CN111239053A - Method for detecting critical polymerization concentration of compound by dynamic mass resetting method - Google Patents
Method for detecting critical polymerization concentration of compound by dynamic mass resetting method Download PDFInfo
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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
Technical Field
The invention relates to the application field of optical sensors, in particular to a method for detecting a dynamic mass reset signal of a compound by using a resonant waveguide grating sensor to judge whether the compound is polymerized.
Background
Polymerization of compounds is a common physical phenomenon. When the concentration of the compound in the solution reaches a certain value, namely critical micelle concentration (CACs), the compound aggregates to form micelles, and various properties change. Therefore, the determination of CAC is of great importance in practical applications. In the field of drug determination, after a compound is polymerized to form a gel, non-specific adsorption can be generated on an enzyme or a receptor, so that a false positive result of a biochemical experiment or a cell experiment is caused. At present, the main physical methods for detecting the polymerization of the compound are a scanning electron microscope method, a dynamic light scattering method and a nuclear magnetic resonance hydrogen spectrometry, and the methods have the problems of low flux, high cost and the like and have high requirements on experimental conditions and equipment. The CAC value of the sample is measured by a dynamic mass resetting method, and the method has the advantages of simplicity, time saving, high efficiency and the like, and therefore, the method can be used as an effective method for detecting compound polymerization.
The principle of the Dynamic Mass Redistribution (DMR) is to detect the change of the refractive index of the sensor surface by the change of the resonance angle (or wavelength) by using a resonant waveguide grating biosensor. In particular, Resonant Waveguide Grating (RWG) biosensors use a diffraction grating to resonantly couple light into a waveguide, resulting in total internal reflection at the solution-surface interface, which in turn produces an evanescent wave at the interface that decays exponentially from the sensor surface to a distance of 1/e of the initial value, referred to as the penetration depth. When the waveguide is illuminated with a constant angle of composite polarized light, only light of a particular wavelength will propagate along the waveguide, the wavelength being related to the refractive index within the depth of penetration; when the mass of the material within the depth of penetration is reset, the refractive index changes and the change in wavelength propagating along the waveguide can be recorded. When a sensor is illuminated with monochromatic light, the information recorded is the change in the resonant coupling angle (or wavelength) of the light. In theory, any change in mass density throughout the depth of penetration can cause dynamic mass resetting. Using this principle, the dynamic mass resetting method has been widely used to detect changes in substances in cells.
This principle can also be used to detect polymerization of the compound itself. When a stable state is reached (namely after the compound finishes the diffusion in the solution, the polymerization and depolymerization rates are close to each other, the concentration of the polymer is not changed any more, and the surface is in a static equilibrium state), the molecules in a non-polymerization state are in a uniformly distributed state in the solution, and the mass density of the uniformly distributed molecules cannot be obviously changed along with the time, so that a dynamic mass reset signal cannot be generated; the molecules in the polymerized state are not uniformly distributed in the solution, and the mass density of the molecules is greatly changed along with the change of time, so that the dynamic mass resetting is caused. The critical micelle concentration can be calculated by measuring the dynamic mass response signal of the compound at different concentrations.
Disclosure of Invention
The invention discloses a method for detecting compound polymerization by using a dynamic mass resetting method of a resonant waveguide grating sensor.
The resonant waveguide grating sensor may include a substrate, a waveguide layer embedded with a grating or periodic structure.
The detection method of the critical micelle concentration is as follows:
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 in the determination time, 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.
The resonant waveguide grating sensor comprises a substrate, a waveguide thin layer embedded with a grating or a periodic structure.
The sample comprises a single compound or composition.
Dissolving a single compound which is an organic micromolecule or macromolecule by adopting a blank solution, wherein the concentration is any concentration which does not generate precipitates; 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.
Blank solution, including single solvent, mixed solvent, salt solution.
The single solvent is a solvent which can be endured by sensors and pore plates such as water, methanol, ethanol and the like; 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.
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.
The certain time refers to any time point within the time when the solution reaches a steady state.
The detection platform is a corning third generation Epic imager.
The method is suitable for the compound and the composition, and the solvent can be a single solvent, a mixed solvent and a salt solution.
The invention has the beneficial effects that: the method can be carried out under a micro-scale condition, adopts an automatic operation system, and has the advantages of less sample consumption and high flux; the application range is wide, and samples in various solvents can be measured; the application range of the dynamic mass resetting experiment is expanded, the cell measuring environment can be simulated, the measured critical micelle concentration can provide a reference for false positive judgment for the cell level dynamic mass experiment, and other devices do not need to be purchased.
