Calibration of TPP+ selective electrode
The sensor calibration was performed at various concentrations of TPP+Cl− solution ranging from 10 μM to 10 mM in both respiratory buffer and 0.1 M NaCl solutions at 25 °C while monitoring potential differences between working electrode and reference electrodes. The layer-by-layer sensor design and the fabricated sensor are shown in . Three working electrodes were integrated in the same chip for the future use of parallel measurements with oxygen respiration, demonstrating the potential for the integration of different kind of sensors in the same chip. In the test, one of the three electrodes was used for the measurement. After filling the test chamber with 71 μL of 0.1 M NaCl (or respiration buffer), 2 μL of various concentrations of TPP+Cl− solution were added into the test chamber successively using micropipettes. The measured potential from the fabricated sensors is plotted vs. time in and the arrows indicate the addition of 2 μL TPP+ solution. Upon the addition of solutions, signal spikes were recorded due to the physical impact of dropping solutions into the chamber medium. Once the potential signal stabilized, it maintained a constant level showing that no significant evaporation effect occurred during the test. The signal stabilized within 25 s, which is considered to be the response time (t90) of the electrode.
Sensor design. (a) Illustration of layer-by-layer sensor construction. (b) Photograph of the fabricated microfluidic TPP sensor showing assembled PDMS layers with filling solution reservoirs and membranes on top of the glass substrate.
Fig. 4 Calibration curve of TPP sensor. (a) Characterization was performed in respiration buffer solution (25 °C, pH 7.2) using fabricated TPP+ microelectrode sensors with successive additions (arrows) of 2 μL TPP+ solution (10 μM to (more ...)
Potential differences increased logarithmically with increase in the concentration of TPP+Cl− solution. shows a linear relationship of measured potential on a logarithmic scale from 3 ×10−6 M TPP+Cl− with the slope of 53 mV dec−1, close to Nernstian response of 59 mV dec−1. No interfering effect was found during the calibration, representing the high selectivity of the sensor to TPP+ ions. The TPP+ selective electrode was soaked in 10 mM TPP+Cl− solution overnight before calibration and the reference electrode was conditioned in 3 M KCl solution.
After each measurement, the sensing chamber including TPP+ selective membrane was cleaned with DI water several times. When inhibitors and substrates are used during the mitochondrial measurement, 50% ethanol was used to rinse the chamber to avoid interferences from residues on the membranes.
Measurement of mitochondrial membrane potential with isolated mitochondria
The evaluation of the mitochondrial membrane potential was performed with human mitochondria (Heb7A) in respiration buffer (225 mM mannitol, 75 mM sucrose, 10 mM KCl, 10 mM Tris–HCl, 5 mM KH2
, pH 7.2). The measurements were repeated 4 times with freshly prepared mitochondria to confirm the performance and the reproducibility of the sensor. The results of the measurements showed reproducible responses under similar conditions. We used 25 ng of isolated mitochondria in 85 μL for the test resulting in a final concentration of 0.29 ng μL−1
. The mitochondrial membrane potential (ΔΨm
) can be determined using:17
concentration in the test chamber before the addition of mitochondria and at time t
is the initial buffer volume in the chamber and Vt
represents the final volume in the chamber which includes the total mass (in mg) of mitochondrial protein (P
) added in the assay. For our purposes, the mitochondrial matrix volume (Vm
) was assumed to be equal to 1 μL mg−1
protein. The partition coefficients describe the innate binding and accumulation of the cationic TPP+
ion to the matrix (Ki
) and external (Ko
) faces of the inner membrane and are given values of 7.9 μL mg−1
and 14.3 μL mg−1
The results of a typical assay are shown in . The respiration chamber was filled with an initial volume of 71 μL respiration buffer. Once the plot baselined to zero, we introduced 100 μM TPP+
solution to provide a working concentration. We purposely kept the working concentration of [TPP+
] ≈ 10 μM to prevent inhibition of respiration.19
After stabilization, isolated mitochondria (5 ng μL−1
) were added to the chamber. The fresh mitochondria quickly took in TPP+
from the chamber due to its value of ΔΨm
, resulting in a lower TPP+
concentration in the chamber as measured by the ISE. However, as the mitochondria consumed substrates in the respiration buffer, the substrate concentration became depleted, and the magnitude of ΔΨm
began to decrease slowly as a result, causing a slow increase in [TPP+
] in the chamber.
Fig. 5 Measurement of [TPP+] and inferred ΔΨm with isolated mitochondria. Arrows indicate successive addition of 100 μM TPP+Cl−, 5 μL of 5 ng μL−1 isolated mitochondria (mito), 10 mM pyruvate (P), 5 mM (more ...)
This slow decrease in the magnitude of ΔΨm was temporarily halted by the addition of complex I substrate PM, which allowed the mitochondria to increase the magnitude of ΔΨm through consumption of these substrates. While there are transients in the data, the slow decrease in the magnitude of ΔΨm is clearly halted by the addition of PM. The complex I inhibitor Rot halts the mitochondrial consumption of PM, leading again to a slow decline in the magnitude of ΔΨm (hence an increase in [TPP]).
The addition of complex II substrate Suc allows the mitochondria again to increase the magnitude of ΔΨm (hence decreasing [TPP]). The addition of complex II inhibitor Mal stops the consumption of Suc, causing again a slow decrease in the magnitude of ΔΨm (thus increasing [TPP]).
These measurements clearly demonstrate the ability of the on-chip ISE to assay meaningful mitochondrial responses to various biochemical stimulants in a controlled, microfluidic environment. We turn next to the device reliability, reproducibility, and sensitivity.
Device-to-device variations, drift, and sensitivity
In , we plot the measured calibration curve for 5 different devices made using identical fabrication techniques. The figure shows there are some device to device variations, but that qualitatively the calibration curves match. Thus, there is still a need to calibrate each devices for each measurement, but the general properties are very reproducible from device to device.
(a) Calibration curves of 5 separate devices. (b) Two different calibration curves for same device indicating drift.
The sensors remain fully functional for over 2 months and after 20 tests. In , we show that the calibration curve has drifted slightly during these experiments. These drifts indicate that it is important to calibrate the sensor before each experiment if absolute measurement of ΔΨm are important. However, in many experiments it is the changes in ΔΨm that one wants to assay in response to various chemical stimuli, which we discuss next.
While we do not have a complete theoretical accounting of the various statistical noise sources, we can easily quantify the fluctuations in the measured sensor voltage. Using this method, we find the measurement of the sensor voltage gives a statistical uncertainty (standard deviation under steady state conditions) of ~1 mV. This gives rise statistical uncertainty of ~0.1–1 μM on the TPP+ concentration, which corresponds to a statistical uncertainty of ~1 to 10 mV on the inferred value of ΔΨm.
The data have a statistical uncertainty somewhat larger than that of systems with larger electrodes, larger sample volumes, and higher analyte concentration. At present we believe the increased statistical noise is related to the small sensor size, and this will be investigated further in future work. The sensitivity is more than adequate for most biochemical assays, but since this is the first ever on-chip TPP+ sensor, we anticipate that further improvements in sensitivity are possible with additional effort.