Assessment of mitochondrial membrane potential using an on-chip microelectrode in a microfluidic device

Abstract

The mitochondrial membrane potential is used to generate and regulate energy in living systems, driving the conversion of ADP to ATP, regulating ion homeostasis, and controlling apoptosis, all central to human health and disease. Therefore, there is a need for tools to study its regulation in a controlled environment for potential clinical and scientific applications. For this aim, an on-chip tetraphenylphosphonium (TPP + ) selective microelectrode sensor was constructed in a microfluidic environment. The concentration of isolated mitochondria (Heb7A) used in a membrane potential measurement was 0.3 ng μL −1 , four orders of magnitude smaller than the concentration used in conventional assays (3 μg μL −1 ). In addition, the volume of the chamber (85 μL) is 2 orders of magnitude smaller than traditional experiments. As a demonstration, changes in the membrane potential are clearly measured in response to a barrage of well-known substrates and inhibitors of the electron transport chain. This general approach, which to date has not been demonstrated for study of mitochondrial function and bio-energetics in generally, can be instrumental in advancing the field of mitochondrial research and clinical applications by allowing high throughput studies of the regulation, dynamics, and statistical properties of the mitochondrial membrane potential in response to inhibitors and inducers of apoptosis in a controlled (microfluidic) chemical environment.

Wafer-scale mitochondrial membrane potential assays

“…(For a 10 ms integration time, this would correspond to a 5 mV standard deviation on the inferred membrane potential.) This corresponds to an order of magnitude improvement over our first generation technology [11]. In addition to rapid, random fluctuations due to statistical variations, we also observed permanent, progressive changes in the measured [TPP + ] values, which we attribute to the actual biochemistry of the mitochondria which respond on a much longer time scale compared to the measurement time per point of one second.…”

“…Then, changes in the mitochondrial membrane potential were recorded as OXPHOS complex I substrates (pyruvate and malate) and ADP were added to the sensor chamber. These measurements were converted to membrane potential using a procedure similar to our previous publication [11] (discussed in supplemental information). …”

“…Previously, we reported on an electrochemical mitochondrial functional assay using a miniaturized on-chip mitochondrial membrane potential sensor equipped with TPP + ion selective electrodes[11]. This first generation planar-type mitochondrial assay sensor showed good sensitivity and a fast response to ΔΨ m by monitoring the activity of TPP + in the sample solution.…”

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

“…This quantity is much smaller than the number of mitochondria that would be included in more conventional solution biochemistry assays, ranging from 3–20 μg mitochondrial protein for assays of cytc release21, single mitochondria membrane potential22, and respiration studies23. In addition, this work pushes the minimal mitochondrial quantity requirement even further compared with our previous reports2425, which used 0.75 μg of mitochondrial protein per assay. Using our typical yield of 140 μg mitochondrial protein from 10 7 HeLa cells, 0.1 μg of mitochondrial protein amounts to around 7000 cells worth of mitochondria, a slight improvement to the 10,000-cell requirement imposed for plate-based assays of mitochondrial functions17.…”

Mitochondrial Oxidative Phosphorylation

“…In intact isolated mitochondria D Y m values of 180-220 mV are generally measured O'Brien et al 2008 ;Lim et al 2010 ) . In contrast, D Y m values of 100-140 mV have been determined in living cells (Zhang et al 2001 ;Wan et al 1993 ; for a review see .…”