Assessment of the Maximal Activity of Complex IV in the Inner Mitochondrial Membrane by Tandem Electrochemistry and Respirometry

Abstract

Assessment of activities of mitochondrial electron transport enzymes is important for understanding mechanisms of metabolic diseases, but structural organization of mitochondria and low sample availability pose distinctive challenges for in situ functional studies. We report the development of a tandem microfluidic respirometer that simultaneously tracks both the reduction of mediators on the electrode and the ensuing reduction of O₂ by complex IV in the inner mitochondrial membrane. The response time of O₂ consumption to multiple alternating potential steps is of approximately 10 s for a 150 μm-thick sample. Steady O₂ depletion shows good quantitative correlation with the supplied electric charge, Pearson’s r = 0.994. Reduction of mediators on biocompatible gold electrodes modified with carbon ink or fumed silica can compete with the oxidation of mediators by mitochondria, yielding an overall respiratory activity comparable to that upon chemical reduction by ascorbate. The dependence of O₂ consumption on mediator and mitochondrial suspension concentrations shows that mass transport between the electrode and mitochondria does not limit biological activity of the latter. The mediated electrochemical approach is validated by the radiometric measurements of simulated changes in the intrinsic mitochondrial activity upon partial inhibition of complex IV by NaN₃. This approach enables the development of O₂-independent, biomimetic electrochemical assays narrowly targeting components of the electron transport chains in their native environments.

Graphical Abstract

Mitochondrial abnormalities are increasingly recognized as a key factor in accelerated aging and in chronic diseases such as cancer and neurodegenerative (Alzheimer’s and Parkinson’s) and metabolic (diabetes) diseases.^1–4 In spite of extensive efforts, mechanisms of mitochondrial damage are still to be elucidated, mostly because of the limitations of existing respirometric techniques that use either polarography^5,6 or fluorescence quenching⁷ to measure O₂ consumption as a net probe of metabolic activity. Although multiple combinations of substrates and inhibitors have been developed to evaluate changes in individual complexes of the mitochondrial electron transport chain (ETC),⁸ such protocols still sample total electron flow to cytochrome c oxidase (COX)⁹ along a complex redox chain, complicating interpretation.

This analysis is further compounded by low permeability of the inner mitochondrial membrane (IMM), whose integrity is critical for preserving chemiosmotic gradient and control over ETC. Only a few native substrates can cross the IMM without disrupting it.^10,11 There is also growing evidence that native supramolecular interactions are critical for optimal ETC function. Therefore, any sample manipulations that can disturb native mitochondrial organization, including alterations of the outer mitochondrial membrane (OMM), must be minimized. Thus, there is a need for a method that can yield quantitative, complex-specific information about the mitochondrial function that would lead to new insights into the role of mitochondrial dysfunction in chronic diseases.

The intrinsically electrochemical nature of the ETC makes mitochondria unique eukaryotic organelles. This opens an intriguing possibility that some, or all, of the ETC steps can be supported from an electrode without the use of consumable chemical substrates. Such an approach would have multiple advantages over traditional chemical titrations, including differential sampling, ease of quantification, unlimited electron supply, testing of maximal activity of individual ETC sections, scalability, and remote manipulations. Unfortunately, spatial separation imposed by the OMM effectively precludes direct electron transfer between the electrode and the ETC.^12,13 Removal of the OMM to form a mitoplast allows us to measure activity of COX following oxidation of cytochrome c (CytC),¹⁴ for example, but this disrupts native mitochondrial compartmentalization and is undesirable. Small mediator molecules can be used instead to shuttle electrons between the electrode and the ETC. An ideal reversible redox mediator should freely cross the OMM, without partitioning into the lipid bilayer, and be selective for or native to a specific ETC complex. If the net electrochemical current via such a mediator is controlled by its redox partner ETC complex(s), it can be used to extract direct information about the said partner.

To achieve this, the electrochemical method should ensure that the overall rate limiting step, including mass transport of the mediator, is a function of the ETC enzyme(s) of interest. In the studies on the coenzyme Q pool, for example, the limitations of mass transport were circumvented by immobilizing mitochondria on the electrode,^15,16 although this could affect the integrity of the OMM or the signaling pathways.¹⁰ The proximal and distal sides of an immobilized mitochondrion may also exhibit different electrode kinetics. Microfluidic electrochemistry is a valuable alternative to immobilization in curtailing mass transport limitations. Every mitochondrion in a microfluidic suspension is accessible to the mediator(s) within the diffusion limit of the electrode, while reducing sample demand and improving integrity with a shorter preparation time.¹⁷ The large electrode area per sample volume also ensures adequate heterogeneous catalytic capacity to sustain unrestricted respiration needed for detecting changes in COX activity.

