A microfluidic device for immuno-affinity-based separation of mitochondria from cell culture

Abstract

In this work, we present a method to isolate mitochondria of mammalian cells after cell disruption on microscale. The device is composed of linear microchannels cast in PDMS (polydimethylsiloxane). Specific antibodies against the translocase outer membrane protein of the mitochondria are immobilized on the surface of the substrate using an avidin-biotin sandwich construct. The mitochondria can be captured in the channel, whereas the remains of the cell lysate flow out the chip unhindered. The captured mitochondria can be observed directly on chip. A successful immobilization of pre-isolated mitochondria was shown at a flow rate between 0 and 5 μl min(-1) (≈0-2.5 mm s(-1)). After fluorescence staining, we demonstrated that the mitochondria covered around 3% of the channel surface. The mitochondria appeared in a distinct spherical shape with a diameter of around 0.8-1.2 μm. Further validation of the microfluidic device using non-treated cell lysate was done at 2 μl min(-1). The immobilized mitochondria were smaller with a diameter of around ≈490 nm. We observed a surface coverage of around 4%. The immobilized mitochondria were active and stable for over 2 h without cooling and were shown to be able to produce ATP under stage 3 respiration on chip.

Cristae remodeling causes acidification detected by integrated graphene sensor during mitochondrial outer membrane permeabilization

“…While pyrene exhibits a strong pi-pi interaction with the graphene, NHS provides a terminal for amide bonding of antibodies. Since TOM20 is a subunit of the translocase of outer membrane, anti-TOM20 antibody can be used to attract mitochondria19. Anti-TOM20 antibodies were incubated and allowed to bond with the pyrene-NHS linker.…”

A Mini Summary of Affinity-based Separation Methods for Sample Preparation

“…In a specific example [4], a method to separate mitochondria from mammalian cells after microscale cell-destruction is proposed. The device to approach this is constructed by linear microchannels cast in polydimethylsiloxane (PDMS) as shown in Figure 2.…”

Micro‐ and Nano‐Devices for Studying Subcellular Biology

“…[ 8 ] Finally, in addition to trapping organelles based solely on physical features, subcellular structures can also be immobilized upon binding to antibody‐coated fluidic channels. [ 71 ] Combining these approaches offers the potential to bolster the specificity of organelle isolation by targeting surface biochemistry unique to the organelle of interest. In addition to enabling insight into the biophysical and chemical details of individual organelles, future work using similar micro‐ and nano‐traps could potentially be used to study interorganelle interactions through visualization of supramolecular structures tethered to or associated with the trapped organelle.…”