The study of single cell is of great significance in the fields of cell heterogeneity, genetic metabolism, genetic engineering, and toxicity detection. To identify the functional characteristics of individual cells, individual cell capture must first be achieved. However, most trapping methods require a constant force to keep the cell trapped. When the force decreases or disappears, the cells easily revert to a disordered state, which is very harmful to subsequent characterization and analysis. Therefore, a method for capturing individual cells stably without the use of constant external forces is expected to open up a new avenue for single cell research. This paper proposes a method of rapid capture of single cell during the self-assembly process of micropillars based on femtosecond laser multiphoton processing technology and the principle of capillary force self-assembly. It has fast capture, convenient operation, and a broad application range, and has a lot of potential in bioengineering, drug analysis, and other fields.
High aspect ratio micropillars were prepared by single-pulse femtosecond laser multiphoton polymerization. The height and space of the micropillars can be adjusted by moving the micro/nano translation stages vertically and horizontally. The laser-processed sample was developed upside down by a developer for 6 min to remove the photoresist in the unprocessed area. After the sample was developed, it was washed in isopropanol solution for 10 min to remove any residual developer. To prevent the self-assembly of the sample after developing and cleaning, we put it into a PBS solution immediately. The sample was sealed and degassed for 8 h before being disinfected. Drop the cell solution directly above the micropillar arrays with a density of 1.2×105 cell/mL. After seeding cells, the sample was placed in the cell incubator for 2 h to allow the cells to fully adhere to the Petri dish and fall into the bottom of the micropillar gap. Subsequently, the uncapped cells were washed off with trypsin, and the sample was processed using living dead staining. Using a pipette gun, remove the liquid and allow the residual solution to evaporate naturally. The cells between the micropillars were captured during the self-assembly process of micropillars.
The micropillar structure with a high aspect ratio has a large diameter at the bottom and gradually shrinks to the top, showing a conical shape (Fig. 1). The bottom diameter of the micropillar gradually increases as the height and laser power increase. By shortening the distance between micropillars, increasing the distance between self-assembly structures, and reasonably adjusting the height of micropillars, the micropillars close to each other can be self-assembled based on capillary force, to realize the high-efficiency preparation of largescale three-dimensional (3D) complex patterned self-assembly structures (Fig. 2). The experiment of micropillars’ self-assembly driven by capillary force to capture microspheres shows that the micropillars can still be self-assembled into microcage structures when there are particles in the micropillar gap (Fig. 3). Based on the above methods, a single cell array capture experiment was carried out. The results of fluorescence imaging and scanning electron microscope (SEM) images show that this method can realize high-throughput in situ capture of single cell array simply and efficiently (Fig. 5). Additionally, the cell capture experiment of microcage composed of a different number of micropillars provides a relatively simple method for sorting, capturing, and in situ observation of cells of different sizes (Fig. 6). The four micropillar microscage can only capture cell with similar diameter of the microcage, providing a new method for cells sorting. The six micropillar microscage can capture different sizes of cells, and single cell analysis experiment can be carried out by the micropillars’ gap. The eight micropillar microscage can strictly restrict the captured cell well in the microcage and is expected to be used in domain-limited growth characteristics research of single cell.
To meet the application requirements of high throughput single cell capture, a domain-limited passive capture method of single cell arrays based on self-assembly of a micropillar array is proposed. Based on femtosecond laser single pulse multiphoton polymerization technology and the capillary force self-assembly principle, this method realizes the high-throughput in situ capture of the MCF-7 single cell array. Using femtosecond laser single pulse multiphoton polymerization to achieve the high-efficiency preparation of micropillar arrays with a high aspect ratio. By optimizing the spacing and height of micropillars as well as largescale path planning, high throughput capillary force self-assembly of a largescale 3D complex microcage structure was achieved. The in situ capture of a single cell array based on capillary force self-assembly was successfully realized by matching the structural parameters of the microcage with the size of the cells. Different from the traditional single cell capture methods, such as single cell orientation capture method previously proposed by us, this method provides a relatively simple and efficient method for the sorting, capture, and in situ observation of cells with different sizes without the need for continuous additional external force. It has various application prospects in the fields of bioengineering, pharmaceutical analysis, and other domains.