This study describes the development of an exhaustive microfluidic-mediated electrochemical approach for functional testing of ETC enzymes. Herein, we focus on the activity of COX in mitochondrial suspensions as the most suitable target for direct comparison with the traditional polarography. Using simultaneous respirometric and amperometric analysis of O₂ reduction, we demonstrate quantitative measurement of changes in COX activity using electric current in lieu of O₂ consumption and compare respirometric and electrochemical detection methods using artificial mediators.

MATERIALS AND METHODS

N,N,N′,N′-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and general chemicals of a reagent grade or better were obtained from Sigma-Aldrich (St. Louis, MO) and used as acquired. Pt octaethylporphyrin (PtOEP) was obtained from Frontier Scientific (Logan, Utah).

MICROFLUIDIC CHAMBER

The microfluidic chamber (chip), including the O₂-sensitive optode, was fabricated as described previously for the closed shell microfluidic respirometer with the following modifications to accommodate electrodes.¹⁸ The chamber volume was ~3.6 μL. Optode O₂ measurements were performed using a NeoFox GT phase shift fluorimeter (Ocean Optics, Dunedin, FL) and calibrated against air-equilibrated water (250 μM O₂) and a fresh anaerobic solution of sodium dithionite.

All working electrodes were connected to a reusable manifold (Figure 1A) via a dry pin contact (Figure 1B(f)). The reference and counter electrodes were assembled entirely in the manifold (a and b in Figure 1A, respectively) and connected to the chip via separate sealed capillaries (Figure 1A, blue volumes). A 1.3 mm-diameter graphite pencil lead was utilized as a counter electrode, as described in Figure S1A. A tetramethyl-orthosilicate sol-gel plug was used to prevent mass transfer between the counter electrode and working solution while ensuring ionic continuity. All results are reported versus the saturated KCl Ag/AgCl reference electrode, fabricated as described in Figure S1B. Manifolds with the imbedded electrodes were useable for over a year.

Figure 1.

Tandem microfluidic respirometric and electrochemical analyzer. (A) Exploded view of the manifold (top), including reference (a) and counter (b) electrodes and the fiber optic probe (c), over the microfluidic chip (bottom). (B) Microfluidic chip includes the reference (d) and counter (e) capillaries and dry working electrode connection (f) on the glass substrate and PtOEP optode (g).

WORKING ELECTRODES

The glass substrate was coated with 40 nm Au (Research and PVD Materials, Lewisburg, WV) over 5 nm Ti by chemical vapor deposition (CVD). The geometry of the Au electrode was defined photolithographically using a positive S1813 photoresist prior to CVD. The bare Au electrodes were typically modified to prevent biological fouling.¹⁹ The Au/carbon ink composite electrodes (AuCi) were prepared by spin-coating a 2 g/mL mixture of carbon ink (Ercon Inc., Wareham, MA) in Ercon ET60 solvent thinner over Au at 1200 rpm for 30 s. The electrodes were dried at 75 °C and then at 120 °C for 60 min each. AuCi was activated in the assembled chamber immediately before every experiment by a potential of +2 V over 120 s in 50 mM potassium phosphate buffer, pH 7.0.²⁰ With regular cleaning, AuCi chips were used with mitochondrial samples repeatedly for over 30 days without noticeable degradation of performance.

Modification of Au with fumed silica (AuFS) was performed prior to the assembly of the microfluidic chamber as described.²¹ All AuFS electrodes were stored in MilliQ water.

Glassy carbon (GC) electrodes were freshly polished using 1 μm MicroPolish alumina powder (Buehler, Lake Bluff, IL) three times, rinsed with MilliQ water, and dried before each use.

ELECTROCHEMICAL ASSAYS

Electrochemical measurements were performed using a CHI830C potentiostat (CH Instruments, Austin, TX). Electrode characterizations were performed using CVs of Fe(CN) ^3−/4− and Ru(NH₃)₆ ^2+/3+ in 0.5 M KCl in a conventional three-electrode cell with the carbon rod counter electrode and a standard saturated KCl Ag/AgCl reference electrode. The electrochemically active surface area of the electrode was determined from the Randles-Sevcik equation (Table S1) at varying scan rates and the reported diffusion coefficients of Fe(CN)₆ ^3−/4− and Ru(NH₃)₆ ^2+/3+.^22,23

Measurements in the fully assembled microfluidic chip were carried out under static conditions after sample injection using a syringe (~400 μL) or a pipet (<50 μL). The chip was thoroughly rinsed between measurements using water−50% ethanol in water-water sequence.

