Hollow Nanoneedle-Electroporation System To Extract Intracellular Protein Repetitively and Nondestructively

Techniques used to understand the dynamic expression of intracellular proteins are critical in both fundamental biological research and biomedical engineering. Various methods for analyzing proteins have been developed, but these methods require the extraction of intracellular proteins from the cells resulting in cell lysis and subsequent protein purifications from the lysate, which limits the potential of repetitive extraction from the same set of viable cells to track dynamic intracellular protein expression. Therefore, it is crucial to develop novel methods that enable nondestructive and repeated extraction of intracellular proteins. This work reports a hollow nanoneedle-electroporation system for the repeated extraction of intracellular proteins from living cells. Hollow nanoneedles with ∼450 nm diameter were fabricated by a material deposition and etching process, followed by integration with a microfluidic device. Long-lasting electrical pulses were coupled with the nanoneedles to permeate the cell membrane, allowing intracellular contents to diffuse into the microfluidic channels located below the cells via hollow nanoneedles. Using lactate dehydrogenase B (LDHB) as the model intracellular protein, the nanoneedle-electroporation system effectively and repeatedly extracted LDHB from the same set of cells at different time points, followed by quantitative analysis of LDHB via standard enzyme-linked immunosorbent assay. Our work demonstrated an efficient method to nondestructively probe intracellular protein levels and monitor the dynamic protein expression, with great potential to help understanding cell behaviors and functions.