Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels

Abstract

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function.

Silicon Nitride-Based Micro-Apertures Coated with Parylene for the Investigation of Pore Proteins Fused in Free-Standing Lipid Bilayers

“…The conductance level of such pores amounts to approximately 15 pS at 200 mV and 40 pS at −300 mV. Moreover, the conductance value of the small pore is similar to the reported value found in the literature [ 41 , 68 ].…”

“…The conductance level of such pores amounts to approximately 15 pS at 200 mV and 40 pS at −300 mV. Moreover, the conductance value of the small pore is similar to the reported value found in the literature [41,68]. Although the above-presented Gramicidin D experiments were conducted at room temperature, it is important to know that temperature has a significant effect on the activity and lifetime of Gramicidin.…”

A Review of Optical Imaging Technologies for Microfluidics

“…To solve these problems, the researchers proposed to use the spinning disc model to solve the x-y scan limitation, which can offer much faster scanning, as shown in Figure 3b [80]. To solve the sample control problems, the microfluidic platform combined with confocal microscope is currently widely used in the research of fluid [81][82][83], giant unilamellar vesicles (GUVs, as shown in Figure 3c [84]) [85][86][87][88][89], lipid membranes [90,91], and biological tissues. (b) the spinning disc model, reprinted with permission from ref.…”

A Low-Cost 3-in-1 3D Printer as a Tool for the Fabrication of Flow-Through Channels of Microfluidic Systems

“…The transfer of the manufacturing process from cleanroom to desktop facilitated, for instance, the involvement of innovative strategies of microfluidic channel and pillar creation using instruments that are available in stationery or hardware stores and sold for other purposes. Tactics already tried include xurography [ 2 ], paper-based preparations [ 3 , 4 ], three-dimensional (3D) printing [ 5 , 6 , 7 , 8 ], computer numerical control (CNC) micro-milling [ 5 , 9 , 10 , 11 , 12 ] and laser engraving [ 12 ], many of which have found application in biomedicine [ 13 , 14 ], biosensing [ 15 , 16 , 17 ], bioprocessing [ 18 ], point-of-care (POC) diagnosis [ 19 , 20 , 21 ], industrial biotechnology [ 22 ] and other industries.…”