Simultaneous respirometric and chronoamperometric measurements started with an open circuit potential (no applied potential) followed by the applied potential alternating between 0.35 and −0.15V for oxidizing and reducing conditions, respectively. An additional oxidizing step at the end of the measurement was used to calculate background O₂ concentration changes. Data analysis and correlation calculations were performed using Igor Pro 8 (Wavemetrics Inc., Portland, OR).

MITOCHONDRIAL ISOLATIONS

Rabbit heart mitochondria were isolated by differential centrifugation²⁴ with minor modifications. Tissue was homogenized on ice using a Scilogix D-160 homogenizer with a saw-tooth probe, first three times at 8000 RPM and then 15,000 RPM for 30 s intervals until a uniform color was observed. Dimethyl sulfoxide was added as a cryoprotectant (5% v/v), and CCCP was added as an uncoupler (5 μM) before isolated mitochondrial suspensions were flash frozen in liquid nitrogen and stored at −80 °C. Protein concentration of mitochondrial stock was measured following Bio-Rad Bradford protein assay protocol in a 96-well plate using a Tecan Infinite M1000 plate reader.

RESPIROMETRIC MEASUREMENTS

Mitochondrial measurements were performed in 10 mM Tris-HCl, pH 7.5, containing 125 mM KCl, 1 mM EGTA, and 1 mM potassium phosphate. Exogenous CytC was added to the final concentration of 25 μM after thawing as a precaution for possible permeabilization of the OMM during isolation and freezing to improve sample uniformity and to ensure efficient electron transfer from TMPD to COX.^25,26 No additional treatments of the IMM or OMM were performed.

During polarographic measurements, Na ascorbate and mitochondria were added sequentially to the sample chamber followed by cumulative additions of 100 mM TMPD stock in the same buffer, pH 5.56, to the indicated concentrations. The autoxidation O₂ consumption rate (OCR) in the presence of. Na ascorbate and CytC was measured separately for varying TMPD concentrations. Linear dependence of autoxidation OCR on O₂ concentration²⁷ was used to correct rates to air-equilibrated conditions (Figure S2). Linear interpolation to the average O₂ concentration was subsequently used to determine the autoxidation OCR during each mitochondrial measurement.

Autoxidation rates under microfluidic conditions were measured using control samples containing TMPD and CytC (Figure S3) immediately before the corresponding mitochondrial measurements. Because both measurements started at 250 μM O₂, interpolation of autoxidation OCRs was not performed.

RESULTS

Traditional polarographic respirometry involves sequential additions of substrates, uncouplers, or inhibitors to large (1–3 mL) enclosed volumes of mitochondrial suspensions. Such additions are cumulative and cannot be reversed, as illustrated in Figure 2A for TMPD, to facilitate reduction of CytC by ascorbate, and the titration with NaN₃, to inhibit COX. Measurements in the presence of excess inhibitor (NaN₃ or KCN) probe background O₂ consumption and must be performed as the last step in the experiment.

Figure 2.

Comparison of chemical and electrochemical control of mitochondrial respiration. (A) Clark electrode measurement of mitochondrial suspension in an ~2 mL chamber in the presence of 8 mM sodium ascorbate. Sequential additions of 1 mM TMPD and indicated concentrations of NaN₃ are shown. (B) Electrochemical measurement in a ~3.6 μL microfluidic chamber in the presence of 1 mM TMPD. Reduction and oxidation of TMPD was controlled by alternating −150 mV reducing (−) and +350 mV oxidizing (+) potential. Data are shown on the identical scales. Protein concentrations were 0.175 and 0.094 mg/mL for panels A and B, respectively.

Electrochemical control over mitochondrial respiration is illustrated in Figure 2B, where TMPD is reduced on the electrode and oxidized by COX via CytC. Oxidized TMPD is re-reduced on the electrode in contrast to Figure 2A, where it is reduced by the ascorbate. Small thickness of the microfluidic sample ensures fascicle access of TMPD to the electrode and limits the sample volume. Only 0.34 μg of mitochondrial protein was used in the microfluidic chamber in Figure 2B, which is three orders of magnitude less than the 0.35 mg used in the polarographic measurement (Figure 2A).

