: 4.4-5 mL, and the concentration of the chloroauric acid aqueous solution is 0.5-1 M.

[0009] Further, the pH is adjusted within the range of 12-12.5, and the reagent used is a 0.5-1 M NaOH aqueous solution.

[0010] Further, the reducing agent is an NaBH4 aqueous solution with a concentration of 6-8 mg / L. The BH4 dissociated in the NaBH4 aqueous solution - has strong reducibility and can reduce AuCl4 - to gold atoms and form crystal nuclei. The quenching method is as follows: adjusting to pH 2.5-3 with 0.5-1 M hydrochloric acid; the stirring reaction time is 12-18 h.

[0011] Further, the buffer solution is a PBS buffer solution with a pH of 6.5-7.2 and a concentration of 10 mM.

[0012] Further, in the supramolecular gold nanometer fluorescent probe solution, the concentration of the supramolecular host material is 60-80 μM, and the concentration of the gold nanomaterials is 66.5-70 μg / mL.

[0013] Further, the supramolecular host material is supramolecular cucurbituril Q8.

[0015] Further, in step (2), the volume ratio of the supramolecular gold nanometer fluorescent probe solution to the pancuronium bromide solution with different concentrations is 1:1-2; the fluorescence intensity is the fluorescence intensity value at an excitation wavelength of 515 nm and an emission wavelength of 630 nm; the linear equation obtained from the standard curve is Y = 450145X - 600404, R 2 = 0.9935, where Y is the change value of the fluorescence intensity and X is the log value of the pancuronium bromide concentration.

[0016] Principle of the invention: In the present invention, the mixing of the supramolecular host material and the gold nanomaterials will cause an enhancement of the gold nanometer fluorescence. After adding the analyte guest pancuronium bromide, since the guest will compete with the phenylalanine of the polypeptide chain containing phenylalanine for binding to the supramolecular host material, it will thus be released from the macrocyclic cavity, causing quenching of the fluorescence intensity.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following remarkable effects: The supramolecular gold nanometer fluorescent probe provided by the method of the present invention can be self-assembled in a buffer solution, with simple operation. The detection of pancuronium bromide can be completed within only a few minutes, with low cost, good specificity, and can be used for the rapid detection of pancuronium bromide in actual biological samples. Description of the Drawings

[0018] Figure 1 It is the TEM image of FGGC-AuNPs prepared in Example 1;

[0019] Figure 2 It is the standard curve graph established at different pancuronium bromide concentrations in Example 1;

[0020] Figure 3 It is the fluorescence image measured in Example 1 under other interfering substances;

[0021] Figure 4 It is the schematic diagram of the principle for detecting pancuronium bromide by the supramolecular gold nanometer fluorescent probe in the present invention. Detailed Embodiments

[0022] The present invention will be further described in detail below in conjunction with the embodiments and the drawings.

[0023] Example 1: In this example, the detection of pancuronium bromide in urine samples was achieved by constructing a supramolecular gold nanometer fluorescent probe FGGC-AuNPs@Q8, and the process is as follows:

[0024] 1. Preparation of FGGC-AuNPs

[0025] Take 9 μL of 0.5 M chloroauric acid aqueous solution and 5 mg of polypeptide FGGC and dissolve them in 4.4 mL of ultrapure water. Mix them evenly at a rotation speed of 500 rpm, then add 1 M NaOH dropwise to adjust the pH of the solution to 12. Add 250 μL of freshly prepared 6 mg / L NaBH4 in batches. The solution gradually turns orange-red, indicating the formation of crystal nuclei. Subsequently, add an appropriate amount of 1 M hydrochloric acid to adjust the pH to 2.5 to quench the excessive reducing agent, and stir and react for 12 h. The prepared gold nanoparticles are collected and washed with a 3KD ultrafiltration centrifugal tube. The obtained FGGC-AuNPs are freeze-dried and stored at 4°C. Figure 1 It is the TEM image of the prepared FGGC-AuNPs. The size of the gold nanoparticles prepared by this method is about 2 nm, and the shape is nearly spherical.

