1. Electrode measuring principle
ABL800 FLEX
Electrode measuring principle
RTC, December 2004
Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: ,

2. Agenda Parameters General construction Measuring principles
The potentiometric measuring principle
Reference electrode
pH electrode
Electrolyte electrodes
pCO2 electrode
The amperometric measuring principle
pO2 electrode
Metabolite electrodes
Summary
Electrode signal updating

3. Location
The electrodes are placed in the electrode modules easily accessible under a window on the front of the analyzer

4. Parameters pH, pCO2, pO2, cK+, cNa+, cCa2+, cCl– , cGlucose, cLactate
There are 10 electrode slots in the two electrode modules
One electrode slot for the reference electrode
Nine other electrode slots available for the following:
pH, pCO2, pO2, cK+, cNa+, cCa2+, cCl– , cGlucose, cLactate

5. Electrode modules Aspiration Continues
Blood aspirated into Lyt/Met module, rinse solution removed
Measurement of pH/BG begins
Probe moves back up
Remove syringe, close inlet

6. General construction The term ‘electrode’ refers to whole sensor unit
Cordless – limiting electrical noise
Amplifiers positioned in each module to amplify the electrical signal
Preamplifier
Electrical contact
Color-coded ring
Electrode jacket (removable)
Electrolyte solution
Membrane

7. Two measuring principles
The potentiometric measuring principle is used for
The amperometric measuring principle is used for
pH, pCO2, cK+, cNa+, cCa2+, cCl–
pO2, cGlucose, cLactate

8. The potentiometric measuring principle
Electrodes measure a change in voltage due to a change in ion concentration across a membrane
pH, pCO2, cK+, cNa+, cCa2+, cCl– V
Voltmeter
Reference electrode
Electrode
Electrolyte solution
Electrolyte solution
Electrode
Electrode
Sample
Liquid junction
Membrane

9. ( ) E = E - E + E Measuring a potential
Each link in the circuit exhibits a potential
The only unknown potential is the one between the membrane and the sample, ESample
The potential of the whole circuit is measured, Evoltmeter, and the unknown potential can be calculated: ( ) E = E - E + E
Sample
voltmeter
Ref pH

10. Reference electrode
Provides a stable, fixed potential against which other potentials can be measured
The potential at the reference electrode is not altered by sample composition
A stable, fixed potential is obtained by maintaining constant conditions
The reference electrode is used in the measurement of pH and the electrolyte parameters

11. Reference electrode – E1001
Electrode contact
Electrolyte jacket - The rubber ring seals the electrode in the jacket to prevent evaporation or leakage of the electrolyte solution
Electrolyte solution
- A 4M sodium formate (HCOONa) adjusted to pH 5.5 with hydrochloric acid. Acts as salt-bridge solution that maintains an electrochemical contact between the coated Ag wire and the sample.
Ag rod coated with AgCl
Membrane
Consists of three separate membrane layers:
- Inner: Limits diffusion through the membrane and stabilizes the whole membrane system
- Middle: Prevents protein interference
- Outer: Reduces the interchange of sample or rinse solution and HCOONa solution

12. pH electrode
Ag rod coated with AgCl - provides electrical contact to inner buffer solution - Ag/AgCl equilibrium maintains a stable potential
Air bubble - allows expansion of solution at 37 °C
Inner buffer solution - has a constant and known pH
pH-sensitive glass membrane - changes in potential only due to changes in pH of sample

13. pH-sensitive glass membrane
Inner buffer solution with known and constant pH
air bubble
Ag/AgCl H+
Sample
Glass membrane
Constant exchange of H+ ions
Varying exchange of H+ ions – dependent on sample pH
The difference in potential across the glass membrane arises when there is a change in charge balance at the membrane.
A difference in ion exchange on either side of the membrane occurs if the H+ concentrations (and therefore pH) on both sides are unequal. The number of positive and negative ions are no longer equal, so the potential difference across the membrane changes.
In other words, the sample concentration of H+ will trigger the difference in ion exchange and cause a change in the potential across the membrane.

14. Nernst equation, pH electrode
Varying exchange of H+ ions between sample and glass membrane gives rise to a change in potential (voltage)
Change in potential can be converted to a change in concentration by the Nernst equation:
The analyzer automatically converts activity (effective concentration) into concentration K E = E +
log a n H +
E = Measured potential E0 = Standard potential K = Temperature-dependent constant n = Charge on ion (+1) aH+ = Activity of H+

15. Electrolyte electrodes
Ion-selective-membrane electrodes
The main difference between the electrolyte electrodes is the selectivity of the membranes with respect to which anions and cations can pass and how. The membranes are selective for a single ion species only.
The membrane potential is determined against the Reference electrode

16. Electrolyte electrodes design
Ag rod coated with AgCl - provides electrical contact to buffer solution - Ag/Ag+ equilibrium maintains a stable potential
Electrolyte solution
Porous pin - absorbs electrolyte solution - holds PVC membrane
PVC membrane - contains a K+ ion exchanger
The three ions are measured with ion-selective electrodes whose sensing element is a PVC membrane containing either a potassium/calcium or a chloride-neutral ion carrier
Cellophane membrane - protects PVC membrane - prevents protein build-up - held in place by white plastic gasket
Potassium is used as an example