Unlike reactions with chemical reductants, electron transfer on the electrode can be controlled by the applied potential and result in reversible reduction or oxidation of TMPD. This, in turn, controls TMPD-supported O₂ reduction by COX and correlates with changes in the OCRs between negative and positive potential steps (Figure 2B). A short delay, observed between the potential step and the ensuing OCR response, is attributed to the propagation of the changes in redox states between the electrode and the optode.

Results shown in Figure 2B suggest that the OCR can be assessed by either following changes in the O₂ concentration or from the charge supplied from the electrode. Quantitative correlation between electric charge and the electrochemically induced O₂ depletion (optode OCR) was examined using a simplified chemical model (eq 1).

$${\text{O}}_2+4{\text{H}}^{+}+4{\text{e}}^{-}\to 2{\text{H}}_2\text{O}$$ (1)

Importantly, detection of the electric charge and the changes in O₂ concentration took place at the opposite walls of the chamber, which represents the worst-case scenario for the diffusion of the analyte across a sample of thickness d = 125 μm (Figure 3A). No significant change in the O₂ concentration was reported by the optode (Figure 3B, top) under an open potential and during the initial oxidizing potential step (+350 mV). The simultaneously recorded amperogram showed a minimal oxidation current after the initial surface charging (Figure 3B, bottom). A subsequent decrease in the applied potential (−450 mV) resulted in a steady depletion of O₂ from the solution with simultaneously detected sustained cathodic current. Both processes continued until the reversal of the applied potential during the second cycle. Such a pseudo-respiration pattern, which mimics mitochondrial results shown in Figure 2B, was observed over multiple alternating cycles.

Figure 3.

Simultaneous measurement of charge transfer and oxygen reduction in the microfluidic electrochemistry. (A) Schematic of the cross section of the microfluidic chamber with layer thickness d. O₂ reduction on the electrode is detected by the PtOEP optode upon its depletion on the opposite side. (B) Tandem measurement of O₂ concentration (a) and the simultaneously measured amperogram (b) under alternating potentials. Open circuit (open), 350 mV (+), and −450 mV (−) potentials were applied as shown in the gray bar. Applied potentials were held constant for the duration of steps shown by vertical lines. (C) OCR reported by the O₂ optode (circles) and equivalent electric charge (squares). Measurements were performed in 0.1 M HCl and 0.5 M KCl on the AuCi electroded.

The differential optode OCR was calculated from the slope of O₂ concentration over time during the reduction step against the average of such slopes during two flanking oxidation steps. A similar correction of the electric current was not necessary because the oxidation current was small relative to the reduction current. The total electric charge for each reduction step was converted into the electrochemical OCR using the chamber design volume and the equivalent amount of consumed O₂ (eq 1). The OCR values obtained using two methods were in good agreement with each other, especially at the beginning of the measurement, (Figure 3C) and exhibited a similar decrease over successive cycles, as expected with net O₂ depletion. Two detection methods showed linear correlation with Pearson’s r = 0.994 (Figure S4). The average relative standard deviations over six steps were ±9.6 and ±2.3% for the OCRs measured simultaneously using optode or amperometric detection, respectively.

It was noted that the optode reported an increasingly negative OCR during oxidation steps as O₂ gradients increased over successive cycles. At the same time, the electrode OCR and the average optode O₂ concentration exhibited evidence of saturation, resulting in a correlation slope of 0.456 ± 0.024 when the transferred charge was converted into the amount of consumed O₂ per eq 1 (Figure S4). The rebound of O₂ concentration during oxidation steps was the most noticeable with small working electrodes and the least noticeable when electrodes were several folds larger than the optode. This suggests that the O₂ rebound (Figure 3B, top) and increasing difference between optode and electrode OCRs (Figure 3C) are contributed by O₂ diffusion along the electrode. Larger electrode size requires longer diffusion distance from the periphery of the chamber to the optode (distance w in Figure 3A). This further implies that O₂ concentration varies in the plane of the electrode and that caution must be exercised in quantitative measurements involving large changes in concentrations of O₂ or other substrates.