[0026] 2. Establishment of the standard curve for detecting pancuronium bromide by the supramolecular gold nanometer fluorescent probe

[0027] Preparation of supramolecular gold nanoparticle fluorescence probe FGGC - AuNPs@Q8: Take a certain volume of Q8 and FGGC - AuNPs and dissolve them in a 10 mM PBS buffer system with pH 6.8. The final concentration of Q8 is 60 μM, and the final concentration of FGGC - AuNPs is 66.5 μg / mL. Mix them evenly and set aside.

[0028] Take 350 μL of the above probe solution, add the same volume of pancuronium bromide solution with known concentration, so that the final concentrations are 0 ng / mL, 31.25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL and 1000 ng / mL respectively. Measure the fluorescence intensity values at an excitation wavelength of 515 nm and an emission wavelength of 630 nm. Each concentration is measured 3 times repeatedly. Use the log value of the pancuronium bromide concentration as the abscissa and the change value of the fluorescence intensity as the ordinate to plot a standard curve, and obtain the linear equation Y = 450145X - 600404, R 2 = 0.9935, Figure 2 which is the fitted standard curve graph.

[0029] 3. Pretreatment of urine samples

[0030] Restore the urine samples to room temperature before detection, take the supernatant after standing, and dilute the urine samples at a dilution factor of 1:5.

[0031] 4. Detection of pancuronium bromide in urine samples

[0032] Take 350 μL of the supramolecular gold nanoparticle fluorescence probe, add the same volume of the diluted urine samples, vortex and mix evenly, measure the fluorescence intensity values at an excitation wavelength of 515 nm and an emission wavelength of 630 nm. Each sample is measured 3 times repeatedly. Calculate the content of pancuronium bromide in the urine samples according to the change value of the fluorescence signal.

[0033] 5. Investigation of method accuracy

[0034] Take 5 blank urine samples, restore them to room temperature, take the supernatant after standing, treat the urine samples with the diluent at a ratio of 1:5, and then add the pancuronium bromide solution with known concentration to make the concentrations 0 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL and 800 ng / mL respectively. Calculate the pancuronium bromide concentration by the said method and measure it three times repeatedly. Recovery rate = measured concentration / added concentration × 100%. Finally, calculate that the recovery rate is between 95.70% and 102.25%, verifying the accuracy of the inventive method.

[0035] 6. Investigation of method specificity

[0036] Take 350 μL of the supramolecular gold nanometer fluorescent probe, add the same volume of pancuronium bromide and various interfering substances including urea, ascorbic acid, glucose, acetylcholine, glutathione, and cholesterol, with a concentration of 500 ng / mL. Measure the fluorescence intensity value at an excitation wavelength of 515 nm and an emission wavelength of 630 nm. Each sample is measured 3 times to evaluate the selectivity of the probe. From Figure 3 As can be seen, only pancuronium bromide can observe obvious fluorescence changes, indicating that the developed supramolecular gold nanometer fluorescent probe has high selectivity for pancuronium bromide.

[0037] Such as Figure 4 As shown, the detection principle of the present invention is as follows: After FGGC-AuNPs binds to the supramolecular cucurbituril Q8, the intramolecular vibration and non-radiative transition of gold nanoparticles are restricted through host-guest encapsulation, resulting in the aggregation of gold nanoparticles themselves and enhanced fluorescence. The presence of pancuronium bromide will cause phenylalanine in FGGC to dissociate from the macrocycle of cucurbituril Q8, weakening the aggregation induction effect of the macrocycle on gold nanoparticles, thereby quenching the fluorescence. The logarithmic value of the concentration of pancuronium bromide is positively correlated with the quenched fluorescence intensity, thus achieving the purpose of detection.

[0038] Comparative Example 1: The difference from Example 1 is that in the preparation process, the polypeptide chain FGGC in the gold nanomaterial is replaced with FGGGGGGC, and the full Chinese name of FGGGGGGC is phenylalanine-glycine-glycine-glycine-glycine-glycine-glycine-cysteine.

