CN111208104A - Preparation of fluorescent polydopamine nanoparticles with controllable particle size and detection of trypsin - Google Patents

Abstract

The invention discloses preparation of fluorescent polydopamine nanoparticles with controllable particle sizes and application of the fluorescent polydopamine nanoparticles to trypsin detection. The method is characterized in that dopamine is stirred under alkaline condition to self-polymerize into poly-dopamine nano-particles, and then H is added₂O₂The time and the temperature are controlled to decompose the polydopamine nano-particles into fluorescent polydopamine nano-particles with the diameter less than 5 nm. The preparation method disclosed by the invention is simple in steps, uses a green and environment-friendly source material, and has controllable particle size of the product. The surface of the fluorescent polydopamine nano particle is rich in negative charges, has good fluorescence performance, quenches fluorescence with cationic protamine through electrostatic adsorption and hydrogen bond aggregation, realizes the detection of trypsin only through simple mixing operation and according to the recovery of the fluorescence of the polydopamine nano particle due to the specific hydrolysis of the protamine by the trypsin, and the minimum detection limit of the trypsin can reach 6.4 ng/mL.

Description

Preparation of fluorescent polydopamine nanoparticles with controllable particle size and detection of trypsin

Technical Field

The invention relates to the application field of fluorescent polydopamine nanoparticles, in particular to a fluorescence method for detecting trypsin based on aggregation induction quenching of fluorescence of the polydopamine nanoparticles under the action of protamine.

Background

(1) A protease is a proteolytic enzyme involved in a variety of biological and physiological processes, including digestion, coagulation, apoptosis and a range of other cellular activities. Trypsin (TRY), one of the most important digestive proteases, plays an important role in regulating the exocrine pancreatic function. TRY is overexpressed in acute pancreatitis, cystic fibrosis, and pancreatic cancer. Thus, TRY has been considered as a biomarker for the above-mentioned diseases.

(2) There are many methods for assaying trypsin, including enzyme-linked immunosorbent assays, colorimetric methods, chemiluminescent methods, electrochemical immunoassays, photoelectrochemical methods, and fluorescent methods. Among them, the fluorescence method has the advantages of simplicity, high sensitivity, rapid implementation, real-time detection and the like, and therefore, has more attraction and prospect. To date, TRY fluorescence detection methods based on gold nanoparticles, conjugated polymer dots and organic dyes have been reported. Most of these methods are designed based on the modulation of the interaction between the fluorescent probe and the protein by TRY to induce fluorescence changes through fluorescence resonance energy transfer or internal filtering effects or aggregated interaction modalities. Therefore, the development of a suitable interaction system between the protein and the specific probe is the key to trypsin detection.

(3) The quantum dots with specific fluorescence properties are established, the interaction between the quantum dots and proteins is realized through reasonable design of size and surface properties, the adjustable luminescence change is realized, the fluorescence adjusted according to the enzyme digestion of trypsin is changed again, and a novel trypsin determination method through simple mixing operation is expected to be established.

(4) Dopamine, a biological neurotransmitter, can become poly-dopamine (PDA) with morphologic modulation through covalent bonds, hydrogen bonds and pi-pi interactions. PDA has become a new biopolymer material for biomedical applications, surface coatings, sewage treatment, etc. due to its excellent biocompatibility. Compared to common large-size non-fluorescent polydopamine probe carriers used for bioanalysis, decomposition of PDA into polydopamine nanoparticles with fluorescence (F-PDA) by certain controlled synthesis methods translates into bioanalytical applications. However, few analytical strategies have been established for the interaction of F-PDA with biological macromolecules (e.g., proteins).

Disclosure of Invention

(1) In view of the above, the present invention aims to restore fluorescence of PDNPs by adding trypsin to specifically hydrolyze protamine (Pro) bound to PDNPs, thereby establishing a fluorescence method for detecting trypsin, which is simple in operation and high in sensitivity.