Preceding results demonstrate that mediated electron flux between mitochondria and the electrode can control the activity of ETC complexes. To make amperometric data (Figure 3) meaningful in the context of biological measurements (Figure 2B), electric currents must show quantitative correlation with the intrinsic mitochondrial activity and, more importantly, changes in such activity. To achieve this, the overall process must not be limited by either heterogeneous redox reactions or the mass transfer steps (k₁ and k₂–k₄ in Figure S5, respectively). In addition to short diffusion distances afforded by microfluidics, this requires biocompatibility of the electrodes with a large effective surface area. Three modified Au electrodes were examined here for electrochemical efficiency and resilience to fouling by proteins.²⁸ AuFS and activated AuCi showed effective surface area close to the geometric surface area, while that of the as-deposited AuCi was 36% lower when probed by CV of Fe(CN)₆ ^3−/4− (Table S1). AuFS exhibited a lower effective area with Ru(NH₃)₆ ^2+/3+ than with Fe(CN)₆ ^3−/4−, indicating differences in surface morphology.²⁹ There were no significant difference between Fe(CN)₆ ^3−/4− and Ru(NH₃)₆ ^2+/3+ for carbon ink electrodes.

Considering its lower efficiency, raw AuCi was not used in further studies. AuFS and activated AuCi electrodes showed comparable capacity to sustain catalytic conversion, while differences in morphology may be favorable for some mediators. An ionic Fe(CN)₆ ^3−/4−, for example, cannot cross the IMM but can oxidize ETC at the CytC level. TMPD is commonly used to facilitate reduction of CytC by ascorbate, including mitochondrial studies owning to its small molecular weight and the ability to cross the OMM. Both Fe(CN)₆ ^3−/4− and TMPD exhibited well-defined CVs on either AuFS or AuCi (Figure S6). Small peak separations observed for TMPD indicate that minimal overpotentials are sufficient for effective electrochemical mediation, while a small increase in the low potential transition on AuFS makes a more efficient oxidant of ETC.

Biocompatibility of the modified Au electrodes was assessed using 0.5 mM CytC as an electrochemically active fouling agent which can also be used as a mediator upon permeabilization or removal of the OMM. CV measurements on CytC were performed in the microfluidic chamber as opposed to the conventional cell used for Fe(CN)₆ ^3−/4− and TMPD above. Well-defined oxidation and reduction peaks were observed for CytC on AuFS, irrespective of the presence of TMPD as a secondary mediator (Figure S7, top). Small CV peak separation and little evidence of mass transfer or other kinetic limitations^30,31 are characteristic of a relatively uniform thin layer microfluidic sample with fast and direct redox transitions. In contrast, CytC did not exhibit redox transition on AuCi unless a small amount of TMPD was added (Figure S7, bottom). The lack of redox activity of CytC on AuCi suggests unfavorable electrostatic interactions compounded by high ionic strengths.³² Once the initial redox activity of CytC was demonstrated, AuFS and AuCi electrodes were further incubated with 0.5 mM CytC for >1 h at room temperature, to simulate exposure to biological samples, and CV measurements were repeated using CytC, TMPD, and Ru(NH₃)₆ ^2+/3+. Neither AuCi nor AuFS showed decreased peak currents or increased peak separations that would indicate fouling.²⁸

Excessively negative electrode potentials can facilitate reduction of mediators but may also lead to nonbiological O₂ consumption in aerobic samples, which can indirectly affect the metabolic state of the sample and directly interfere with OCR measurements. Surface dependence of direct O₂ reduction is shown in Figure 4 for AuFS and AuCi using simultaneously detected electric current (bottom) and optode OCR (top) across the potential range. To simplify the direct comparison, the conductive surface of an oversized AuCi electrode was cut by a CO₂ laser to the same geometry and area (3.1 mm²) as the AuFS electrode prepared separately by photolithography. Both the optode OCR (Figure 4, top) and total transferred charge (bottom) were higher for AuCi (filled markers) than for the AuFS surface, especially in the range of −0.3 to −0.2 V, where O₂ reduction on AuFS was low. This extends the advantage of AuFS over AuCi in facile reduction of TMPD and CytC (Figures S4 and S5) by permitting faster electron supply to the solution at larger reducing overpotentials without a concomitant increase in the background O₂ reduction.

Figure 4.

Potential-depended O₂ reduction on modified electrodes. Optode-detected O₂ reduction (top) and simultaneously measured electric charge (bottom) measured at various static applied potentials in a microfluidic cell over a small area AuFS (empty markers) or AuCi (filled markers) electrodes in 0.5 M KCl. All measurements were recorded over one-minute long intervals. Pearson’s correlation coefficient between OCR and charge is r = 0.993 for both materials.

