General
The Stern-Volmer-equation
A relation exists between the oxygen concentration in the sample and the luminescence intensity as well as the luminescence lifetime which is described in the Stern-Volmer-equation (1). Here, τ0 and τ are the luminescence decay times in absence and presence of oxygen (l0 and l are the respective luminescence intensities), [O2] the oxygen concentration and KSV the overall quenching constant
l: Luminescence intensity in presence of oxygen
l0: Luminescence intensity in absence of oxygen
τ: Luminescence decay time in presence of oxygen
τ0: Luminescence decay time in absence of oxygen
KSV: Stern-Volmer constant (quantifies the quenching efficiency and therefore the sensitivity of the sensor)
[O2]: oxygen content
(A) Luminescence decrease in the presence of oxygen. (B) Stern-Volmer plot.
Indicator dyes quenched by oxygen are, for example polycyclic aromatic hydrocarbons, transition metal complexes of Ru(II), Os(II) and Rh(II), and phosphorescent porphyrins containing Pt(II) or Pd(II) as the central atom.
It is important to mention that the quenching effect is highly specific for molecular oxygen. Effects of varying pH-values, ionic species and other solutes in the sample are avoided by embedding the oxygen sensitive indicator in an ion-impermeable matrix.
In contrast to the amperometric oxygen electrodes, optodes are in a thermo-dynamical equilibrium and not in a steady state. Therefore, no oxygen is consumed during the measurement and the signal is independent from changes in flow velocity. The calibration function of oxygen optodes only depends on the composition of the sensing layer. So it is possible to design sensors with a tailored oxygen sensitivity.
How does an oxygen microsensor work?
The principle of measurement is based on the effect of dynamic luminescence quenching by molecular oxygen. The following scheme explains the principle of dynamic luminescence quenching by oxygen.
Principle of dynamic quenching of luminescence by molecular oxygen:
(1) Luminescence process in absence of oxygen
(2) Deactivation of the luminescent indicator molecule by molecular oxygen
The collision between the luminophore in its excited state and the quencher (oxygen) results in radiationless deactivation and is called collisional or dynamic quenching. After collision, energy transfer takes place from the excited indicator molecule to oxygen which consequently is transferred from its ground state (triplet state) to its excited singlet state. As a result, the indicator molecule does not emit luminescence and the measurable luminescence signal decreases.
What is the difference between tapered and flat-broken sensor tips?
All the sensors mounted in different housings are available with two different glass-fiber tips, (A) a < 50 µm tapered tip and (B) a 140 µm flat-broken tip.
Advantages of micro-optodes with a tapered tip high signal intensity
- high spatial resolution (< 50 µm)
- very fast response times (up to 1 s)
Disadvantage of micro-optodes with a tapered tip:
- fragile display photobleaching
Advantages of micro-optodes with a flat-broken tip:
- more photostable than tapered ones
- long-term stable
- tougher
Disadvantages of micro-optodes with flat-broken tip:
- response times in the scale of 40 s
- lower signal intensity
Can the same instrument be used for pH, oxygen or CO2 sensors?
No!
All instruments - except SDR and SFR - are either oxygen, pH or CO2 meters. They can only be used for the respective parameter. The SDR can detect either oxygen or pH, depending on the SensorDish used. The SFR measures oxygen and pH simultaneously.
Can I use the same instrument for microsensors, non-invasive sensors and invasive probes?
Our microsensors for pH and oxygen are based on 140 ?m fibers. Therefore, they can not be used with transmitters compatible with non-invasive sensors and invasive probes as those are based on a 2 mm fiber. (For detailed information please refer to our product matrix.)
In which vessels can I integrate a sensor spot?
The sensor spots can be integrated in any kind of transparent, non-fluorescent vessel (glass or plastic). Slightly milky vessels are also appropriate. The thickness of the vessel wall should not extend 8 mm / 0.3 inch (sensor spot O2) and 4 mm / 0.15 inch (sensor spot pH), respectively.
Examples: shake flasks, T-flasks, spinner flasks, tubes, bottles, bioreactors, plates, Petri dishes, etc.
Where do I need sensor spot sizes different from the common diameter of 5 mm (approx. 0.2 inch)?
A bigger diameter is needed if the thickness of your vessel wall is larger than 2 mm (approx. 0.08 inch): The excitation light coming out of the light guide (POF) hits a larger area.
For spots with a diameter below 5 mm (approx. 0.2 inch), please contact us.
Which substances can interfere with the measurement?
- High concentration of fluorescent substances can interfere. Please contact us for solutions solving this issue.
- Our sensors are made for aqueous solutions. Higher concentrations of organic solvents should be avoided. Oxygen sensors do even stand pure ethanol.
- pH and CO2 sensors show cross sensitivity for ionic strength (osmolarity). This can be compensated by a one point re-calibration.
Do the sensors work in turbid solutions?
Can I measure inside a tissue?
How do I import data into excel from a text file containing measurement data?
First of all, please note that the described procedure is not software specific!
