CN111239053A - A method for detecting critical polymerization concentration of compounds by dynamic mass reset method - Google Patents

Abstract

The invention discloses a method for determining critical polymerization concentration of a compound based on a dynamic mass reset signal. The device used is a resonant waveguide grating and the signal detected is the dynamic mass change of the substance near the sensor surface. When the solution reaches a more stable state, the non-polymerized molecules do not undergo dynamic mass rearrangement, whereas the polymerized molecules are capable of causing dynamic mass rearrangement. The critical micelle concentration can be deduced by detecting a series of dynamic mass reset signals of different concentrations of the compound. The method has the advantages of small sample dosage, high flux, wide application range and the like, and can provide a new way for the polymerization determination of the compound.

Description

A method for detecting critical polymerization concentration of compounds by dynamic mass reset method

technical field

The present invention relates to the application field of optical sensors, in particular, the present invention creates a method for detecting a dynamic mass reset signal of a compound by using a resonant waveguide grating sensor to judge whether the compound produces polymerization.

Background technique

Compound polymerization is a common physical phenomenon. When the concentration of compounds in the solution reaches a certain value, that is, critical aggregation concentrations (CACs), the compounds will aggregate to form micelles, and at the same time, various properties will change. Therefore, the determination of CAC is of great significance in practical applications. In the field of drug determination, after the compound polymerizes to form micelles, it will produce non-specific adsorption to enzymes or receptors, resulting in false positive results in biochemical experiments or cell experiments. At present, the main physical methods for detecting compound polymerization are scanning electron microscopy, dynamic light scattering, and hydrogen nuclear magnetic resonance spectroscopy. These methods have problems of low throughput, high cost, and high requirements for experimental conditions and equipment. Determination of sample CAC value by dynamic mass reset method has the advantages of simplicity, time saving and high efficiency, so it can be used as an effective method to detect compound polymerization.

The principle of dynamic mass redistribution (DMR) is mainly to use the resonant waveguide grating biosensor to detect the change of the refractive index of the sensor surface through the change of the resonance angle (or wavelength). Specifically, the resonant waveguide grating (RWG) biosensor utilizes diffraction gratings to resonantly couple light into the waveguide, resulting in total internal reflection at the solution-surface interface, which in turn generates an evanescent wave at the interface. The sensor surface decays exponentially, and the distance at which it decays to 1/e of the initial value is called the penetration depth. When the waveguide is irradiated with compound polarized light at a constant angle, only light of a specific wavelength will propagate along the waveguide, and the wavelength is related to the refractive index in the penetration depth; when the mass of the material in the penetration depth is reset, the refractive index occurs Changes in wavelengths propagating along the waveguide can be recorded. When the sensor is illuminated with monochromatic light, the information recorded is the change in the resonant coupling angle (or wavelength) of the light. Theoretically, any change in mass density throughout the depth can cause dynamic mass reset. Using this principle, dynamic mass reset methods have been widely used to detect changes in substances in cells.

This principle can also be used to detect the polymerization of the compound itself. When a relatively stable state is reached (that is, after the compound completes its own diffusion in the solution, the polymerization and depolymerization rates are close to each other, the concentration of the polymer does not change, and the surface is in a static equilibrium state), the molecules in the non-polymerized state are in the equilibrium state. The solution is in a state of uniform distribution, and the mass density of the uniformly distributed molecules will not change significantly with time, so no dynamic mass reset signal will be generated; while the molecules in the aggregated state are not uniformly distributed in the solution, and change with time. , its mass density will change greatly, thus causing dynamic mass reset. By measuring the dynamic mass response signal at different concentrations of the compound, the critical micelle concentration can be deduced.

SUMMARY OF THE INVENTION

The invention discloses a method for detecting compound polymerization using a dynamic mass reset method of a resonant waveguide grating sensor.

The resonant waveguide grating sensor may include a substrate, a thin waveguide layer embedded with gratings or periodic structures.

The detection method of critical micelle concentration is as follows:

1) Add a blank solution to the microplate with the resonant waveguide grating sensor embedded in the bottom of the detection platform, and balance the baseline to be stable;

2) Rebalance the baseline, add a series of concentration samples, and measure for a certain period of time;

3) Select a certain time point in the measurement time, and read the signal value corresponding to each concentration sample under this time point;

4) Taking the logarithm of the sample concentration as the abscissa, and the change correlation of dynamic mass reset as the ordinate, fitting each coordinate with a horizontal line and an oblique line, and the intersection between the horizontal line and the oblique line is the critical micelle concentration.

