CN114292753A

CN114292753A - Method for separating cells and discharging nanoparticles from cells

Info

Publication number: CN114292753A
Application number: CN202111588767.7A
Authority: CN
Inventors: 缪爱军; 黄彬; 张佳欣; 郭文博; 赵雅彤; 王川; 杨柳燕
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-04-08
Anticipated expiration: 2041-12-23
Also published as: CN114292753B

Abstract

The invention discloses a method for separating cells and discharging nanoparticles from the cells, which is characterized in that a Rogowski solution is used for separating the cells and the nanoparticles discharged from the cells, and the total concentration of iodine in the Rogowski solution is not lower than 100 g/L. In particular to a preparation containing I₂And a Rugowski solution of KI, and storing in dark place; adding the Rugowski solution into a sample to be separated, and uniformly mixing; standing the uniform sample for a period of time to precipitate the cells, and removing the supernatant solution by suction; resuspending the precipitated cells in a culture medium without nanoparticles, centrifuging, and discarding the supernatant; the suspension was repeated at least twice and the pelleted cells were isolated.

Description

Method for separating cells and discharging nanoparticles from cells

Technical Field

The invention belongs to the field of cell biology, and particularly relates to a method for separating cells and discharging nanoparticles from the cells.

Background

The cumulative concentration of nanoparticles within the cell is an important factor affecting their toxic effects. It depends mainly on the balance of both absorption and expulsion. Since most nanoparticles are not degraded inside the cell, excretion is considered to be the most effective way to reduce their toxicity. In addition, nanoparticles accumulated in aquatic organisms (especially single-celled organisms) can be ingested by their predators, and thus the rate of removal of nanoparticles from organisms can also be linked to their migration in the food chain. The process of biologically discharging the nano particles is deeply understood, and the method has very important significance for understanding the toxicity mechanism and the biogeochemical behavior of the nano particles in the water environment. However, research in this regard is still quite limited. The reason is partly that the discharged nanoparticles are difficult to separate from the cells, and it is difficult to quantify the discharged nanoparticles, making it difficult to study the discharge kinetics.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for effectively separating cells and nanoparticles discharged by the cells aiming at the defects of the prior art, and provide a basis for accurately researching the discharge dynamics of the nanoparticles in the cells.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for separating cells from cell-shed nanoparticles, wherein a Rogowski solution is used for separating the cells from the cell-shed nanoparticles, and the total concentration of iodine in the Rogowski solution is not lower than 100 g/L.

Preferably, the Rogowski solution is prepared from I₂And KI in ultrapure water, I₂Concentration of (2)Not less than 40g/L, and the KI concentration is not less than 60 g/L.

The cells include but are not limited to phytoplankton (such as green algae, blue algae, and the like), protozoa (such as tetrahymena, paramecium, and the like), and the like.

The nano-particles include, but are not limited to, nano-gold, nano-silver, nano-iron oxide, nano-titanium oxide, nano-silicon oxide, nano-plastic, nano-copper oxide, nano-zinc oxide, and the like.

Further, the method for separating the cells and discharging the nanoparticles from the cells specifically comprises the following steps:

(1) preparation of a composition containing₂And a Rugowski solution of KI, and storing in dark place;

(2) adding the Rugowski solution obtained in the step (1) into a sample to be separated, and uniformly mixing;

(3) standing the uniform sample in the step (2) for a period of time to precipitate cells, and sucking a supernatant solution;

(4) resuspending the pelleted cells from step (3) in a medium without nanoparticles, centrifuging, and discarding the supernatant;

(5) repeating the step (4) at least twice, and separating the precipitated cells.

Preferably, in the step (2), the volume ratio of the Rugowski solution to the sample solution is 1: 100-1: 5, preferably 1: 20.

Preferably, in the step (3), the standing time is 5-30 min, and the cell sedimentation efficiency is ensured and is shortened as much as possible.

Preferably, in step (4), the culture medium without nanoparticles includes, but is not limited to, physiological saline or phosphate buffer.

Preferably, in the step (4), the rotating speed and time are controlled during centrifugation, the integrity of cells is not damaged, the rotating speed is controlled to be 1000-4500 rpm, and the centrifugation time is 5-10 min.

Preferably, in the step (5), the number of times of the suspension washing is reduced as much as possible on the basis of ensuring the washing efficiency.

Has the advantages that:

the method utilizes common cheap and easily available Rugowski solution as the fixing agent, and enhances the separation effect of the fixing agent on cells and nano particles and improves the application universality by optimizing the parameters such as the concentration, the addition ratio, the settling time and the like of the fixing agent. The process has the advantages of simple operation, easily obtained raw materials and low cost.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 the integrity of the cells was changed at different times after addition of a 1:20 volume ratio of the Rogowski solution.

FIG. 2 shows a comparison of the separation effect of cells from expulsed nanoparticles in different ways after different volume ratios of the Rogowski solution are added.