(2) In order to achieve the purpose, the preparation method of the fluorescent polydopamine nanoparticle with the controllable particle size is characterized by comprising the following steps: mixing Dopamine (DA) and Tris (hydroxymethyl) aminomethane (Tris), stirring, self-polymerizing to form poly-dopamine nanoparticles (PDA nanoparticles), and adding H₂O₂And NaOH is heated and refluxed, and the final product is dialyzed by a dialysis bag with the molecular weight cutoff of 3500 Da to obtain the fluorescent poly-dopamine nano particles (PDNPs).

Adding HCl into the Tris (hydroxymethyl) aminomethane (Tris) to adjust the pH of the solution to 8.5, and stirring for 20 hours.

The preparation method is characterized in that H is added₂O₂Heating and refluxing the mixture and NaOH for 30 min to prepare PDNPs.

The maximum excitation wavelength of the PDNPs is 340 nm, and the maximum emission wavelength of the PDNPs is 435 nm.

The fluorescent poly-dopamine nanoparticles (PDNPs) are prepared by the preparation method.

The method for detecting trypsin by using fluorescent polydopamine nanoparticles is characterized in that a solution used in a fluorescence spectrophotometry is formed by mixing a buffer solution, the fluorescent polydopamine nanoparticles (PDNPs) as claimed in claim 5, protamine and trypsin.

In a buffer solution, PDNPs, protamine and trypsin solution, the concentration of protamine (Pro) is optimized within the range of 10-80 mu g/mL, and the reaction time of the PDNPs, the protamine and the trypsin is optimized within the range of 0.1-20 minutes.

The detection method is characterized in that the optimal Pro concentration is selected to be 40 mu g/mL, and the optimal reaction time is 20 min.

The detection method is characterized in that the buffer solution is PBS, the concentration of the buffer solution is 10 mM, the pH value is 8.0, and the Trypsin (TRY), the PDNPs and the protamine solution are mixed uniformly and then incubated at room temperature for 10 min, and then fluorescence detection is carried out.

The fluorescence spectrum curve equation drawn by the fluorescence spectrophotometry is as follows: y =0.060+4.298X, R²=0.99203, Y is (F-F)₀)/F₀，F₀And F are fluorescence values of PDNPs-Pro before and after adding trypsin respectively; x is trypsin concentration, and the lowest limit of detection (LOD) = 6.4 ng/mL; the content of each component in the solution is as follows: the concentration of PDNPs was 0.15. mu.g/mL, and the concentration of Pro was 40. mu.g/mL.

Specifically, the invention provides a method for detecting trypsin by using a fluorescence method based on a fluorescence aggregation-induced quenching system (PDNPs-Pro) of fluorescent polydopamine nanoparticles under the action of protamine, which is characterized by comprising the following steps of: preparing trypsin solutions with different concentrations, adding the trypsin solutions into PDNPs-Pro, and respectively detecting the fluorescence of the PDNPs-Pro before and after the trypsin is added. Wherein, the reagent configuration operation is simple, and the reaction conditions are easy to operate.

(3) The prepared reagent is reacted for 20 minutes at room temperature in a dark place, transferred into a fluorescence spectrophotometer, excited at the wavelength of 340 nm, and the fluorescence intensity of 435 nm is read. The reaction and measurement conditions are simple.

(4) In order to obtain a platform detection trypsin for measuring aggregation-induced quenching based on the fluorescent polydopamine nanoparticles, the fluorescence change value of the PDNPs after different concentrations of protamine are added is preferably detected.

(5) Aiming at the prior art, the invention aims to provide a novel application field of fluorescent polydopamine nanoparticles which are simple and convenient to operate, high in sensitivity and capable of effectively measuring trypsin.

(6) In order to achieve the above object, the present invention provides a method for quantitatively detecting trypsin by fluorescence spectrophotometry, wherein a solvent in the fluorescence spectrophotometry is a mixture of a buffer solution, PDNPs, Pro, and trypsin.

