Miniaturized platforms for analyzing mitochondrial bioenergetics
Mitochondrial bioenergetics converges at the mitochondrial inner membrane and encompasses the proton gradient, mitochondrial membrane potential and respiration. Measuring these parameters can assess the mitochondrial functional state and consequently, the effectiveness of pharmacological manipulation of diseases involving mitochondrial dysfunctions. However, available technologies to assay mitochondrial functions are primarily limited to ensemble measurements, which mask the functional dynamics and variability of single mitochondria. These dynamics could provide a better understanding of mitochondrial bioenergetics and diseases related to mitochondrial dysfunctions. This thesis describes three developments to improve mitochondrial functional assays, with an ultimate goal of achieving single mitochondrial resolution.
The first development is single mitochondrial respirometers that require only 1.5 pL of assay buffer and can measure respiration from one mitochondrion. The micro-respirometers consist of micron sized chambers etched out of glass substrates and coated with an oxygen sensitive phosphorescent dye Pt(II) meso-tetra(pentafluoropheny)porphine (PtTFPP). Sealing the chambers
with a polydimethylsiloxane layer coated with oxygen impermeable Viton rubber enables detection of single mitochondrial respiration.
The second development is the fabrication of nanochannels capable of trapping single mitochondria for fluorescence analysis of their membrane potential. The use of these channels significantly reduces background noise and allows the ease of experimental buffer exchange. Experimental results show fluctuations of membrane potential at the single mitochondrial level.
Finally, an integrated platform to detect extra-mitochondrial pH of isolated mitochondria is described. This platform was based on tethering mitochondria to one-atom thin graphene. The mitochondria are tethered via graphene bound antibodies, which recognize the mitochondrial outer membrane protein TOM20. Graphene is an excellent conductor and changes in the pH surrounding the mitochondria can change the graphene conductance and can be detected electrically. Being transparent, the graphene layer also permits optical interrogation of the mitochondria concurrent with the analysis of pH. Hence, this system permits the simultaneous monitoring of changes in extra-mitochondrial pH through graphene conductance and inner membrane potential using fluorescence. In addition, the integrated graphene system offers a unique scalability down to one mitochondrion without loss of electrical sensitivity.