CN114292753B

CN114292753B - Method for separating cells and discharging nanoparticles from cells

Info

Publication number: CN114292753B
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: 2023-06-16
Anticipated expiration: 2041-12-23
Also published as: CN114292753A

Abstract

The invention discloses a method for separating nano particles discharged by cells from the nano particles discharged by cells, which is characterized in that Lu Geshi solution is used for separating the nano particles discharged by cells, and the total concentration of iodine in the Lu Geshi solution is not lower than 100g/L. In particular to a preparation method of the composition containing I ₂ And Lu Geshi solution of KI, stored protected from light; adding Lu Geshi solution into a sample to be separated, and uniformly mixing; standing the uniform sample for a period of time to precipitate cells, and sucking the supernatant solution; re-suspending the precipitated cells with a nanoparticle-free medium, 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 a cell is an important factor affecting its toxic effects. It depends mainly on the balance of both absorption and drainage. Since most nanoparticles are not degradable within cells, expulsion is considered to be the most effective way to reduce their toxicity. In addition, nanoparticles accumulated in aquatic organisms (especially unicellular organisms) can be ingested by their predators, and thus the rate of clearance of the nanoparticles in the organism is also related to their migration in the food chain. The process of biological discharge of nano particles is deeply understood, and has very important significance for understanding the toxicity mechanism and the bio-geochemical behavior of the nano particles in water environment. However, research in this regard is still very limited. In part, the reason is that the ejected nanoparticles are difficult to separate from the cells, and it is difficult to quantify the ejected nanoparticles, making the study of the ejection kinetics difficult to develop.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a method for effectively separating the nano particles discharged by cells aiming at the defects of the prior art, and provides a basis for accurately researching the discharge dynamics of the nano particles in the cells.

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

a method of separating cell from cell-depleted nanoparticles, using a Lu Geshi solution to separate cell from cell-depleted nanoparticles, the Lu Geshi solution having a total concentration of iodine of no less than 100g/L.

Preferably, the Lu Geshi solution is prepared from the components I ₂ And KI in ultrapure water, I ₂ The concentration of (C) is not lower than 40g/L, and the concentration of KI is not lower than 60g/L.

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

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

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

(1) Preparing the composition containing I ₂ And Lu Geshi solution of KI, stored in dark place；

(2) Adding the Lu Geshi solution in the step (1) into a sample to be separated, and uniformly mixing;

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

(4) Re-suspending the precipitated cells of step (3) with a nanoparticle-free medium, centrifuging, and discarding the supernatant;

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

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

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

Preferably, in step (4), the nanoparticle-free medium includes, but is not limited to, physiological saline or phosphate buffer.

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

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

The beneficial effects are that:

the invention uses the common, cheap and easily available Lu Geshi solution as the fixative, and enhances the separation effect of the fixative on cells and nano particles by optimizing parameters such as concentration, adding proportion, sedimentation time and the like, thereby improving the application universality of the fixative. The process is simple to operate, raw materials are easy to obtain, and the cost is low.

Drawings

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

FIG. 1 shows the change in cell integrity at different times after addition of a 1:20 volume ratio of Lu Geshi solution.

FIG. 2 shows the separation effect of cells from the ejected nanoparticles by different methods after addition of Lu Geshi solutions in different volume ratios.

FIG. 3A is a graph showing the difference in nanoparticle discharge kinetics between the collection of tetrahymena cells by a 1:20 volume ratio Lu Geshi solution addition and the collection of cells by direct centrifugation.

FIG. 4A is a graph showing the change of the nanoparticles discharged from the tetrahymena over time after the cells were collected by a static natural sedimentation method with the addition of a Lu Geshi solution in a volume ratio of 1:20 to expose the nanoparticles at different concentrations.

Detailed Description

The invention will be better understood from the following examples.

In the following examples, lu Geshi solution is prepared from I ₂ And KI in ultrapure water, I ₂ The concentration of (C) was 40g/L and the KI concentration was 60g/L.

Example 1: after addition of a 1:20 volume ratio of Lu Geshi solution, the integrity of the cells was changed at different times.