The last requirement for a direct correlation between the catalytic activity in the solution and the supporting electric current is small contribution of mass transfer into the overall rate limitation (k₂–k₄ in Figure S5). Mass transfer is negligible when respiration is supported by homogenous reaction between TMPD and ascorbate in the traditional assays, which results in proportionality between the mitochondrial suspension density and OCR at each given TMPD concentration. This is an established protocol that can adequately report changes in the activity of COX in mitochondria.

In contrast, mass transfer of TMPD between the electrode and the endogenous mitochondrial CytC (Figure 5C) may limit the overall turnover rate. Electrochemically driven respiration (Figure 5B) showed ~2.5-fold lower OCR than that using ascorbate as a chemical reductant (Figure 5A) for the same mitochondrial stock. All measurements reported in Figure 5 were performed using the same large stock of mitochondrial suspension in an alternating order over multiple days.

Figure 5.

Electrochemically supported respiration in mitochondrial suspension. OCRs observed upon either chemical reduction of TMPD (A) or TMPD reduced on the microfluidic electrode (B) are shown across the range of TMPD concentrations. Respiration occurring in the presence of 4 mM sodium ascorbate as a reductant (A) was followed using the Clark electrode in 3 mL of stirred mitochondrial suspension. Respiration occurring upon electrochemical reduction of TMPD at −150 mV (B) was followed using the PtOEP optode. (C) Schematic diagram of microfluidic respiration. All measurements were conducted using dilutions of common mitochondrial stock at 1× (circles), 2× (squares), and 4× (triangles) densities using both instruments on different days. The average protein concentrations were 0.17 and 0.14 mg/mL for panels A and B, respectively, which correspond to the total amounts of protein of 0.34 mg and 0.50 μg used in polarographic and microfluidic chambers, respectively. All OCRs are shown as an increase over background O₂ reduction in the absence of mitochondria at the corresponding TMPD concentrations. Separate controls were measured under polarographic and microfluidic conditions.

Pairwise comparison between the OCR observed upon electrochemical reduction and that using ascorbate as an electron donor at identical TMPD concentrations of ≤1 mM showed linear correlation with the average Pearson’s r = 0.974 ± 0.022 between three different densities of mitochondrial suspensions (Figure S8). At higher TMPD concentrations OCRs first plateau and then decline. Microfluidic OCRs measured upon reduction on the electrode reached maximal activity at lower TMPD concentrations than the corresponding polarographic measurement using ascorbate as a reductant, leading to the loss of correlation at ≥1.5 mM TMPD (Figures 5 and S8). Furthermore, pronounced background reduction of O₂ was observed at concentrations of TMPD above 1 mM, hindering measurements of dilute mitochondrial suspensions.

Mitochondrial suspensions used herein were treated with an uncoupler (CCCP) for maximal COX turnover rates to exasperate any kinetic limitations associated with the mass transport of TMPD. Two observations indicate that mass transport was sufficient to support O₂ reduction in uncoupled mitochondria and, hence, under more physiological conditions where electron demand is lower. First, electrode-driven OCR remained proportional to the concentration of mitochondrial suspension at all TMPD concentrations (Figure S9). Second, maximal activity was observed at comparable, albeit not identical, TMPD concentrations. Together, these results indicate that the overall reaction in the electrochemical assay (Figure 5B) is still controlled by the same rate laws as in the traditional method (Figure 5A), even if the maximal OCR is somewhat limited by either heterogenous reduction or the diffusion of the mediator.

Inherent variability of biological samples in traditional respirometry is offset using activity ratios versus well-defined metabolic reference states.⁹ Therefore, the electrochemically driven metabolic assay must also accurately report changes in the activity of a given mitochondrial sample. Importantly, it must detect variations in the intrinsic activity of ETC components at a constant suspension density rather than changes in concentrations of bimolecular reactants. For example, inhibition of COX by the chemiosmotic potential is a critical characteristic of the IMM (coupling), while changes in the bimolecular reaction rate between endogenous CytC and TMPD are not.

