OROBOROS INSTRUMENTS

high-resolution respirometry

Oxygraph-2k

www.oroboros.at

ISE for TPP+ and High-Resolution Respirometry

Zuzana Sumbalová, Mario Fasching, Erich Gnaiger

OROBOROS INSTRUMENTS, Innsbruck, Austria

Tetraphenylphosphonium (TPP+) accumulates in the mitochondrial matrix as a function of the mitochondrial membrane potential.

The TPP+ electrode is an ion selective electrode (ISE). The signal [V] of the TPP+ electrode is linearly dependent on the logarithm of free [TPP+].

The ISE system (OROBOROS INSTRUMENTS) consists of separate measuring and reference electrodes. The ISE are inserted through the stoppers into two chambers of the Oxygraph-2k and connected to the potentiometric channels of the O2k-MultiSensor system, for simultaneous recording of oxygen and TPP+.

Development of a TPP+ electrode

Tetraphenylphophonium chloride

Figure 2: Calibration test of a TPP+ electrode performed in Oxygraph-2k chamber in MiRO6, 37ºC with stirring speed 750 rpm. The calibration solution (1 mM TPPCl in 100 mM KCl) was injected automatically with TIP2k. A calibration range 0.1 -10 µM TPP+ was covered in 17 steps at 5 min intervals, using data recording intervals of 1 s.

Table 1: Typical performance parameters of OROBOROS TPP+ electrodes. Values are calculated from non-amplified signal.The critical sensitivity is defined at 40 mV/decade [mV/dec]. At this sensitivity, the critical [TPP+] was 0.7 µM. The absolute value of drift was measured in all calibration runs in the range 2-5 µM TPP+.

A long-term study over 3.5 months To evaluate the long-term stability of 10 TPP+ electrodes without change of membranes with rinsing procedure necessary for removal of inhibitors and uncouplers: water, 99% ethanol, water

10 TPP+ electrodes were followed for 3.5 months after mounting a new membrane:

● one calibration run (Fig. 2) per electrode every 7-14 days ● 10 runs each electrode

Ethanol: 5 electrodes rinsed with water, 99% ethanol, waterWater: 5 electrodes rinsed with water only

Table 2: Performance parameters of TPP+ electrodes in the course of the study. Values shown as mean ± SD are calculated from non-amplified signal. Ethanol: electrodes rinsed with water, ethanol, water. Water: electrodes rinsed with water only, *P<0.05 vs. 1st run in the same group, #P<0.05 vs. water group in the same run.Ethanol group:One electrode needed a new membrane after 7th run

Summary Characteristics of the electrodes:1. High sensitivity: > 40 mV/dec for [TPP+] >0.7 µM

2. Low detection limit: < 0.2 µM TPP+

Long-term performance of the electrodes:1. The zero signal, sensitivity, and critical [TPP+] did not change significantly within 3.5 months (Table 2).

2. The response time increased slightly in the course of the study, there is a trend of a faster increase in response times of ethanol-rinsed vs. water-rinsed electrodes (Figure 5).

Conclusions

1. Improved performance parameters of the OROBOROS TPP+ electrode are the basis for application in mitochondrial physiology with high-resolution respirometry.

2. The low detection limit allows for using low [TPP+] to avoid inhibition of mitochondrial respiration.

3. Rinsing with ethanol is possible, the same membrane can be used on a TPP+ electrode for many experiments. The membranes can be changed at intervals to ensure high performance.

Refferences:

1. Buck RP, Lindner E (1994) Recommendation for nomenclature of ion-selective electrodes (IUPAC recommendations 1994). Pure Appl. Chem. 66: 2527-2536.

Acknowledgement:

This work was supported by FEMtech, a program of the Federal Ministry for Transport, Innovation and Technology (BMVIT) to promote equal opportunities in research and technology.

Figure 3: The performance parameters of a TPP+ electrode over 3.5 months. a) Non-amplified raw signal [V] as a function of log[TPP+]. The parameter of a linear fit slope [mV/dec] represents the sensitivity of ISE. b) Sensitivity [mV/dec] of non-amplified signal (calculated for each point) as a function of log[TPP+]. The red arrow shows the detection limit of 0.2 µM, the black arrows show critical [TPP+] of 0.7 µM (see Table 1).