[0039] Compare the detection performances of the products prepared from two different polypeptide chains, and investigate the influence of the polypeptide chain on the detection of pancuronium bromide by the supramolecular gold nanometer fluorescent probe. Under the action of pancuronium bromide with the same concentration of 1000 ng / mL, the sensitivity of the gold nanometer fluorescent probe prepared with FGGGGGGC in Comparative Example 1 is lower, and the fluorescence intensity only decreases by 10.7%. In contrast, the polypeptide chain FGGC in Example 1 shows more obvious fluorescence changes, decreasing by 40.6%. This may be due to the increase in the length of the polypeptide chain affecting the aggregation of the supramolecular gold nanometer fluorescent probe, resulting in a decrease in its detection performance.

Claims

1. A method for detecting pancuronium bromide based on a supramolecular gold nanoparticle fluorescent probe, characterized in that, It includes the following steps: (1) Mix an aqueous solution of chloroauric acid and a polypeptide chain containing phenylalanine in an aqueous solution, adjust the pH to alkaline, add a reducing agent, then adjust the pH to acidic to quench the excess reducing agent, react at room temperature, concentrate with a microcentrifugal filtration tube after the reaction is completed, and freeze-dry to obtain a gold nanomaterial powder; Mix the obtained gold nanomaterial powder with a supramolecular host material in a PBS buffer system for self-assembly to prepare a supramolecular gold nanometer fluorescent probe solution; (2) Add the supramolecular gold nanometer fluorescent probe solution to a solution containing pancuronium bromide with different concentrations, mix well and measure the fluorescence intensity, then use the logarithm value of the pancuronium bromide concentration as the abscissa and the change value of the fluorescence intensity as the ordinate to draw a standard curve, and further realize the detection of pancuronium bromide by the supramolecular gold nanometer fluorescent probe.

2. The method according to claim 1, wherein In step (1), the chain length of the polypeptide chain containing phenylalanine is 4 - 7 amino acids; the solvent is ultrapure water.

3. The method according to claim 2, wherein The polypeptide chain containing phenylalanine is FGGC, and its full Chinese name is phenylalanine - glycine - glycine - cysteine.

4. The method according to claim 1, wherein In step (1), the dosage ratio of the aqueous solution of chloroauric acid, the polypeptide chain containing phenylalanine and the solvent is 9 μL: 5 mg: 4.4 - 5 mL, and the concentration of the aqueous solution of chloroauric acid is 0.5 - 1 M.

5. The method according to claim 1, wherein In step (1), the pH adjustment range is 12 - 12.5, and the reagent used is a 0.5 - 1 M NaOH aqueous solution.

6. The method according to claim 1, wherein In step (1), the reducing agent is an aqueous solution of NaBH4 with a concentration of 6 - 8 mg / L; the quenching method is: adjust to pH 2.5 - 3 with 0.5 - 1 M hydrochloric acid; the stirring reaction time is 12 - 18 h; the buffer solution is a PBS buffer solution with a pH of 6.3 - 7.2 and a concentration of 10 mM.

7. The method according to claim 1, characterized in that, In step (1), in the supramolecular gold nanometer fluorescent probe solution, the concentration of the supramolecular host material is 60 - 80 μM, and the concentration of the gold nanomaterial is 66.5 - 70 μg / mL.

8. The method according to claim 1, characterized in that In step (1), the supramolecular host material is supramolecular cucurbit[8]uril.

9. The method according to claim 1, characterized in that, In step (2), the volume ratio of the supramolecular gold nanometer fluorescent probe solution to the solution containing pancuronium bromide with different concentrations is 1:1 - 2; the fluorescence intensity is the fluorescence intensity value at an excitation wavelength of 515 nm and an emission wavelength of 630 nm.

10. The method according to claim 1, characterized in that, In step (2), the linear equation obtained from the standard curve is Y = 450145X - 600404, R 2 = 0.9935, where Y is the change value of fluorescence intensity and X is the log value of pancuronium bromide concentration.

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