A Triune In Situ Integrated Nanoprobe with Controlled Exposed Tip for Reliable Single-Neuron Analysis

The development of nanoprobes targeting single neurons is crucial for elucidating the intrinsic mechanisms of the nervous system. However, limitations of existing nanoprobes in terms of structural design, functional expansion, and tip exposure methods significantly hinder accurate analysis and in-depth research on single-neuron behaviors. Here, we develop a nanoscale tip-processing strategy based on atmospheric plasma jet branch self-focusing (APJBSF) for achieving <i>in situ</i> integration of outer surface sensing, inner self-reference and inner delivery functions in the effective working area of the nanoprobe tip, enabling the three functions to collaboratively analyze single-neuron behaviors. For the outer sensing channel, protective layers with nanoscale thickness are selectively removed using the ABJBSF technique. This process allows for the controlled exposure levels of functional tip surfaces at nanoscale dimensions, while simultaneously broadening the range of applicable protective coatings for nanoprobes, which facilitates reliable recordings at intracellular specific sites. For the inner self-reference and delivery channels, intracellular delivery experiments indicate that the <i>in situ</i> reference enhances the accuracy and stability of intracellular recordings and the triune <i>in situ</i> integrated nanoprobe (TIINP) enables accurate monitoring of intracellular pH changes induced by <i>in situ</i> delivery. The proposed TIINP structure and the tip-processing technique for nanoprobes provide powerful tools for enhancing the functionality and reliability of single-neuron analysis.