(7) The method has the advantages that PDNPs and Pro are reacted, the PDNPs and Pro are subjected to aggregation induction quenching, and then the fluorescence of the PDNPs is recovered by adding trypsin to specifically hydrolyze Pro. The trypsin is detected by adding the trypsin to determine the change degree of the fluorescence intensity of the PDNPs and establishing a fluorescence detection method for the change of the fluorescence signal of the PDNPs, so that a good effect is obtained, the operation is convenient, the sensitivity is high, and the lowest detection limit of the trypsin can reach 6.4 ng/mL.

(8) Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a transmission electron micrograph of PDNPs;

FIG. 2a is a UV spectrum of PDNPs;

FIG. 2b is a fluorescence spectrum of PDNPs;

FIG. 3 is an emission spectrum obtained from PDNPs at an excitation wavelength of 310 nm to 440 nm;

FIG. 4 is a graph showing the results of PDNPs solution under natural light irradiation and ultraviolet irradiation, wherein a is natural light irradiation and b is 365 nm ultraviolet excitation;

FIG. 5 is a fluorescence intensity profile of PDNPs at different pH;

FIG. 6 is a fluorescence plot of PDNPs with excitation at 340 nm for 1 hour;

FIG. 7 is a plot of fluorescence recovery of PDNPs after aggregation quenching after Pro addition and after trypsin addition;

FIG. 8 is a plot of fluorescence intensity of PDNPs with different concentrations of Pro added;

FIG. 9 is a transmission electron micrograph of the degree of aggregation of PDNPs with addition of 30. mu.g/mL Pro and with addition of 40. mu.g/mL Pro, wherein a is the PDNPs with addition of 30. mu.g/mL Pro and b is the PDNPs with addition of 40. mu.g/mL Pro;

FIG. 10 is a plot of fluorescence intensity of PDNPs after addition of trypsin of various activities;

FIG. 11 is a graph of the linear relationship between the fluorescence intensity recovery efficiency of PDNPs and the trypsin concentration;

FIG. 12 is a graph comparing the recovery efficiency of PDNPs fluorescence under different interfering substances.

Detailed Description

(1) The present invention will be described in detail with reference to the following detailed description and accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

(2) Example (feasibility analysis)

A sample of the feasibility test of this example was prepared by:

the following solutions were prepared: the solution 1 is PDNPs; solution 2 is protamine (Pro); the solution 3 is PDNPs + Pro; solution 4 was PDNPs + Pro + trypsin. Wherein the PDNPs concentration is 0.15 mu g/mL, the Pro concentration is 40 mu g/mL, the trypsin concentration is 0.1 mu g/mL, and the three component solutions are all prepared by phosphate buffer solution (the buffer solution is Na) used in the embodiment of the invention₂HPO₄∙12H₂O、NaH₂PO₄∙2H₂O, NaCl) was added to the solution, and the pH of the buffer solution was 8.0. The fluorescent poly-dopamine nanoparticles (PDNPs) are prepared by dissolving 0.05 g of dopamine hydrochloride and 0.0606 g of Tris (hydroxymethyl) aminomethane (Tris) in 40 mL, stirring for 20H, and then adding H₂O₂(20 wt%, 15 mL) and NaOH (2.5M, 10 mL) were heated under reflux for half an hour, and HCl was added to Tris (hydroxymethyl) aminomethane (Tris) to adjust the pH of the solution to 8.5 as necessary.

(3) The PDNPs prepared in this example were tested:

1) FIG. 1 is a transmission electron microscope image of PDNPs prepared in this example, as shown in FIG. 1, the particles of PDNPs are uniformly distributed, and the average size is 4.83 nm.

2) FIG. 2a shows that the UV-VIS absorption spectrum of aqueous PDNPs shows a broad absorption band at 200 to 450 nm, indicating that the PDNPs contain a low proportion of 5, 6-dihydroxyindole and dopamine units. FIG. 2b shows that the maximum excitation wavelength and the emission wavelength of the PDNPs aqueous solution are 340 nm and 435 nm respectively, which are basically symmetrical.