FIG. 3 shows the difference in the kinetics of nanoparticle expulsion compared to direct centrifugation of collected cells by the natural sedimentation method with the addition of 1:20 volume ratio of Rogowski solution.

FIG. 4 the change over time of tetrahymena efflux nanoparticles after exposure to different concentrations of nanoparticles is determined by collecting cells by natural sedimentation with addition of 1:20 volume ratio of Rogowski solution left undisturbed.

Detailed Description

The invention will be better understood from the following examples.

In the following examples, the Rugoer's solution is prepared from₂And KI in ultrapure water, I₂The concentration of (2) is 40g/L, and the concentration of KI is 60 g/L.

Example 1: the integrity of the cells changed at different times after addition of the 1:20 volume ratio of the Rogowski solution.

The cell number was counted by adding the Rogowski solution to the Dryl's medium for culturing Tetrahymena thermophila at a volume ratio of 1:20, allowing to stand for various times (0,5,15,30min) (FIG. 1). It can be found that the number of cells added with 1:20 volume of Rogowski solution has no obvious change in 5-30 min, indicating that the cells remain intact.

Example 2: different methods compare the separation effect of cells from the expelled nanoparticles after addition of different volume ratios of the Rogowski solution.

Tetrahymena thermophila, exposed for 2h to nano-iron oxide, was transferred to Dryl's medium without nanoparticles for an experiment of 24 h. The effect of separation of the cells from the discharged nanoparticles was observed in the tetrahymena cells collected after standing for natural sedimentation after addition of 1:5 and 1:20 volumes of the Rogowski solution, centrifugation (3000rpm, 5min) and comparison with the cells collected by direct centrifugation (3000rpm, 5min) (FIG. 2, gray and white represent the signals of tetrahymena and iron oxide, respectively). It was found that the separation of the collected cells from the discharged nanoparticles by natural sedimentation after addition of 1:20 volume of the Rogowski solution was the best.

Example 3: adding a Rugowski solution with the volume ratio of 1:20, standing, and collecting tetrahymena cells by a natural sedimentation method, and comparing the kinetic difference of the discharge of the nanoparticles with the kinetic difference of the direct centrifugal collection of the cells.

Tetrahymena thermophila was exposed to nano-titania (1.32mg-Ti/L) for 2h and transferred to Dryl's medium without nanoparticles for 24h of experiment. The nanoparticle discharge kinetics were determined by comparing the tetrahymena cells collected after direct centrifugation (3000rpm, 5min) and addition of 1:20 volumes of Rogowski solution followed by natural sedimentation and centrifugation (3000rpm, 5min) washing (FIG. 3, black and red indicate the collection of tetrahymena by natural sedimentation after direct centrifugation and Rogowski fixation, respectively, and the intracellular titanium oxide content was determined). It was found that the method of collecting cells by sedimentation after addition of 1:20 volume of Rogowski solution could better detect the expulsion of nanoparticles by Tetrahymena.

Example 4: and (3) collecting cells by a standing natural sedimentation method by adding a 1:20 Rogowski solution, and measuring the change of nanoparticles discharged by tetrahymena along with time after exposing the nanoparticles with different concentrations.

Tetrahymena thermophila was exposed to varying concentrations of nano-titania (0.4,1.32,4,13.2mg-Ti/L) for 2h and transferred to Dryl's medium without nanoparticles for 3h of experiment. The nanoparticle discharge kinetics were determined by adding 1:20 volumes of Rogowski solution, allowing to settle naturally, harvesting the cells after washing by centrifugation (3000rpm, 5min) (FIG. 4).

The present invention provides a method for separating cells and discharging nanoparticles, and a method and a device for implementing the method, which are many embodiments, and it should be noted that, for those skilled in the art, various modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. A method for separating cells from cell-shed nanoparticles, characterized in that a cell-shed nanoparticles is separated from cell-shed nanoparticles using a rogowski solution in which the total concentration of iodine is not less than 100 g/L.

2. The method of claim 1, wherein the Rogowski solution is prepared from I₂And KI in ultrapure water, I₂The concentration of the KI is not lower than 40g/L, and the concentration of the KI is not lower than 60 g/L.

3. The method of separating cells from cell-draining nanoparticles of claim 1, comprising the steps of:

4. The method for separating the cells from the cell-discharging nanoparticles as claimed in claim 3, wherein the volume ratio of the Rogowski solution to the sample solution in the step (2) is 1:100 to 1: 5.

5. The method for separating cells from cell-discharged nanoparticles according to claim 3, wherein the standing time in step (3) is 5 to 30 min.

6. The method for separating cells from cell-shed nanoparticles according to claim 3, wherein in the step (4), the culture medium without nanoparticles is physiological saline or phosphate buffer.

7. The method for separating cells from cell-discharging nanoparticles as claimed in claim 3, wherein in the step (4), the rotation speed and time are controlled during centrifugation, the integrity of the cells is not damaged, the rotation speed is controlled to be 1000-4500 rpm, and the centrifugation time is 5-10 min.