17. Ion sensitive membranes
constant K+ exchange - constant potential
PVC membrane containing specific ion-carrying molecules
Electrolyte Solution K+ K+ K+ K+ K+ K+ K+ K+ K+
Cellophane membrane K+ K+ K+ K+
Sample K+ K+
varying K+ exchange - changing potential - dependant on cK+ in sample
Other electrolyte electrodes have different ion carriers
No ion-specific PVC membrane in Na electrode; instead the pin is made of special ion-carrying material

18. pCO2 electrode
Consists of a pH electrode and an internal reference electrode in one complete unit
Reference electrode – Ag/AgCl
Electrolyte solution
- The bicarbonate electrolyte solution also contains glycerol to prevent collection of air bubbles in the jacket, thus improving electrode stability
pH electrode – Ag/AgCl
- Inner solution with known and constant pH
Air bubble
This is the electrode contact seen from above. Note the thick gold ring which is the contact for the reference system in the electrode. Be careful when membraning the electrode, not to accidentally smear the gold ring with glycerol (from the electrolyte solution in the jacket onto your fingers etc.) and thereby cause electrode instability.
pH-sensitive glass membrane
Silicone membrane on nylon net - Silicone is permeable to CO2 - Nylon net traps electrolyte solution
Electrode from above
- The gold ring is the contact of the reference system in the electrode

19. [ ] pCO2 measuring method K E = E + log a n
Glass membrane
CO2 permeates the silicone membrane and dissolves in the electrolyte solution trapped in the nylon net
Carbonic acid is produced, a pH change occurs
This pH change is measured by change in potential at the pH-sensitive glass membrane.
The potential reading is converted into a pH value by the Nernst equation.
The pH value is related to the partial pressure of CO2 in the sample by the Henderson-Hasselbalch equation
Nylon net with electrolyte solution
Silicone membrane
Sample + - CO + H O H CO H +
HCO 2 2 2 3 3 K E = E +
log a n H +
The nylon net reinforces the silicone membrane and serves as a spacer in order to trap a layer of electrolyte between the membrane and the glass tip of the electrode.
The dissolved CO2 from the sample diffuses across the membrane into the thin layer of bicarbonate electrolyte solution until equilibrium is reached.
The release of H+ ions changes the H+ concentration and thus the pH of the solution on one side of the pH-sensitive glass membrane [ ]
HCO - pH = pK +
log 3 a a p CO CO 2 2

20. The amperometric measuring principle
Electrodes measure the current produced during an electrochemical reaction at an electrode
pO2, cGlucose, cLactate
Applied voltage
Ammeter - measures current
Cathode - reduction
Anode - oxidation
Electrolyte solution - buffered
Sample

21. pO2 and metabolite electrodes
The pO2 and metabolite electrodes are designed to measure the current produced during an electrochemical reaction
The flow of electrons (the current) is proportional to the concentration of the substrate (the pO2/Glu/Lac)
An electrochemical reaction: - a reaction where electrons are transferred
A+ + e-  A

22. pO2 electrode design
The pO2 electrode is designed to measure the current produced during an electrochemical reaction
Anode (+) – Ag rod coated with AgCl - oxidation of Ag
Electrolyte solution - provides electrical contact between anode and cathode
Cathode (–) – Pt wire encased in glass - reduction of O2
Platinum black catalyst (thin black layer) conversion of H2O2 to H2O and O2
Polypropylene membrane - permeable to O2

23. pO2 measuring method 2H O + ® Ag Ag+ e- +
Pt cathode
Oxygen from the sample diffuses across the membrane into the electrolyte solution and is reduced at the cathode
Reduction of O2:
The H+ ions come from the electrolyte solution and e- comes from the silver anode
H2O2 produced from O2 not completely reduced
This is then immediately decomposed and catalyzed by Pt black to O2 which again is reduced at the cathode
To complete the electrical circuit, Ag is oxidized at the silver anode
Electrolyte solution (contains H+)
Pt black
O2 -permeable membrane
Sample O
4H+
4e- 2H 2 + 2H O + 2 Pt Ag
Ag+ e- +

24. pO2 measuring method
The reduction of oxygen produces a flow of electrons the size of the current, I, which is proportional to the amount of oxygen and measured by the ammeter in pico Ampere (pA) I
pO2
I = Sens(pO2) x pO2 + I pA
The current measured during this process is automatically converted to a pO2 value by the analyzer

25. Metabolite electrodes
The electrode is an amperometric electrode consisting of a silver cathode with a AgCl reference band and a platinum anode, all protected by an electrode jacket filled with electrolyte solution
At the tip of the jacket is a multilayer membrane
The electrodes are identical in design

26. Metabolite electrodes design
Glucose and lactate electrodes are identical in construction, the major difference is the enzyme in the membrane layer
Cathode – Ag rod coated with AgCl - reduction of Ag+
Electrolyte solution - provides electrical contact between anode and cathode
Anode – Pt wire - oxidation of H2O2
Differences are the enzymes and the outer membranes
Multilayer membrane - outer layer permeable to glucose/lactate - middle enzyme layer (production of H2O2) - inner layer permeable to H2O2
Glucose is used as an example

