We have created a novel system for assessing functional changes in mammalian mitochondria. Our system employs an optically transparent, single atom thin graphene layer onto which the mitochondria are tethered using a graphene-bound antibody against the outer membrane protein TOM20 to which isolated mitochondria adhere. Because the graphene electrode is optically transparent, single bound mitochondria can be functionally analyzed using analytical fluorescent dyes at the same time that the mitochondrial outer membrane can be monitored in real time by changes in the conductance of the graphene. The electrical properties of the graphene have been found to detect subtle changes in the proton concentration on the outside of the mitochondrial outer membrane as the mitochondrial inner membrane electrochemical gradient changes. The acute sensitivity of this system is permitting the dissection of alterations in the mitochondrial membrane status during various mitochondrial processes such as uncoupled depolarization of OXPHOS and the induction of the intrinsic pathway of apoptosis initiated by the pro-apoptotic peptide BIM-BH3.

Using our graphene biosensor platform, we have been able to make three important observations pertaining to the mitochondrial inner membrane electrochemical gradient and its changes during the BIM-BH3 initiated intrinsic pathway of apoptosis. First, we found that CCCP addition to respiring mitochondria causes buffer alkalization along with membrane potential depolarization. Second, we observed that treatment of respiring mitochondria with BIM-BH3 results in external buffer acidification and membrane potential decline. Third, we found that the BIM-BH3-induced acidification and membrane potential decline of respiring mitochondria can be blocked by the addition of exogenous cytc to the external mitochondrial buffer.

Our first observation of buffer alkalization can be attributed to CCCP allowing protons to rapidly enter the matrix. When immersed in isotonic buffer containing the mitochondrial respiratory chain complex II substrate, succinate, and the complex I inhibitor, rotenone, mitochondria rapidly oxidize the succinate passing the electrons from complex II, to coenzyme Q, to complex III, to cytc, to complex IV, and finally to O 2 to reduce it to H 2 O. The flux of electrons through complex III and IV is coupled to the transport of protons from the mitochondrial matrix out across the mitochondrial inner membrane to the inter membrane space rendering the outside of the mitochondrial inner membrane acidic and positively charged and the inside negatively charged and alkaline39. Addition of uncouplers to the respiring mitochondria result in the rapid flux of protons back into the mitochondrial matrix, rendering the inter-membrane space more alkaline40,41,42 (Fig. 6). Given that the mitochondrial outer membrane voltage dependent ion channel43 (VDAC or porin) is freely permeable to protons this would result in the pH of the surrounding buffer to become more alkaline, exactly as we observed, a result consistent with Mitchell’s chemiosmosis theory44 for oxidative phosphorylation (OXPHOS) coupling.

Figure 6 Schematic diagrams of membrane morphology and compartmental pHs for the three experiments performed. The steady state of the mitochondria is shown at the top with buffer pH (indicated as measured) and the other pHs as inferred from the literature61,62. In the case of CCCP, pH is equilibrated across the three compartments. The bottom shows the qualitative changes in pH vs. time (not to absolute scale) with the bolded line being the measured variable while the other lines are inferred. For the next two cases, the top two figures correspond to our proposal that the cristae remodel and release protons and cytc from the invaginations and the absence of such a mechanism when exogenous cytc is added. The bottom figures indicate again the qualitative assessment of the pHs. Full size image

We propose that buffer acidification with BIM-BH3 in our second observation is caused by release of protons from the lumina created by the invaginations of the mitochondrial inner membrane. At first glance, the acidification of the surrounding mitochondrial buffer on treatment of respiring mitochondria with the inducer of the intrinsic pathway of apoptosis, BIM-BH3, would seem contrary to the chemiosmosis theory of OXPHOS. However, recent discoveries9,11,45 about structure and function of the mitochondrial inner membrane and the changes that occur during the induction of the intrinsic pathway of apoptosis render the observation of acidification explicable.