1. Open excel.
2. Go to the main menu and click File > Open or "Ctrl+O".
3. Choose the text file from the dialog box opened.
4. This will lead to a wizard; in step 1 activate the checkbox "Delimited" as shown below.
5. In a second step, activate the checkboxes "Tab" and "Semicolon" under the "Delimiters".
6. As final step, press the "Finish" button, which shows all data from the text file.
Is the glue or the spot biocompatible - which tests were done? Are there any studies on leachables and extractables?
The combination of pH sensor spot HP5 and glued oxygen sensor spot PSt3 was tested according to USP class VI. Test reports are available on request.
Which glue can I use?
Can I sterilize the sensors?
Compensation of pressure/salinity?
Our instruments do NOT have an automatic compensation for salinity or pressure changes. If a compensation for salinity or pressure changes is necessary, please refer to the compensation formulas given in the appendix of the corresponding manual.
What is the time of delivery?
Delivery time for items in stock should be a maximum of 10 working days for processing plus 2 days for shipment. The precise delivery time is stated on the order confirmation.
Which lenghts of the light guides / POFs (polymer optical fiber) are available?
We offer the POFs (polymer optical fiber, light guide) in different lenghts of up to 25 meters (approx. 68 feet).
Oxygen Sensors
What are the response times for the oxygen sensors based on the 2 mm fiber like non-invasive oxygen sensors and oxygen probes?
The response time is defined as t90 (90 % of the signal is reached within this period).
- 8 s for the gas phase, 30 s for the liquid phase without optical isolation of the sensor (NOP order code)
- 10 s for the gas phase, 40 s for the liquid phase with optical isolation of the sensor (YOP order code)
Which side of the sensor spot should face the medium?
In case of optical isolated dissolved oxygen (DO) sensors (code - YOP) the black side of the sensor spot has to be in contact with the medium, the pink side has to be glued to the vessel wall.
In case of DO sensors without optical isolation (code - NOP) the shiny side has to be glued to the vessel wall.
How can I prepare the calibration solutions cal0 and cal100?
Preparation of calibration solution 0 (oxygen-free water)
- For preparing calibration solution 0 (oxygen-free water) add 1 g sodium sulfite (Na2SO3) and 50 µL cobalt nitrate solution (Co(NO3)2, 500 mM to the vessel and label it cal 0.
- Dissolve the salt mixture in 100 mL water. The water becomes oxygen-free due to a chemical reaction of oxygen with Na2SO3. Additional oxygen, diffusing from air into the water, is removed by surplus of Na2SO3. Cobald is used as catalyst to accelerate and complete the reaction of sulfite with oxygen.
- Close the vessel with a screw top and shake it for approximately one minute to dissolve Na2SO3 and to ensure that the water is oxygen-free.
Keep the vessel closed after calibration with a screw top to minimize oxygen contamination. The shelf life of cal 0 is about 96 hours provided that the vessel is closed with the screw top.
Prepartion of calibration solution 100 (air-saturated water)
- For preparing calibration solution 100 add 100 mL water to a suitable vessel and label it cal 100.
- To obtain air-saturated water, blow air into the water using an air-pump with a glass-frit (airstone, normally used in aquariums), creating a multitude of small air bubbles, while stirring the solution. The bubbling also ensures mixing in the glass so that oxygen gradients do not form in the water.
- After 20 minutes, switch off the air-pump and stir the solution for another 10 minutes to ensure that the water is not supersaturated.
How do I verify whether the sensor is giving correct readings?
If you want to prove the performance of the last measurement, please verify the calibration values by inserting the sensor in the 'cal 0' and 'cal 100' calibration standards once you finished your measurement. If the device indicates 0 % air-saturation measuring the 'cal 100' standard, the sensor worked perfectly during the entire measurement.
What factors will affect the oxygen reading?
Temperature (which is already compensated using the temperature probe), salinity and pressure affect the oxygen sensor. The two latter parameters are easily compensated for by simple formulas which are common for all sensor types PSt1, PSt3 and PSt6.
I use a glass vessel with an integrated autoclavable oxygen sensor. How do I remove this sensor?
In order to guarantee optimal sensor characteristics, PreSens uses a very strong glue. It is not possible to fully remove this sensor.
How does temperature effect the oxygen measurement?
Like all physical parameters the fluorescence decay time and the Stern-Volmer quenching constant (= collision frequency) are dependent on temperature. The temperature behaviour of these parameters is known and compensated. Beside this, the solubility of oxygen in water is temperature-dependent and can be described using the Bunsen absorption coefficient α(θ) and the oxygen partial pressure p(O2).With increasing temperature, the solubility of oxygen in water decreases.
cS(p,θ): temperature-dependent solubility of oxygen in water, given in (cm3(O2)/cm3)
p(O2): oxygen partial pressure
pN: standard pressure
α(θ): Bunsen absorption coefficient, given in (cm3(O2)/cm3)
The following table gives oxygen solubilities in mg/L for temperature intervals of 0.1°C from 0 - 40°C. The calculated value for cs at a temperature of 20.0°C agrees with the tabulated value of 9.08 mg/L.
