A lab-on-a-chip for hypoxic patch clamp measurements combined with optical tweezers and spectroscopy- first investigations of single biological cells

Abstract

The response and the reaction of the brain system to hypoxia is a vital research subject that requires special instrumentation. With this research subject in focus, a new multifunctional lab-on-a-chip (LOC) system with control over the oxygen content for studies on biological cells was developed. The chip was designed to incorporate the patch clamp technique, optical tweezers and absorption spectroscopy. The performance of the LOC was tested by a series of experiments. The oxygen content within the channels of the LOC was monitored by an oxygen sensor and verified by simultaneously studying the oxygenation state of chicken red blood cells (RBCs) with absorption spectra. The chicken RBCs were manipulated optically and steered in three dimensions towards a patch-clamp micropipette in a closed microfluidic channel. The oxygen level within the channels could be changed from a normoxic value of 18% O 2 to an anoxic value of 0.0-0.5% O 2. A time series of 3 experiments were performed, showing that the spectral transfer from the oxygenated to the deoxygenated state occurred after about 227 ± 1 s and a fully developed deoxygenated spectrum was observed after 298 ± 1 s, a mean value of 3 experiments. The tightness of the chamber to oxygen diffusion was verified by stopping the flow into the channel system while continuously recording absorption spectra showing an unchanged deoxygenated state during 5400 ± 2 s. A transfer of the oxygenated absorption spectra was achieved after 426 ± 1 s when exposing the cell to normoxic buffer. This showed the long time viability of the investigated cells. Successful patching and sealing were established on a trapped RBC and the whole-cell access (Ra) and membrane (Rm) resistances were measured to be 5.033 ± 0.412 M Ω and 889.7 ± 1.74 M Ω respectively.

Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions

“…By doing so, a level of 5% O 2 can be reached—which cannot be conserved as truly hypoxic. In a previous study, these technical problems had been solved by maneuvering optically trapped biological cells in a closed system towards a fixed micropipette by moving the xyz-microscope stage in 3D [ 12 ]. The system proved to be successful, however, it was (1) intricate to fix the micropipette; (2) the optical steering of the cells emerged to be very time consuming and (3) it was impossible to measure adhesive cells—such as PASMCs.…”

Advances in In Vitro Models of Neuromuscular Junction: Focusing on Organ‐on‐a‐Chip, Organoids, and Biohybrid Robotics

“…Resistance measurements of the sealed patches suggested that the system can be used in the future to perform complete patchclamp measurements on different biological cells in a controlled environment. [91] Jouhanneau et al developed in vivo two-photon targeting using multiple whole-cell patch clamp recordings of shallow neurons to monitor single synaptic connections in vivo. This approach can be used to investigate the synaptic mechanisms of action potential generation and to assess the link between single synaptic transmission and cortical function on a millisecond timescale.…”

Optically Controlled Dissolution Kinetics of Vaterite Microcapsules: Toward Novel Crystal Growth Strategies

“…Prepulled glass micropipettes are must-have tools for diverse scientific needs, from biological engineering , and microinjection , to electrophysiology. − In a broad sense, these instruments are beneficial for their ability to build a bridge between micrometer-sized objects (bacteria, microparticles, or long DNA chains) and macroscopic controlling tools (stepper motors for mechanical manipulations, an electronic amplifier for ionic current registration, microsyringe pumps for drug injections).…”