The mitochondrial inner membrane is highly invaginated and with the tips of the invaginations closest to the outer membrane held together by the mitochondrial inner membrane integral membrane protein OPA110 located within the mitochondrial contact site and cristae organizing system (MICOS). This creates multiple internal lumina in which the interior of each lumen is equivalent to the outside of the mitochondrial inner membrane. Hence, within these lumina the cytc and proton concentration can become very high.

When the mitochondrial membrane potential declines or the intrinsic pathway of apoptosis is activated mitochondrial inner membrane proteases including OMA1 become activated and cleave OPA1 resulting in the opening of the inner membrane lumina to the mitochondrial intermembrane space9,11. Since most of the cytc is encompassed within the lumina of the inner membrane46,47 this process is central to the release of cytc from the mitochondria observed as the intrinsic pathway of apoptosis progresses. Because protons are much smaller in diameter than cytc, it follows that as OPA1 is cleaved, the first event is the escape of the protons into the mitochondrial inter membrane space and out through the permeabilized outer membrane and into the surrounding buffer (Fig. 6). This would cause the gradual acidification of the surrounding medium detected by the graphene electrodes. Treatments of the mitochondria with BH3 peptides often require 60–90 min incubation17,48 before any functional measurements can detect a change. This time frame is consistent with the completion of the pH change that we detected with the graphene sensor which occurred at about 60 minutes.

Given that the decline in the mitochondrial inner membrane potential is an important prerequisite for the activation of inner membrane proteases and the cleavage of OPA1, it follows that the release of cytc from the mitochondrial inter membrane space following BIM-BH3 induced BAK activation could locally activate the proteases that cleave OPA1. This possibility follows from the conceptualization of the distribution of the respirasomes across the mitochondrial inner membrane. The respirasomes would fall into two categories: those that are located on the mitochondrial inner membranes that form the cristae invagination-generated lumina and pump protons into the cristae lumina and those respirasomes that are located in the inner membrane which faces the intermembrane space. This later set of respirasomes would be responsible for maintaining the pH acidification of the intermembrane space and thus would control the activation of the outer membrane proteases that can cleave OPAI. When BIM-BH3 activates BAK to open the outer membrane, the first cytc to be released would be that in the inter membrane space resulting in localized drop in the mitochondrial membrane potential, activating OMA1, and initiating the cleavage of OPA1. Once OPA1 is cleaved the invaginated cristae lumina would open to the inner membrane space and the remaining cytc would be released stopping all further respiration. Addition of exogenous cytc to the medium surrounding the mitochondria would sustain the respiration required for maintaining the intermembrane space pH and membrane potential thus stabilizing OPA1 and blocking detectable changes in proton and cytc release (Fig. 6), which is our third observation.

Although previous studies have shown that cytosolic acidification is associated with apoptosis in whole cells12,13,49, its role, timing, and mechanism has remained until now a mystery. One proposed mechanism (ATP synthase reversal14) has been ruled out on thermodynamic grounds50. Our observation of acidification even in the presence oligomycin (which blocks ATP synthase activity) also rules out ATP synthase involvement. Furthermore, our results provide a plausible mechanism that is consistent with recent discoveries of OMA1/OPA1 based cristae remodeling, Although it is generally believed that mitochondria remodel during apoptosis51, to directly measure cristae structure during apoptosis is technically challenging, and beyond the scope of this work. Our hypothesis that cristae remodeling causes buffer acidification is the most consistent hypothesis with our data, as well as all of the data in the literature on this subject51.

Furthermore, it is still highly plausible that the physiological role of pH change is to enhance caspase activities during apoptosis, as the caspases are known to have pH-dependent activation49. These questions have led to a “renaissance” of interest in mitochondrial pH52. In addition, because the tumor microenvironment also exhibits significant pH alterations, studying mitochondrial mediated pH changes would make for fruitful future studies.

