Robotic Intracellular Electrochemical Sensing for Adherent Cells

Abstract

Nanopipette-based observation of intracellular biochemical processes is an important approach to revealing the intrinsic characteristics and heterogeneity of cells for better investigation of disease progression or early disease diagnosis. However, the manual operation needs a skilled operator and faces problems such as low throughput and poor reproducibility. This paper proposes an automated nanopipette-based microoperation system for cell detection, three-dimensional nonovershoot positioning of the nanopipette tip in proximity to the cell of interest, cell approaching and proximity detection between nanopipette tip and cell surface, and cell penetration and detection of the intracellular reactive oxygen species (ROS). A robust focus algorithm based on the number of cell contours was proposed for adherent cells, which have sharp peaks while retaining unimodality. The automated detection of adherent cells was evaluated on human umbilical cord vein endothelial cells (HUVEC) and NIH/3T3 cells, which provided an average of 95.65% true-positive rate (TPR) and 7.59% false-positive rate (FPR) for in-plane cell detection. The three-dimensional nonovershoot tip positioning of the nanopipette was achieved by template matching and evaluated under the interference of cells. Ion current feedback was employed for the proximity detection between the nanopipette tip and cell surface. Finally, cell penetration and electrochemical detection of ROS were demonstrated on human breast cancer cells and zebrafish embryo cells. This work provides a systematic approach for automated intracellular sensing for adherent cells, laying a solid foundation for high-throughput detection, diagnosis, and classification of different forms of biochemical reactions within single cells.

The New Era of High-Throughput Nanoelectrochemistry

“…The SICM-current feedback allows automated nanopipette vertical positioning on cell membrane while cell membrane penetration and intracellular ROS sensing is enabled by continuous monitoring of the ROS current. 147 .A multiplexed localized electroporation device in conjunction with automated image segmentation and analysis based on artificial intelligence (AI) swiftly improved molecular delivery and transfection conditions for a variety of adherent and suspension cell types. The AI pipeline provides automated measurements of delivery/transfection efficiency and assists in the analysis of crucial cell morphological traits to pinpoint factors that promote high efficiency while also maintaining cell viability and health.…”

“…Their system is based on a computer-vision algorithm for automatic detection of cell nuclei and lateral nanopipette positioning. The SICM-current feedback allows automated nanopipette vertical positioning on cell membrane while cell membrane penetration and intracellular ROS sensing is enabled by continuous monitoring of the ROS current …”

“…The SICM-current feedback allows automated nanopipette vertical positioning on cell membrane while cell membrane penetration and intracellular ROS sensing is enabled by continuous monitoring of the ROS current. 147 …”

Optoelectronic tweezers: a versatile toolbox for nano-/micro-manipulation

“…165,166 There are other methods to distinguish cells based on their morphology and intrinsic physical properties such as size. 167 OET can also perform label-free sorting of different cells based on their sizes. Shown in Fig.…”

The new era of high throughput nanoelectrochemistry

“…The SICMcurrent feedback allows automated nanopipette vertical positioning on cell membrane while cell membrane penetration and intracellular ROS sensing is enabled by continuous monitoring of ROS current. 148 A recent study demonstrated the importance of the development of alternative technologies to perform routine biological procedures (e.g., transfection) to increase the throughput of biological assays. 149 A multiplexed localized electroporation device in conjunction with automated image segmentation and analysis based on artificial intelligence (AI) swiftly improved molecular delivery and transfection conditions for a variety of adherent and suspension cell types.…”