The Lu Geshi solution was added to the Dryl's medium for culturing Tetrahymena thermophila at a volume ratio of 1:20, and left for various times (0, 5,15,30 min) to count the number of cells (FIG. 1). It was found that there was no significant change in the number of cells in the 5-30 min period with the addition of 1:20 volumes of Lu Geshi solution, indicating that the cells remained intact.

Example 2: after adding Lu Geshi solutions in different volume ratios, the different methods compare the separation effect of cells from the expelled nanoparticles.

The thermophilic tetrahymena exposed to 2h of nano-iron oxide was transferred to the nanoparticle-free xyl's medium and tested for 24h. The separation effect of cells from the expelled nanoparticles was observed by comparing the collected tetrahymena cells after washing with 1:5 and 1:20 volumes of Lu Geshi solution, which were left to stand for natural sedimentation, centrifugation (3000 rpm,5 min) and direct centrifugation (3000 rpm,5 min) (fig. 2, grey and white represent the signals of tetrahymena and iron oxide, respectively). It was found that cells collected by natural sedimentation after addition of 1:20 volumes of Lu Geshi solution separated best from the exiting nanoparticles.

Example 3: and (3) adding a Lu Geshi solution with the volume ratio of 1:20, standing, and collecting tetrahymena cells by a natural sedimentation method, and comparing the tetrahymena cells with the collected cells by direct centrifugation, wherein the nano-particle discharge kinetics difference is compared.

The thermophilic tetrahymena was exposed to nano titanium oxide (1.32 mg-Ti/L) for 2h and transferred to the Dryl's medium without nanoparticles for 24h. Nanoparticle discharge kinetics were determined by comparing tetrahymena cells collected after washing with direct centrifugation (3000 rpm,5 min) and resting for natural sedimentation after addition of 1:20 volume of Lu Geshi solution, centrifugation (3000 rpm,5 min) (fig. 3, black and red represent collection of tetrahymena by direct centrifugation and natural sedimentation after fixation with Lu Geshi, respectively, and intracellular titanium oxide content was determined). It was found that the removal of nanoparticles by tetrahymena could be better detected by sedimentation of the collected cells after addition of 1:20 volume of Lu Geshi solution.

Example 4: the change of the tetrahymena discharge nanoparticles with time after exposing the nanoparticles at different concentrations was measured by collecting cells by a static natural sedimentation method with the addition of a 1:20 Lu Geshi solution.

The thermophilic tetrahymena was exposed to various concentrations of nano titanium oxide (0.4, 1.32,4,13.2 mg-Ti/L) for 2h and transferred to the Dryl's medium without nanoparticles for 3h. After adding 1:20 volumes of Lu Geshi solution, standing for natural sedimentation, centrifuging (3000 rpm,5 min) washing, the cells were collected and the nanoparticle discharge kinetics were determined (FIG. 4).

The present invention provides a method for separating cells from cells and discharging nanoparticles, and a method for specifically implementing the technical scheme, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A method for separating nanoparticles from cell discharge, characterized in that a Lu Geshi solution is used to separate nanoparticles from cell discharge, the total concentration of iodine in the Lu Geshi solution being not less than 100 g/L;

the method specifically comprises the following steps:

(1) Preparing the composition containing I ₂ And Lu Geshi solution of KI, stored protected from light;

(5) Repeating the step (4) at least twice, and separating out the precipitated cells;

in the step (2), the volume ratio of the Lu Geshi solution to the sample solution is 1:100-1:5;

the cells include phytoplankton or protozoan cells.

2. The method of separating cells from cells and draining nanoparticles according to claim 1, wherein said Lu Geshi solution is prepared from I ₂ And KI in ultrapure water, I ₂ Is not less than 40g/L and KI is not less than 60g/L.

3. The method for separating cells from cells and discharging nanoparticles according to claim 1, wherein in the step (3), the standing time is 5 to 30 min.

4. The method of claim 1, wherein in step (4), the nanoparticle-free medium is physiological saline or phosphate buffer.

5. The method for separating cells from cells and discharging nanoparticles according to claim 1, 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.