High‐Throughput DNA Tensioner Platform for Interrogating Mechanical Heterogeneity of Single Living Cells

Abstract

The heterogeneities among individual cells lead to different cellular behaviors and responses against the stimulation. [3] In given scenarios, abnormal regulations of mechanical force in specific cell subtypes may introduce disorders to the organism, such as tumorigenesis, [4] drug resistance, [5] and metastasis. [6][7][8] The understanding of the mechanical properties at the single-cell level is an important index to explore the initiation and development of diseases, and further contribute to the advancement of treatment strategies.Recent years have seen emerging technologies for cell mechanical analysis, which can be categorized into two subgroups in terms of their format: i) the first group, including atomic force microscopy (AFM), [9][10][11] optical tweezers, [12] and magnetic tweezers, [13,14] focuses on the measurement of relevant force curves to obtain basic cell mechanical information, such as Young's modulus and stiffness. [15][16][17] These methods generally apply a mechanical stimulus to the cells by manipulating beams Cell mechanical forces play fundamental roles in regulating cellular responses to environmental stimulations. The shortcomings of conventional methods, including force resolution and cellular throughput, make them less accessible to mechanical heterogeneity at the single-cell level. Here, a DNA tensioner platform is introduced with high throughput (>10 000 cells per chip) and pN-level resolution. A microfluidic-based cell array is trapped on "hairpin-structured" DNA tensioners that enable transformation of the mechanical information of living cells into fluorescence signals. By using the platform, one can identify enhanced mechanical forces of drug-resistant cells as compared to their drug-sensitive counterparts, and mechanical differences between metastatic tumor cells in pleural effusion and nonmetastatic histiocytes. Further genetic analysis traces two genes, VEGFA and MINK1, that may play deterministic roles in regulating mechanical heterogeneities. In view of the ubiquity of cells' mechanical forces in the extracellular microenvironment (ECM), this platform shows wide potential to establish links of cellular mechanical heterogeneity to genetic heterogeneity.

Sensitive Interrogation of Enhancer Activity in Living Cells on a Nanoelectroporation-Probing Platform

“…The total detection process by the NP platform can be accomplished within 1 h, achieving considerable time-saving benefits to clinical application. Given that heterogeneity is the main culprit for tumor recurrence, in future work, we will focus on engineering the NP platform with single-cell analysis techniques, such as a single-cell array chip, to explore the cause of the difference in enhancer activity at the single-cell level . Furthermore, delivering targeted genetic drugs into tumor cells through the nanoelectroporation platform enables us to alter the activity of key enhancers in tumorigenesis, thus achieving precision medicine in cancer treatment …”

“…The siRNA experiment was carried out according to the reported experimental conditions . Briefly, the cells (1.5 × 10 5 ) were cultured in a six-well plate.…”

“…The siRNA experiment was carried out according to the reported experimental conditions. 32 Briefly, the cells (1.5 × 10 5 ) were cultured in a six-well plate. When the cell density reached 60%, the cell culture medium was replaced with a fresh medium containing 70 pmol siRNA and 8 μL of the transfection reagent (GenePharma).…”

Single-Molecule Mechanochemical Sensing

“…Another approach to achieve high throughput with reduced cost is to perform mechanochemical sensing on DNA chips. As demonstrated by Gaub, , force can be determined for arrays of DNA on chip. Such a setup can be harnessed for massively parallel detection of analytes.…”

A Wireless, Regeneratable Cocaine Sensing Scheme Enabled by Allosteric Regulation of pH Sensitive Aptamers

“…These methods, however, rely on additional chemical reagents for surface cleaning followed by refunctionalization of the devices and, therefore, are not compatible with building in vivo biosensors for continuous monitoring. , Removal of adsorbed biomarkers through electrochemical reactions/electrical repulsion serves a promising strategy for in situ regeneration but may require delicate design of the sensing interfaces consisting of multiple functional layers such as molecularly imprinted polymers and redox-active nanoreporters . In vitro selected aptamers enabled by the systematic evolution of ligands by exponential enrichment (SELEX), ,− on the other hand, provide attractive opportunities for developing reusable sensors due to the reconfigurable nature of DNA sequences. However, while their noncovalent interactions with the corresponding targets (e.g., hydrogen bonds, electrostatic bonds, van der Waals forces) are reversible in nature, most methods that previous works report for dissociating targets from aptamers exploit extreme conditions, such as with heating, ultraviolet light exposure, , and concentrated chemicals .…”