As with any new bio-assay technology, it is important to benchmark results against known standards, and to compare and contrast advantages and disadvantages of the new vs. traditional methods. For pH sensing, there are two traditional approaches: Electrochemical sensors and genetically encoded pH sensitive fluorescent proteins such as pHluorin, SypHer, and pHred. We have chosen to benchmark our results against a conventional pH meter (Oakton Instruments pH meter and an OrionTM ROSS pH electrode), and found the results to be consistent. We found this to be more expedient than engineering a separate strain with pH sensitive fluorescent proteins. However, the conventional pH meter has barely sufficient pH resolution (0.01 pH) and a slow response time, as compared to our integrated graphene detectors. Consequently, we had to use significantly more sample (140 μg vs 0.1 μg) with the pH meter, and probably because of 0.01 pH resolution we could only detect pH changes after 60 min of mitochondrial incubation with BIM-BH3. In contrast, the intimate contact between the graphene and the mitochondria allows our graphene devices to detect an estimated 4.0 × 10−4 relative change in pH unit (Supplementary Information) with 0.2 s temporal resolution. The enhanced sensitivity comes with a drawback that the pH calibration for the graphene devices is not absolute (Supplementary Information).

In principle, a genetically engineered mitochondrial pH sensor based approach has the advantage that an absolute calibration can be performed for pH sensing. However, this approach has resulted in some uncertainties in the literature about the proton motive force dynamics during apoptosis14,49. Still, recent advances in live cell imaging have enabled detailed (and calibrated) studies of mitochondrial and cytosolic pH under different metabolic conditions53,54. While advantageous in many ways, genetically encoded mitochondrial pH sensors do have some disadvantages, including restricted applicability to established transformed cell lines.

What our ultrasensitive graphene system for analyzing isolated mitochondria has revealed is a model where there are two totally different mitochondrial inner membrane electrochemical gradients. One of these is the proton gradient between the mitochondria matrix and the intermembrane space. This gradient appears to be an important mediator for the regulation of OMA1 and the resulting regulation of OPA1 cleavage, the important factor in linking the opening of MOMP with the initiation of the intrinsic pathway of apoptosis. However, the second inner membrane electrochemical gradient is between the mitochondrial matrix and the enclosed lumina of the cristae invaginations which are isolated from the intermembrane space electrochemical gradient by closure of the cristae lumina by MICOs and OPA1. This structure potentially permits a much higher concentration of protons to be generated, since the pH cannot be buffered by the cytoplasm surrounding the mitochondrion. Furthermore, by confining the protons in a small volume, the electrostatic potential can be much higher increasing the energetic capacity of the system.

In conclusion, by taking advantage of our ability to study isolated mitochondria using our graphene sensor, we have been able to separate two fundamentally different classes of mitochondrial inner membrane, respirosomes, and electrochemical gradients, resulting in a strikingly new perspective of the role of the mitochondrial electrochemical gradient and thus of the biophysics associated with the Mitchell’s chemiosmosis hypothesis44.

The development of the graphene-based system for monitoring changes in the mitochondrial function permitted us to investigate events associated with mitochondrial OXPHOS coupling and the intrinsic pathway of apoptosis. However, we have only begun to monopolize on the exquisite time and sensitivity resolution that our new system permits. Further miniaturization of the technology from millimeter to micrometer dimensions (a straightforward process using photolithography) could enable assays on single mitochondria. In addition to the studies presented herein, this platform could enable new studies of mitochondrial biology and medicine that were heretofore inaccessible. The first possibility is the parallelization from one to thousands of on-chip devices. Such a system could permit for the first time the analysis of 1) mitochondrial heterogeneity within a single cell, say as mtDNA mutations in heteroplasmy55 leading to functional heterogeneity, an unexplored frontier in mitochondrial biology; 2) massively parallel screening for peptides and drugs that could modulate mitochondrial respiration or the intrinsic pathway of apoptosis. For example, it has recently been shown that mitochondrial sensitivity to apoptosis/MOMP/cytc release in response to chemotherapies in tumor biopsies (in vitro) is directly correlated with patient survival rate56. Further investigation has led to a proposed “profile” of each tumor, defined as its qualitative response (defined as cytc release) to a panel of peptides, entitled “BH3 profiling”17. Another potential application is high temporal resolution, high pH resolution studies of mitochondrial flashes54,57. The role of these flashes, and their relationship to signaling pathways, metabolism, bioenergetics, apoptosis and the mitochondrial permeability transition pore is only now beginning to be understood.