Kovacs Boglarka, Novak Szabolcs, Sallai Igor, Magyarodi Beatrix, Szekacs Inna, Selmeczi David, Szabo Balint, Horvath Robert
Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, HUN-REN Centre for Energy Research, Budapest, Hungary.
Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, HUN-REN Centre for Energy Research, Budapest, Hungary; Doctoral School of Electrical Engineering, Budapest University of Technology and Economics, Budapest, Hungary.
Colloids Surf B Biointerfaces. 2025 Dec;256(Pt 1):114972. doi: 10.1016/j.colsurfb.2025.114972. Epub 2025 Jul 22.
Single-cell manipulations are a limiting factor in single-cell omics (genomics, transcriptomics, proteomics), in vitro fertilization, and cloning. Cellular adhesion force often plays a pivotal role in various biological contexts, spanning from lower organisms to the human body. Investigating the mechanism of adhesive interactions at the individual cell level holds significant importance. We used a computer-controlled piezoelectric micropipette (NanoPick) built onto an inverted microscope, offering subnanoliter precision liquid handling in the range of 0.1-600 nanoliters with a temporal resolution of 1 millisecond. In contrast to previous pipette-based cell manipulations, in our device, phase contrast and fluorescent imaging of the microscope was not limited by the micropipette. Moreover, this compact setup efficiently enabled single-cell detection, targeting, picking, and isolation without fluidic tubes and syringes. We investigated the integrin-mediated adhesion between an RGD (Arg-Gly-Asp) motif displaying surface and the HeLa Fucci tumor cell line. Using a 70 µm inner diameter micropipette, we found that increasing the pipetting speed (voltage ramp rate applied on the piezoelectric head) improved the cell picking success rate to almost 100 %. Although the more strongly attached unmodified HeLa cells could not be picked up even at the highest flow rates. However, vibrating the fluid in the micropipette successfully detached fully flattened cells without any biochemical treatment. This vibration micropipetting method enabled the detachment of 79.9 % of the strongly adherent HeLa cells, preserving mechanical integrity for downstream omics analyses despite a loss in viability. Compared to valve-controlled systems, NanoPick demonstrated higher efficiency and precision, particularly in handling THP-1 cells. Its rigid design minimized transient delays, allowing time-dependent flow profiles and enhanced detachment at lower flow rates. Our method allows adhesion measurements on hundreds of cells and offers precise control over fluid volume and timing, suitable for manipulating adherent cells or larger objects such as organoids, spheroids, oocytes, or larvae. The introduced vibration micropipetting method could be employed for the mechanical stimulation of single cells.
单细胞操作是单细胞组学（基因组学、转录组学、蛋白质组学）、体外受精和克隆中的一个限制因素。细胞粘附力在从低等生物到人体的各种生物学环境中通常起着关键作用。在单个细胞水平上研究粘附相互作用的机制具有重要意义。我们使用了一种安装在倒置显微镜上的计算机控制压电微吸管（NanoPick），它能在0.1至600纳升范围内提供亚纳升精度的液体处理，时间分辨率为1毫秒。与以前基于吸管的细胞操作不同，在我们的设备中，显微镜的相差和荧光成像不受微吸管的限制。此外，这种紧凑的设置有效地实现了单细胞检测、靶向、挑选和分离，无需流体管和注射器。我们研究了显示RGD（精氨酸 - 甘氨酸 - 天冬氨酸）基序的表面与HeLa Fucci肿瘤细胞系之间的整合素介导的粘附。使用内径为70μm的微吸管，我们发现提高移液速度（施加在压电头上的电压斜坡率）可将细胞挑选成功率提高到近100%。尽管即使在最高流速下，附着更强的未修饰HeLa细胞也无法被挑选出来。然而，振动微吸管中的液体成功地分离了完全扁平的细胞，无需任何生化处理。这种振动移液方法能够分离79.9%的强粘附HeLa细胞，尽管细胞活力有所损失，但仍保留了用于下游组学分析的机械完整性。与阀控系统相比，NanoPick表现出更高的效率和精度，特别是在处理THP - 1细胞时。其刚性设计最大限度地减少了瞬态延迟，允许随时间变化的流量分布，并在较低流速下增强分离效果。我们的方法允许对数百个细胞进行粘附测量，并能精确控制液体体积和时间，适用于操作贴壁细胞或更大的物体，如类器官、球体、卵母细胞或幼虫。所引入的振动移液方法可用于对单个细胞进行机械刺激。