A resonant waveguide grating sensor includes a substrate, a thin layer of waveguides with embedded gratings or periodic structures.

A sample includes a single compound or composition.

The single compound is an organic small molecule or macromolecule and is dissolved in a blank solution, and the concentration is any concentration that does not produce precipitation; the composition is a combination of two or more small molecules or macromolecules, and is dissolved in a blank solution, The concentration is any concentration at which precipitation does not occur.

Blank solution, including single solvent, mixed solvent, salt solution.

The single solvent is water, methanol, ethanol and other solvents that the sensor and the orifice plate can tolerate; the mixed solvent is the solvent that the sensor and the orifice plate composed of two or more single solvents can withstand, and the ratio between the solvents is acceptable. Any ratio of mutual dissolution; the salt solution is a single solvent or mixed solvent containing one or more inorganic salts, and the inorganic salt concentration is any concentration that does not precipitate.

A certain time refers to the time for the solution to reach stability, specifically, the time domain of 5-10 minutes, 10-20 minutes, 20-50 minutes, and 50-120 minutes after adding the solution.

A certain time refers to any time point within the time when the solution reaches a steady state.

The detection platform is Corning's third-generation Epic imager.

This method is applicable to compounds, compositions, and the solvent can be single solvent, mixed solvent, or salt solution.

Beneficial effects of the invention: it can be carried out under micro conditions, adopts an automated operating system, and has the advantages of less sample consumption and high throughput; wide application range, can measure samples in various solvents; enlarged dynamic mass reset The application range of the experiment can simulate the cell measurement environment, and the measured critical micelle concentration can provide a reference for false-positive judgments for cell-level dynamic quality experiments, without the need to purchase other devices.

Description of drawings

Fig. 1 The principle of determination of compound polymerization by dynamic mass reset method, (A)-non-polymerized state, (B)-polymerized state.

Figure 2 (A)-Dynamic mass reset signal of salvianolic acid C, (B)-Dynamic mass reset signal of procyanidin, (C)-Dynamic mass reset signal of niacin, (D) Rhodiola rosea extract The dynamic quality of the reset signal.

Figure 3 (A) - Determination of critical polymerization concentration of salvianolic acid C, (B) - Determination of critical polymerization concentration of procyanidins, (C) - Determination of critical polymerization concentration of nicotinic acid, (D) - Extraction of Rhodiola rosea Determination of the critical polymerization concentration of the material.

Detailed ways

Below in conjunction with the accompanying drawings, the embodiments of the present invention are described in detail: the embodiments are implemented on the premise of the technical solutions of the present invention, and provide detailed embodiments and specific operation processes, but the protection scope of the present invention is not limited to the following example.

Example 1: Determination of the critical polymerization concentration of salvianolic acid C, procyanidins and niacin

The detection platform is Corning's third-generation Epic imager. The test is carried out in Corning Epic microplates. The bottom of the microplate contains resonant waveguide gratings. The detected signal is the wavelength shift change caused by dynamic mass reset.

The first is sample configuration. Accurately weigh 1 mg of salvianolic acid C powder, dissolve with HBSS (1×Hanks balanced salt solution, add 20 mM Hepes, pH 7.2) to obtain a solution of 400 μg/mL. Next, take 100 μL of the 400 μg/mL solution for gradient dilution, and finally obtain 400 μg/mL, 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.125 μg/mL mL, 1.5625μg/mL, 0.7813μg/mL, 0.3907μg/mL, 0.1953μg/mL, 0.0977μg/mL, 0.0488μg/mL, 0.0244μg/mL, 0.0122μg/mL series of solutions. Proanthocyanidins and niacin are formulated in the same way.

The second is the experimental determination. First, 30 μL of HBSS (1×Hanks Balanced Salt Solution, 20 mM Hepes, pH 7.2) is added to each well, and the baseline is equilibrated for 10 min. Next, re-equilibrate the baseline for 2 min, add salvianolic acid C solutions of each gradient concentration into the microplate, 10 μL per well, and measure for 60 minutes. Each concentration was measured three times (because 30 μL of HBSS buffered saline solution was added to the well plate in advance, the sample concentration in the actual measurement was one-fourth of the concentration in the configuration). Select the DMR signal value at 10 min and take the average value. Taking the logarithm of the sample concentration as the abscissa, and the change of dynamic mass reset as the ordinate, each point was fitted with a horizontal line and an oblique line, and the intersection between the horizontal line and the oblique line was the critical micelle concentration. Proanthocyanidins and niacin were subjected to the same experiment and data analysis. Finally, the critical polymerization concentrations of salvianolic acid C and proanthocyanidin obtained in the experiment were 0.49 μg/mL and 1.65 μg/mL, respectively, while niacin did not polymerize in this system.

