Quantitative Visualization of Molecular Delivery and Uptake at Living Cells with Self-Referencing Scanning Ion Conductance Microscopy-Scanning Electrochemical Microscopy

Abstract

A multifunctional dual-channel scanning probe nanopipet that enables simultaneous scanning ion conductance microscopy (SICM) and scanning electrochemical microscopy (SECM) measurements is demonstrated to have powerful new capabilities for spatially mapping the uptake of molecules of interest at living cells. One barrel of the probe is filled with electrolyte and the molecules of interest and is open to the bulk solution for both topographical feedback and local delivery to a target interface, while a solid carbon electrode in the other barrel measures the local concentration and flux of the delivered molecules. This setup allows differentiation in molecular uptake rate across several regions of single cells with individual measurements at nanoscale resolution. Further, operating in a "hopping mode", where the probe is translated toward the interface (cell) at each point allows self-referencing to be employed, in which the carbon electrode response is calibrated at each and every pixel in bulk for comparison to the measurement near the surface. This is particularly important for measurements in living systems where an electrode response may change over time. Finite element method (FEM) modeling places the technique on a quantitative footing to allow the response of the carbon electrode and local delivery rates to be quantified. The technique is extremely versatile, with the local delivery of molecules highly tunable via control of the SICM bias to promote or restrict migration from the pipet orifice. It is expected to have a myriad of applications from drug delivery to screening catalysts.

Multifunctional scanning ion conductance microscopy

“…The theta configuration is used most and is simple to make. SECM-SICM has recently been used to detect the delivery and uptake of molecules to a cellular surface, with the SECM barrel used to monitor the flux of a molecule of interest, delivered from the SICM barrel, which also tracked the substrate topography [141]. A self-referencing hopping mode was used in which the SECM and SICM channels were calibrated at each pixel.…”

Recent Advances in Scanning Electrochemical Microscopy for Biological Applications

“…A micropipette is designed to be loaded with solution containing the species of interest. After specific species have been delivered to the target cells or biofilm by the micropipette, SECM can be used to monitor the response or quantify the uptake of the target biological system [29,30]. …”

“…Subsequently, simulations were performed with varying analyte uptake rates at the same height. Using the calibration curve obtained this way, the uptake of hexaammine ruthenium (III) to Zea mays root hair cells can be quantified, and different uptake rates can be obtained for the cell tip and cell body [30]. …”

Advances and Perspectives in Chemical Imaging in Cellular Environments Using Electrochemical Methods

“…For example, Unwin and coworkers [69] used a SECM-SICM probe to deliver hexaammineruthenium (III) ([Ru(NH 3 ) 6 ] 3+ ) to a Zea mays root hair cell ( Figure 4). The [Ru(NH 3 ) 6 ] 3+ migrates out of the SICM barrel of the pipette, controlled by the applied potential to the electrode inside this barrel, and can be reduced at the SECM carbon electrode.…”

“…At each pixel in the image, the probe is calibrated, so that changes in probe response over time can be accounted for, which is especially important for lengthy experiments or measurements performed in living systems that may be more dynamic and change over time [69].…”