0

10

20

30

40

50

60

70

80

1.00E-07 1.00E-06 1.00E-05

[TPP+] M

1th

2nd

3rd

4th

5th

6th

7th

8th

9th

10th

-0.220

-0.200

-0.180

-0.160

-0.140

-0.120

-0.100

-0.080

1.00E-07 1.00E-06 1.00E-05

V

[TPP+] M

1th

2nd

3rd

4th

5th

6th

7th

8th

9th

10th

0.2 µM

Slope [mV/dec]

Design

Figure 4: Evaluation of the response time of a TPP+ electrode. a) 1st calibration run b) 10th calibration run

Results

Figure 5: The response time as a function of number of runs. a) electrodes rinsed with water, 99% ethanol, water b) electrodes rinsed withwater only. The response time was defined as the time after which the slope of the raw signal achieved the value -0.004 mV/s (IUPAC recommendation [1]), measured at the calibration step from 0.8 to 1 µM TPP+. The number of data points for time derivation was 10.

a) b)

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

response time (s)

T84

T89

T92

T96

T97

n of rinsing

Ethanol - rinsed electrodes

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

response time (s)

T86

T88

T93

T94

T95

n of rinsing

Water - rinsed electrodes

a)

b)

a)

b)

Figure 1: Set with 2 TPP+ and 2 reference electrodes

Range [h:min:s]: 1:45:002:20:002:02:301:45:001:27:301:10:000:52:300:35:00

pX C

alib

rate

d (Z

)

-0.12

-0.144

-0.168

-0.192

-0.216

Slop

e pX

(Z) [

mpX

/s]

0.008

0.004

0

-0.004

-0.008

2e-7 4e-7 5e-7 6e-7 8e-7 1.0e-6 1.2e-6 1.4e-6 1.6e-61e-7 2.5e-62e-6 3e-6 4e-6 5e-6 6.98e-6 9.96e-601TIP startP001 TIP end TIP startP001 P002 P003 P004 P005 P006 P007 P008 P009 P010 P011 P012 P013 P014 P015 P016 TIP end

pX R

aw S

igna

l (Z)

[V]

Range [h:min:s]: 0:02:001:27:001:26:401:26:201:26:001:25:401:25:201:25:00

pX C

alib

rate

d (Z

)

-0.154

-0.156

-0.158

-0.16

-0.162

-0.164

Slo

pe p

X (Z

) [m

pX/s

]

0.008

0.004

0

-0.004

-0.008

21P006

pX R

aw S

igna

l (Z)

[V]

Range [h:min:s]: 0:02:002:30:202:30:002:29:402:29:202:29:002:28:40

pX C

alib

rate

d (X

)

-0.14

-0.142

-0.144

-0.146

-0.148

Slo

pe p

X (X

) [m

pX/s

]

0.008

0.004

0

-0.004

-0.008

01P006

pX R

aw S

igna

l (X)

[V]

Features of the OROBOROS TPP+ electrodeISE membrane: + simple mechanical mounting

ISE membrane composition:

+ optimized to increase signal stability

and minimize side effects by chemical

substances such as ethanol and ADPHigh quality inner electrode: + signal stability

PVDF electrode housing:+ minimal O2 storage and diffusion

+ minimal carry-over of inhibitors

Integration into the O2k

chamber :

+ minimal oxygen diffusion

Calibration Rinsing Raw signal Sensitivity Critical [TPP+] |Drift| Response

 test  group @ 0 TPP+ @ 1-3 µM TPP+ @ 40 mV/dec of raw signal time[mV] [mV/dec] [µM] [mV/s] [s]

1st Ethanol (5) -199 ± 10 51 ± 4 0.9 ± 0.5 0.0007 14 ± 3Water (5) -213 ± 18 (3) 52 ± 4 1.0 ± 0.4 0.0006 16 ± 6

5th Ethanol (5) -200 ± 14 56 ± 6 0.6 ± 0.3 0.0004 49 ± 10*#

Water (5) -199 ± 8 54 ± 3 0.7 ± 0.2 0.0002 28 ± 11

10th Ethanol (4) -189 ± 18 54 ± 7 0.9 ± 0.4 0.0006 52 ± 19*Water (4) -198 ± 12 55 ± 5 0.4 ± 0.2* 0.0003 36 ± 14*

[V]

Raw signal @ 0 TPP+ Sensitivity @ 1-3 µM TPP+ Critical [TPP+] @ 40 mV/dec |Drift| of raw signal[mV] [mV/dec] [µM] [mV/s]

Mean ± SD -196 ± 14 53 ± 5 0.7 ± 0.3 0.0007n 96 99 82 99