Nanopipettes for Intracellular Surface-Enhanced Raman Spectroscop

Single cell analytical techniques facilitate early detection of diseases and promote the development of more specific and efficient drugs. Surface-enhanced Raman spectroscopy (SERS) is one of the most promising methods for studying intact cells. SERS allows for high sensitivity analysis of molecular composition and structure. However, the lack of SERS-active nanoprobes, which provide consistently reproducible signals and do not affect cell viability, hinders the advancement of SERS applications for cellular studies. This work focuses on the development of the first SERS-active nanopipette, a tool for intracellular SERS analysis with unprecedented data reproducibility.;SERS-active nanopipettes were designed using a bottom-up approach. Gold nanoparticles, enabling SERS functionality of the nanopipette, were engineered to provide strong SERS enhancement while still being small enough for intracellular applications. Model SERS-active substrates were fabricated to study the effects of nanoparticle surface density and excitation wavelength on resulting SERS activity. Upon the selection of the optimal surface density of nanoparticles, the SERS sensitivity of the substrates was tested on intact cells and major cell organelles isolated into a suspension. The latter was designed to model the intracellular environment.;To fabricate a SERS-active nanopipette, the engineered gold nanoparticles were deposited on a tip of a pulled glass capillary. The interparticle distance was set according to the results obtained on model planar SERS substrates. Three new applications of surface-enhanced Raman spectroscopy were experimentally demonstrated using planar SERS substrates: (a) differentiation between various second calcium messengers (IP3, cADPR, NAADP), (b) detection of NAADP in cell extracts, (c) analysis of protein-protein interaction.;Different types of carbon nanotube-based cellular probes were studied. CNT-tipped pipettes were functionalized with gold nanoparticles and tested as SERS-active probes. A systematic Raman spectroscopic study of synthesis effects on the wall structure and surface chemistry of carbon nanopipettes was performed.;SERS-active nanopipettes were used for highly localized probing of a single living cell. The characteristic fingerprints of major cell compartments, nucleus and cytoplasm, were obtained. For the first time, SERS-active nanopipette enabled in situ detection of living cell function by means of surface-enhanced Raman spectroscopy.