Example 2: Determination of the critical polymerization concentration of Rhodiola rosea extract

The detection platform used was the same as in Example 1.

The first is sample preparation. 10 g of Rhodiola rosea powder was ultrasonically extracted with 100 mL of 70% ethanol for 1 hour, and the solvent was removed by rotary evaporation. Accurately weigh 1 mg of the medicinal material extract, and dissolve it into a 400 μg/mL solution with HBSS (1×Hanks balanced salt solution, adding 20 mM Hepes, pH 7.2). Next, take 100 μL of the 400 μg/mL solution for gradient dilution, and finally obtain 400 μg/mL, 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.125 μg/mL mL, 1.5625μg/mL, 0.7813μg/mL, 0.3907μg/mL, 0.1953μg/mL, 0.0977μg/mL, 0.0488μg/mL, 0.0244μg/mL, 0.0122μg/mL series of solutions.

Followed by the experimental determination, firstly, 30 μL of HBSS (1×Hanks Balanced Salt Solution, supplemented with 20 mM Hepes, pH 7.2) was added to each well, and the baseline was equilibrated for 10 min. Next, re-equilibrate the baseline for 2 minutes, add salvianolic acid C solutions of each gradient concentration into the microplate, 10 μL per well, and measure for 60 minutes. Each concentration was measured three times (because 30 μL of HBSS buffered saline solution was added to the well plate in advance, the sample concentration in the actual measurement was a quarter of the concentration in the configuration). Select the DMR signal value at 10 min and take the average value. Taking the logarithm of the sample concentration as the abscissa, and the change of dynamic mass reset as the ordinate, each point was fitted with a horizontal line and an oblique line, and the intersection between the horizontal line and the oblique line was the critical micelle concentration. The experimentally measured critical micelle concentration of Rhodiola rosea extract was 3.55 μg/mL.

Claims (9)

1. A method for detecting critical polymerization concentration of a compound by a dynamic mass resetting method is characterized by comprising the following steps:

1) adding a blank solution into a microporous plate with a resonant waveguide grating sensor embedded at the bottom of the detection platform, and balancing a base line to be stable;

2) rebalancing the baseline, adding a series of concentration samples, and measuring for a certain time;

3) selecting a certain time point, and reading signal values corresponding to the concentration samples at the time point;

4) and taking the logarithm of the concentration of the sample as an abscissa, taking the change correlation of the dynamic mass resetting as an ordinate, fitting each coordinate by a horizontal line and an oblique line respectively, and taking the intersection point between the horizontal line and the oblique line as the critical micelle concentration.

2. The method of claim 1, wherein: the resonant waveguide grating sensor comprises a substrate, a waveguide thin layer embedded with a grating or a periodic structure.

3. The method of claim 1, wherein: the sample comprises a single compound or composition.

4. The method of claim 3, wherein: the single compound is organic micromolecule or macromolecule and is dissolved by adopting blank solution, and the concentration is any concentration which does not generate precipitate; the composition is a combination of two or more than two small molecules or macromolecules, and is dissolved by adopting a blank solution, wherein the concentration is any concentration which does not generate precipitates.

5. The method of claim 1, wherein: the blank solution comprises a single solvent, a mixed solvent and a salt solution.

6. The method of claim 5, wherein: the single solvent is a solvent which can be endured by sensors and pore plates such as water, methanol and ethanol; the mixed solvent is a solvent which can be endured by a sensor and a pore plate consisting of two or more single solvents, and the proportion of the solvents is any proportion which can be mutually dissolved; the salt solution is a single solvent or a mixed solvent containing one or more inorganic salts, and the concentration of the inorganic salts is any concentration at which precipitation does not occur.

7. The method of claim 1, wherein: the certain time refers to the time for the solution to reach stability, and specifically refers to the time domain of 5-10 minutes, 10-20 minutes, 20-50 minutes and 50-120 minutes after the solution is added.

8. The method of claim 1, wherein: the certain time refers to any time point within the time when the solution reaches a steady state.

9. The method of claim 1, wherein: the detection platform is a Corning third generation Epic imager.

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