3) FIG. 3 is an emission spectrum of PDNPs under the condition of excitation wavelength varying from 310 nm to 440nm, the maximum excitation wavelength of the PDNPs is 340 nm, the maximum emission wavelength is 440nm, and the emission wavelength of the PDNPs shows the characteristic of excitation wavelength dependence.

4) The PDNPs shown in a in figure 4 are brown yellow under natural light irradiation and bright blue fluorescence under the irradiation condition of ultraviolet light (365 nm) are shown in b in figure 3.

5) FIG. 5 shows fluorescence intensities of PDNPs prepared in this example in phosphate buffer solutions with different pH values, and as shown in FIG. 5, the fluorescence intensities of PDNPs remained relatively stable at pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0.

6) As shown in FIG. 6, the fluorescence intensity of PDNPs is always stable after being continuously excited for 1 hour at the wavelength of 340 nm, which indicates that the prepared polydopamine nanoparticles have stable fluorescence and better photobleaching resistance.

7) FIG. 7 is a graph showing fluorescence intensity before and after adding PDNPs having a concentration of 0.15. mu.g/mL to Pro having a concentration of 40. mu.g/mL, which is obtained in this example, and showing significant fluorescence quenching of PDNPs by Pro.

8) As shown in FIG. 8, gradual increase of Pro causes gradual quenching of PDNPs, and fluorescence quenching of PDNPs reaches a plateau after 40. mu.g/mL of Pro. As shown in FIG. 9, the introduction of Pro causes the PDNPs to aggregate, and the higher the Pro concentration is, the more obvious the PDNPs are aggregated, which indicates that the addition of Pro causes the PDNPs to aggregate to induce a fluorescence quenching system, thereby forming PDNPs-Pro.

(4) The invention also provides a trypsin activity detection method, which is used for detecting the trypsin activity by a fluorescence spectrophotometry method, wherein a solvent in the fluorescence spectrophotometry method is formed by mixing a buffer solution, Pro, trypsin and the PDNPs.

① accurate measurement of 10 mu L PDNPs solution, 10 mu L Pro and 10 mu L trypsin with a series of different concentrations, supplement of phosphate buffer solution, keeping the total volume at 200 mu L, and after 10 min of light-shielding reaction, placing the obtained solution in a fluorescence spectrophotometer, wherein all component solutions are prepared by using the phosphate buffer solution, the concentration of PDNPs is 0.15 mu g/mL, and the concentration of Pro is 40 mu g/mL.

② reading the fluorescence intensity value at the emission wavelength of 440nm under the condition of 340 nm of the excitation wavelength, and obtaining the fluorescence intensity value of known Pro according to the fluorescence intensity data to draw a standard curve.

③ in a preferred embodiment of the invention, the content of each component in the solution is specifically that the concentration of PDNPs is 0.15 mug/mL, and the buffer solution (the buffer solution is Na)₂HPO₄∙12H₂O、NaH₂PO₄∙2H₂O, NaCl) was added to the buffer solution, and the concentration was 10 mM, and the pH of the buffer solution was 8.0. The trypsin concentration corresponding to the curve from bottom to top in the graph 10 is 0 mug/mL, 0.01 mug/mL, 0.02 mug/mL, 0.03 mug/mL, 0.04 mug/mL, 0.05 mug/mL, 0.06 mug/mL, 0.1 mug/mL, 0.2 mug/mL, 0.3 mug/mL, 0.4 mug/mL, 0.5 mug/mL and 0.6 mug/mL, so that the fluorescence intensity of the PDNPs is gradually increased along with the increase of the trypsin concentration. The ordinate in FIG. 11 shows the fluorescence recovery efficiency of PDNPs, and the fluorescence intensity of PDNPs gradually increases with the increase in trypsin concentration. As can be seen from the figure, when the concentration of the trypsin to be detected is 0-0.06 mug/mL, the concentration has a good linear relation with the fluorescence recovery value of PDNPs, and the linear equation is as follows: y =0.060+4.298X, R²=0.99203，Y=0.060+4.298X，R²=0.99203, Y is (F-F)₀)/F₀，F₀And F are fluorescence values of PDNPs-Pro before and after adding trypsin respectively; x is trypsin concentration, lowest limit of detection (LOD) = 6.4 ng/mL.