27. The metabolite membrane design
Multilayered membrane
outer layer permeable to glucose/lactate
middle enzyme layer (production of H2O2)
inner layer permeable to H2O2
Inner layer
Outer layer
Middle layer

28. Metabolites – measuring method
Glucose/lactate molecules are transported across the outer membrane to be converted by the enzyme to form H2O2
The H2O2 is then oxidized to oxygen and electrons, a current, which is measured by the ammeter
Glucose + O
Gluconic a
cid H 2
Lactate
Pyruvate H O
2H+ +
2e- 2
The amount of current produced is proportional to the amount of H2O2, which in turn is directly related to the amount of glucose or lactate in the sample

29. Metabolite membrane - outer layer
Has pores of well-defined density and diameter to limit the amount of glucose/lactate entering the enzyme layer
Outer side is treated to prevent protein build-up that could block the pores
Glucose/lactate
Inner layer
Outer layer
Middle layer
Outer layer
Red blood cells
- There has to be a limitation with respect to how much lactate will reach the lactate oxidase membrane since there is a limit of how much the enzyme will convert. If this was not achieved by the outer membrane we would have to dilute the sample
- The poredensity is lower than the glucose
- The membrane is 10 em thick
Blood sample
The sensor is not affected by hematocrit due to the outer low-porous membrane

30. Metabolite membrane – middle enzyme layer
The enzyme glucose or lactate oxidase is immobilized between the outer and inner layer
The enzymes only catalyze the following reactions:
Inner layer
Outer layer
Middle layer
Glucose + O
Gluconic a
cid H
Lactate
Pyruvate 2
The O2 for the reactions is supplied by the Rinse solution and stored in the outer membrane. During rinse cycles, when air and rinse solution are flushing the measuring chamber, the outer membrane gets topped up with a fresh supply of oxygen.
- Only 1–2 em thick
- There is enough physically dissolved in the outer membrane, O2 is also “recycled” from the platinum wire,
- The enzyme is not very stable at 37 C so it has to be immobilized in a way that will increase the stability at that temperature
- We have a unique way of immobilizing the enzyme, giving the very long lifetime of the membrane. This is a big problem on comp. system since their lactate sensor has a lifetime of 7–14 days.
O2 is supplied from the outer membrane to make the process independent of the O2 content in the sample
Long lifetime of membrane
No dependency on sample’s oxygen content

31. Metabolite membrane – inner layer
The H2O2 from the enzyme reaction is transported across the inner membrane to the Pt anode for oxidation
The inner membrane is an interference-limiting membrane. In less than 30 seconds the H2O2 has diffused through the membrane and reached the Pt anode.
Electrochemical substances that can interfere are delayed, e.g. Paracetamol-4-acetamidophenol, and will not pass through the membrane during the 30-second period
Inner layer
Inner layer
Outer layer
Middle layer
Enzyme layer
- Oxidation: liberation of electrons
- With the potential applied other substances than hydrogenperoxide can be oxidized so a limitation is needed
- The inner membrane almost works as a physical filter, keeping substances away from the electrode
- Hepes will be oxidized, and can then be used as a control, which is done during CAL2
- The buffer is an imidazol buffer, which will keep pH within range, despite development of H+
E.g. Paracetamol-4-acetamidophenol

32. Summary The following parameters are measured:
pH, pCO2, pO2, cK+, cNa+, cCa2+, cCl– , cGlucose, cLactate
Two measuring principles
ion-selective electrodes for pH, pCO2 and electrolytes
measurement of current produced from reactions of O2, Glu and Lac
Multilayer interference-limiting membranes for glucose and lactate electrodes
Long lifetime of membrane
No dependency on sample’s oxygen content
No interference from commonly known substances

33. Electrode updating
Electrode signals are registered at second intervals during calibrations and measurements
Updating starts when the sample/calibration solution has reached the measuring modules
Duration and number of updatings (30) of electrode signals are predetermined
The stability of the signals are evaluated before a calibration or a measurement result is accepted

34. pH electrode signal updating
7.70
Calibration
pHupd.30 - pHupd.20  0.005
7.60
7.50 p1 pH
7.40
p30
p20 p21
7.30
7.20 5 10 15 20 25 30
Seconds

35. pO2 calibration electrode signal updating
pO2 upd.30 - pO2 upd.24  0.80
18.80
18.75 p1
18.70
pO2
18.65
p30
18.60
p16
p24 p25
18.55
18.50 5 10 15 20 25 30
Seconds

36. pCO2 calibration electrode signal updating
pCO2 upd.30 - pCO2 upd.24  0.40
5.50
5.40
5.30
pCO2
5.20
p16
p24 p25
p30
5.10 p1
5.00
4.90 5 10 15 20 25 30
Seconds

37. Radiometer Training Center, December 2004
Radiometer Medical ApS, Åkandevej 21, DK-2700 Brønshøj, Tel: ,