Drawings
FIG. 1 dynamic Mass resetting method for determining the principle of polymerization of a compound, (A) -non-polymerized state, (B) -polymerized state.
FIG. 2(A) -the dynamic mass resetting signal of salvianolic acid C, (B) -the dynamic mass resetting signal of procyanidins, (C) -the dynamic mass resetting signal of nicotinic acid, (D) the dynamic mass resetting signal of rhodiola rosea extract.
FIG. 3(A) -determination of critical polymerization concentration of salvianolic acid C, (B) -determination of critical polymerization concentration of procyanidin, (C) -determination of critical polymerization concentration of nicotinic acid, and (D) -determination of critical polymerization concentration of rhodiola rosea extract.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
Example 1: determination of critical polymerization concentration of salvianolic acid C, procyanidin and nicotinic acid
The detection platform is a Corning third generation Epic imager, the test is carried out in a Corning Epic micropore plate, and the bottom of the micropore plate comprises a resonance waveguide grating. The detected signal is a wavelength shift change caused by dynamic mass reset.
First, 1mg of salvianolic acid C powder was accurately weighed and dissolved in HBSS (1 Xhanks Balanced salt solution, 20mM Hepes added, pH7.2) to obtain a solution of 400. mu.g/mL. Then, 100. mu.L of 400. mu.g/mL solution was subjected to gradient dilution to obtain 400. mu.g/mL, 200. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, 1.5625. mu.g/mL, 0.7813. mu.g/mL, 0.3907. mu.g/mL, 0.1953. mu.g/mL, 0.0977. mu.g/mL, 0.0488. mu.g/mL, 0.0244. mu.g/mL, and 0.0122. mu.g/mL. The procyanidin and nicotinic acid are prepared by the same preparation method.
The next is the experimental assay, which first adds 30 μ L HBSS (1 Xhanks Balanced salt solution, 20mM hepes added, pH7.2) per well and equilibrates the baseline for 10 min. Then, the baseline was re-equilibrated for 2min, and the salvianolic acid C solution of each gradient concentration was added to the microplate at 10. mu.L per well for 60 min. Each concentration was measured in triplicate (the actual sample concentration at the time of measurement was one-fourth the concentration at the time of preparation due to the pre-addition of 30 μ LHBSS buffered saline solution to the well plate). And selecting the DMR signal value at 10min, and averaging. And taking the logarithm of the concentration of the sample as an abscissa, taking the change of the dynamic mass resetting as an ordinate, fitting each point 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. The same method is used for the experiment and data analysis of procyanidin and nicotinic acid. Finally, the critical polymerization concentrations of the salvianolic acid C and the procyanidin obtained by the experiment are 0.49 mu g/mL and 1.65 mu g/mL respectively, and the nicotinic acid is not polymerized under the system.
Example 2: determination of critical polymerization concentration of rhodiola rosea extract
The detection platform used was the same as in example 1.
First is sample preparation. 10g of rhodiola root medicinal material powder is extracted by 100mL of 70% ethanol for 1 hour by ultrasonic, and the solvent is removed by rotary evaporation. 1mg of the extract was weighed out accurately and dissolved in HBSS (1 Xhanks Balanced salt solution, 20mM Hepes added, pH7.2) to 400. mu.g/mL. Then, 100. mu.L of 400. mu.g/mL solution was subjected to gradient dilution to obtain 400. mu.g/mL, 200. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, 1.5625. mu.g/mL, 0.7813. mu.g/mL, 0.3907. mu.g/mL, 0.1953. mu.g/mL, 0.0977. mu.g/mL, 0.0488. mu.g/mL, 0.0244. mu.g/mL, and 0.0122. mu.g/mL.
The next is an experimental assay, where 30 μ L of HBSS (1 × hanks balanced salt solution, 20 mheps added, pH7.2) is first added to each well and the baseline is equilibrated for 10 min. Then, the baseline was re-equilibrated for 2min, and the salvianolic acid C solutions of various concentrations were added to the microplate at 10. mu.L/well for 60 min. Each concentration was measured in triplicate (the actual sample concentration at the time of measurement was one-fourth the concentration at the time of preparation due to the pre-addition of 30 μ LHBSS buffered saline solution to the well plate). And selecting the DMR signal value at 10min, and averaging. And taking the logarithm of the concentration of the sample as an abscissa, taking the change of the dynamic mass resetting as an ordinate, fitting each point 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. The critical micelle concentration of the rhodiola rosea extract is measured by experiments to be 3.55 mu g/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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