④ the trypsin determination method provided by the invention has good selectivity, as shown in FIG. 12, only trypsin can recover the fluorescence value of PDNPs-Pro, and other common interferents cannot recover the fluorescence value of PDNPs-Pro.

⑤ the method for determining the trypsin provided by the invention determines the adding standard trypsin in the serum, the adding standard recovery rate is 98.0-109.8% (see table 1 specifically), the relative standard deviation is not more than 1.6%, and the method has good accuracy and reproducibility.

Table 1 shows the recovery of serum spiked and the relative standard deviation of trypsin determined by this method

⑥ the above embodiments are only preferred embodiments of the present invention, and not all embodiments are within the scope of the present invention.

Claims (10)

1. A preparation method of fluorescent polydopamine nanoparticles with controllable particle sizes is characterized by comprising the following steps: mixing Dopamine (DA) and Tris (hydroxymethyl) aminomethane (Tris), stirring, self-polymerizing to form poly-dopamine nanoparticles (PDAnanoparticles), and adding H₂O₂And NaOH is heated and refluxed, and the final product is dialyzed by a dialysis bag with the molecular weight cutoff of 3500 Da to obtain the fluorescent poly-dopamine nano particles (PDNPs).

2. The method of claim 1, wherein Tris (hydroxymethyl) aminomethane (Tris) is added to HCl to adjust the pH of the solution to 8.5 and stirred for 20 hours.

3. The method of claim 1, wherein H is added₂O₂Heating and refluxing the mixture and NaOH for 30 min to prepare PDNPs.

4. The method of claim 1, wherein the PDNPs have a maximum excitation wavelength of 340 nm and a maximum emission wavelength of 435 nm.

5. Fluorescent polydopamine nanoparticles (PDNPs) obtained by the preparation method according to any one of claims 1 to 4.

6. The method for detecting trypsin by using fluorescent polydopamine nanoparticles as claimed in claim 5, wherein fluorescence of PDNPs before and after adding trypsin in different concentrations is detected by a fluorescence spectrophotometry, and a solution used in the fluorescence spectrophotometry is prepared by mixing a buffer solution, the fluorescent polydopamine nanoparticles (PDNPs) as claimed in claim 5, protamine and trypsin.

7. The detection method according to claim 6, wherein in the buffer solution, PDNPs, protamine and trypsin solution, the protamine (Pro) concentration is in the range of 10 to 80 μ g/mL and the reaction time of PDNPs, protamine and trypsin is in the range of 0.1 to 20 minutes.

8. The assay of claim 7 wherein the optimal Pro concentration is selected to be 40 μ g/mL and the optimal reaction time is 20 min.

9. The method of claim 6, wherein the buffer solution is PBS, the concentration of the buffer solution is 10 mM, the pH is 8.0, and the Trypsin (TRY), PDNPs and protamine solutions are mixed and incubated at room temperature for 10 min before fluorescence detection.

10. The detection method according to any one of claims 6 to 9, wherein the fluorescence spectrum curve equation drawn by the fluorescence spectrophotometry is: y =0.060+4.298X, R²=0.99203, Y is (F-F)₀)/F₀，F₀And F is PDNPs-Pro before and after trypsin additionFluorescence value; x is trypsin concentration, and the lowest limit of detection (LOD) = 6.4 ng/mL; the content of each component in the solution is as follows: the concentration of PDNPs was 0.15. mu.g/mL, and the concentration of Pro was 40. mu.g/mL.

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