Partial inhibition of COX in uncoupled mitochondria was used here as a chemically reproducible model to test the ability of the heterogeneous electrochemical assay to report typical changes in the intrinsic activity of ETC complexes (Figure 6). NaN₃ was chosen as an inhibitor of COX over KCN because of its compatibility with the Au conductor layer, even though direct contact with the substrate of the modified electrode is limited. Initial polarographic titrations of NaN₃ in the presence of Na ascorbate showed that 1 mM NaN₃ inhibits respiration to 32 ± 4.5% of the control sample after subtraction of the residual activity observed in the presence of 4 mM NaN₃ (Figure 6, left). A comparable three-to fourfold change in OCR would approximate changes observed during typical respiratory control ratio measurements in the presence of pyruvate and succinate, for example, although different ratios have been reported for other substrates.³³ Parallel microfluidic electrode-driven measurements showed NaN₃-inhibited OCR of 16 ± 3.7 and 16 ± 2.4% when detected simultaneously by the optode (middle) and the charge (right), respectively, after correction for residual activity. Both microfluidic detection methods yielded remarkably close values with smaller variability reported by the electrode than by the optode. A larger extent of inhibition observed in the electrode-driven assay is likely contributed by the differences in sample volumes between the Clark electrode (>1 mL) and microfluidics (<10 μL) that lead to large differences between sample handling protocols.

Figure 6.

Ratiometric assessment of the intrinsic activity of COX in the IMM. OCR of uncoupled mitochondria (solid bars) and its inhibition by 1 mM NaN₃ (hashed bars) were measured using the Clark electrode (left) or microfluidic electrochemistry with simultaneous optode (middle) and electrode (right) detection. All OCRs were normalized to the activity of uninhibited samples after subtraction of the residual OCR (4 mM NaN₃). Samples contained 1 mM TMPD. Polarographic measurements were performed in the presence of 8 mM Na ascorbate. Microfluidic measurements were performed under an applied potential of −250 mV on the activated AuCi electrode. Samples contained 0.175 and 0.094 mg/mL protein in polarographic and microfluidic measurements, respectively.

DISCUSSION

The electrochemical probe of ETC metabolism is designed to examine changes in the intrinsic rate limitations of the particulate sample (organelle suspension) by providing mediated electron flux in excess of the activity of interest. In standalone applications and in tandem with respirometry, a primary advantage of this technique is the ability to make O₂ an optional reporter molecule which gives flexibility in experimental design. In this study, amperometric measurements consistently showed smaller variability than simultaneous microfluidic optode measurements (Figures 3, 6, S4). This offers intriguing possibilities, including tandem studies comparing upstream-mediated electron supply versus electron sink into O₂, direct targeting of individual ETC complexes, and metabolic studies under anoxic conditions. One biomedically relevant example is the ceramide-mediated mitochondrial damage mechanism that was proposed to involve either direct inhibition of CmpIII or an indirect inhibition upon permeabilization of OMM via the Bax/Bcl2 pore and ceramide channel formation, leading to the loss of CytC.^34–37 Both of the mechanisms lead to similar metabolic phenotypes that would be indistinguishable using traditional methods. Another area of potential impact involves metabolic response under hypoxic or anoxic conditions, such as acute ischemia, oxidative stress, and under the conditions of avascular tumor. In addition to the suppression of the detection-induced O₂ depletion, the freedom to control the applied potential permits reversible electron transfers and paves the way to new differential measurement protocols when samples are not physically amenable to traditional chemical manipulations. Furthermore, by creating a minimal environment that mimics cytosolic composition, the use of applied potential to control and manipulate the functional state of mitochondria enables transient investigations in artificial respiration that cannot be afforded using depletable chemical cofactors (Figure 2).

Microfluidics expands these potential applications by reducing sample demand and creating a possibility for studies on scarce samples, including human biopsy tissues, primary cell cultures, and small animal models. A thousand-fold reduction in sample volume from 2–3 mL in conventional mitochondrial polarography to 4 μL or less in our microfluidic respirometer¹⁸ allows us to use more concentrated samples and boost the signal, while keeping the overall sample demand within attainable limits. Stationary sampling conditions, shown here for tandem respirometry, permit longer signal accumulation, improving sensitivity over differential polarographic measurements under flow sampling conditions as long as detection methods do not consume significant amounts of O₂.^7,38 The simple suspension loading approach used herein can be improved by entrapment of organelles in the chamber, curbing sample overhead of the loading capillaries, eliminating the need for sample replacement between measurements, and permitting sampling of the same specimen under multiple experimental conditions. Our recent studies on microfluidic respirometry of adherent cells^18,39 under similar conditions offer an intriguing alternative approach where selective permeabilization of the cellular wall⁴⁰ could, in principle, provide access of mediators to mitochondria without their isolation.

