Most of cellular behaviors and functions are associated with intracellular signal transduction and the signal molecular (protein, miRNA, etc.) interactions. Understanding the signaling cascades and network is one of the core topics in molecular biology. The existing methods have various disadvantages in investigating the signal transduction in living cells so that they cannot provide information about how the signal molecular works in the functional environment in the living cell. The invention of novel tools for probing signaling activity in living cells has been part of the major thrust advancing our knowledge about different aspects of cell function.

To realize a minimally invasive method for in-situ probing of intracellular signaling dynamics in living cells, we developed a nanoneedle-based platform called “intracellular biopsy” (inCell biopsy). The biopsy was based on a diamond-nanoneedle array functionalized with molecular sensors, which were inserted into the cytoplasm domain in a reversible manner upon the centrifugation-controlled supergravity to capture molecular targets in living cells.

To test the applicability of our method, we first applied this platform to transcriptional factor protein (NF-κB) involved in the cellular innate immunity targeting the host-defense response to pathogen molecules such as foreign DNAs. The extracellular pathogen double strand DNA (dsRNA90) was delivered into the cytoplasm in A549 cells along with the poking, activating the pathway involving the stimulator of interferon genes (STING). The signal protein was successfully isolated along with the nanoneedle array retrieval. By using this reversible protocol and repeated probing in living cells for multiple time points, we were able to investigate the singling dynamics in a functional environment inside live cells.

In order to extend the compatibility of our approach, we then applied the platform to study another essential small signaling molecules, the intracellular miRNAs, which plays a very important role in many physiologic and pathogenic activities during whole cell life. A size-dependent double strand RNA (dsRNA) binding protein, P19, was recruited as the functional “hook” to multiplexed “fish” intracellular miRNA in living cells. The hybridization chain reaction (HCR) based isothermal amplification procedure was enabled to amplify the miRNA signal in order to improve the detection limit of our method. Utilizing this intracellular miRNA fishing biopsy, we have realized the multiplexed miRNA isolation and signal quantification within one fishing procedure. The miRNA dynamic variation under different cellular conditions, for instance, different cell cycle stage, could be analysis utilizing this method. Moreover, with the help of some bioinformatic analysis, the multiplexed miRNA signal revealed the cellular heterogeneity within a complexed cell population. These results demonstrated our platform is a novel and versatile tool for dissecting the signaling activity in living cells.