Another major advantage of microfluidics is the suppression of the mass transfer effects when the sample is confined within the diffusion limit of the electrode. For a sample thickness of <150 μm used here, effects of mass transfer were observed only for the first 10 s after the potential step, while the dynamic equilibrium was established. As long as changes in the sample composition remain relatively small during sampling, the effect of mass transfer on the minutes time scale is negligible. Biological relevance of this constraint is supported by a low reported variability of tissue-dependent O₂ concentration over time.⁴¹ Although additional studies are necessary to quantify contributions of individual steps in the electrochemically driven respiration (Figure S5), the partial mitochondrial inhibition model shows that mass transfer does not hinder measurements of relative activities in mitochondrial suspensions (Figure 6). Should the mass transfer impose an overall rate limit under these conditions, subsequent inhibition of COX by NaN₃ would have either no effect on the observed OCR or partially the shift rate limit to COX—in either case, this would yield a smaller apparent inhibition than that observed using chemical reduction. This is contrary to the results presented in Figure 6 even for the worst-case scenario of the most active (uncoupled) mitochondrial samples.

Early studies employed CytC as the only mediator for the electrode-supported mitochondrial respiration.⁴² Although the existence of exogenous CytC pathways has since been proposed,^43–45 it is now accepted that CytC cannot cross the intact OMM, suggesting that initial studies probably used mitochondria with the abnormally permeable OMM. Never-theless, CytC is an attractive mediator for electrometabolic studies on mitoplasts and on physiological or pathological permeability of the OMM in mitochondria.^43,45 In this study, only the AuFS-supported reduction of CytC at small overpotentials (Figure S7, bottom) is in agreement with earlier reports.^46,47 Large overpotentials needed to achieve direct electron transfer on AuCi electrodes (up to ±0.5 V) make them an impractical choice because of the likelihood of side reactions, including O₂ reduction and alteration of biological samples. A small amount of a secondary mediator, such as TMPD, can rescue such redox transition for studies on the mitoplasts, for example, while ensures that its contribution to the overall mass transport is small (Figure 5).

In the course of this study, which is primarily focused on the possibilities and limitations of electrochemical control over particulate samples, we made several practical simplifications that should be considered in translating current results to physiological conditions. Isolation and storage protocols used herein were designed to reduce biological variability associated with multiple mitochondrial isolations and to provide a large amount of chemically uniform suspension sample needed for comparative studies. Although freezing of mitochondria typically increases mitochondrial permeability of the membranes, it does not cause solubilization of COX, thus preserving the most distinct characteristics of mitochondria as a particulate catalyst of O₂ reduction. This is supported by the lack of respiratory activity in the absence of TMPD. Similarly, possible loss of matrix enzymes is not expected to affect activity of COX herein, although this cannot be ignored in the studies involving typical mitochondrial substrates. Finally, mitochondrial respiration is sensitive to temperature. Although temperature control has not been the focus of the present work, small size of the tandem microrespirometer makes it amenable to such control in future studies.

CONCLUSIONS

Sensitivity of respiration, catalyzed by COX in the IMM, to the applied potential demonstrates that mediated electrochemistry is an effective analytical tool for quantitative studies on ETC beyond the existing methods that track O₂ consumption. Tandem assessment using fluorometric respirometry and simultaneous amperometry shows good correlation between charge transferred at the electrode and the resulting O₂ reduction both in the inorganic model and TMPD-mediated catalysis by COX. Large, biocompatible electrodes can sustain near-maximal turnover of the components of the ETC with the support of small molecule mediators. Both the effective electrode surface area and diffusion distances are critical for a fast and exhaustive electrolysis of the mediator(s), which is necessary to exceed the intrinsic mitochondrial activity and enable detection of its changes. High conductivity of the Au layer combined with resilience of modified AuFS or AuCi electrodes to protein fouling resulted in activities that are comparable to the established mitochondrial assays that utilize ascorbate as a reductant.^26,48,49 In addition to >500-fold reduction in sample demand versus conventional respirometry, microfluidics provides a significant method-specific advantage by limiting the diffusion distance of mediators and substrates, thus ensuring relative sample homogeneity. Changes in the redox state of the sample, induced by the applied potential pulse, propagate across the thin layer on the seconds times scale, making diffusion mass transfer along the normal to the electrode insignificant in most biological applications. Mass transfers in the plane of the electrode, however, may affect the experimental observations and must be considered in analysis, but such effects can be minimized by limiting reaction-induced variations in the sample composition. Employment of differential sampling protocols, afforded by the reversible potential steps and the resulting redox transitions in the mediator medium, can improve sensitivity and alleviate sample depletion. Unlimited and controllable supply of the electrons opens the door to transient sample control and novel studies that mimic native metabolic pathways.