Example: cs (20.0°C) = 9.08 mg/L
How does salinity effect the measurement?
The solubility of oxygen in water is dependent on salinity, while the partial pressure and the % saturation of oxygen is not affected by changes in salinity. This means taht in absolute concentration a seawater sample will contain less oxygen than a freshwater sample at the same temperature although the partial pressure is the same.
The table below lists values of the concentration of dissolved oxygen at several temperatures in solutions with various chloride concentrations. Increasing the salt concentration leads to a decrease in oxygen solubility. This behaviour is characteristic for the solubility of many non-electrolytes - this phenomenon is known as salting-out effect.
Instead of chlorinity [Cl‾] - the amount of chloride in parts per thousand - which was used as a measure of the amount of salt in water, the term salinity is often used. If salinity if preferred as a measure of salt concentration, then the conversion from g/L can be readily made using equation 1.
S = 0.1805 [Cl‾] + 0.003 where S is the salinity in [%] or [g/1000g] (1)
The dependence of oxygen solubility on salt concentration can be obtained from equation 2
where θ is the temperature in °C, a - e are the coefficients used in equation 16 and p - t are new constants given in the table below. The values of theses new constants are obtained by fitting the polynomial to experimental dta int he ranges 0 ≤ θ ≤ 30 °C and 0 ≤ [Cl‾] ≤ 0.2 %. To obtain oxygen solubility from the Bunsen absorption coefficient, the same procedure as described previously is used.
Seawater has a typical salinity of 0.35 % (35g / 1000g) or a chloride content of about 0.19 %, and therefore meets the scope of both equations.
pH Sensors
Which side of the sensor spot should face the medium?
In case of pH sensors (HP5) the white side of the sensor spot has to be in contact with the medium, the yellow side has to be glued to the vessel wall.
How can I clean non-invasive pH sensors?
First of all, non-invasive pH sensors are designed as disposables. But you can rinse them carefully with water or buffers. Please do not use any organic solvents, tenside solutions or pH above 9.5.
Can I use Ethanol for disinfecting non-invasive pH sensors?
How to prepare buffers of constant ionic strength and calibrate optical pH sensors?
The most basic version is the application of a PBS buffer system with the following recipe for two solutions: Please put in a beaker glass the acid solution (A) together with an optical pH sensor mounted according to the respective manual and a calibrated pH electrode. Make a note of the pH (of the electrode) and the phase value (displayed at the pH transmitters). By adding basic alkali solution (B) you will set a new pH value and therefore produce subsequent the required different pH-phase results. Make sure that the achieved pH values cover the pH range of interest for your application.
Why does my pH sensor not work in tap water?
The minimum ionic strength of the samples is about 50 mM, the minimum buffer capacity is about 2 mM - both can not be reached in fresh water like tap water.
CO2
What is the operating principle of the CO2 sensor?
The carbon dioxide sensor is an optical-chemical sensor utilizing the acidic nature of CO2 for detection. It consists of a gas-permeable membrane in which a pH-sensitive luminescence dye is immobilized together with a buffer and an inert reference luminescent dye. CO2 permeating into the membrane changes the internal pH of the buffer. With this changes the luminescence of the pH-sensitive dye. Together with the inert reference dye internal referencing is made for detection of the luminescence lifetime of the sensor. The measurement signal detected by the pCO2 mini correlates to the partial pressure of CO2 ambient.
Can I measure CO2 also in a gas phase or only in liquid phase?
The carbon dioxide sensor was developed for measuring partial pressure of dissolved carbon dioxide in physiological solutions. Thus, the sensor membrane requires humidity for operation. However, under certain conditions it is also possible to measure CO2 in the gas phase. The CO2 sensor can be calibrated with CO2 containing gas mixtures of defined constant humidity for measuring CO2 in gas phase at the same humidity level thereafter. Changes in humidity will alter calibration.
What happens if the temperature changes while measuring?
The pCO2 mini is equipped with a PT1000 temperature sensor for measuring the temperature of the sample. The reading is then compensated for these temperature changes according to internal compensation algorithms.
Do I measure CO2 or HCO3-?
The sensor membrane is only permeable to CO2, ionic species like protons or HCO3- do not penetrate through the membrane. Thus, the sensor only responds to changes of pCO2.
Does the pH of the sample affect my reading?
pH does not affect the performance of the chemical-optical CO2 sensor. Of course, pH affects the pCO2 in the sample.
However, the sensor might be affected b volatile acids or bases, depending on low respectively high pH value.
SDR
Do the SensorDishes come sterile?
Which toxicity tests where done with the SensorDish?
They do not show cytotoxic or cytostatic influence to cell cultures according to DIN ISO 10993-5 (1999) and EN 30993-5 (1994).
Can I use the SDR with other formats than the 24-well plates?
The SDR can be used with 24- and with 6-well plates. It is not compatible to other formats like 12- or 48-well plates.
Do cells grow on the sensor inside the SensorDishes?
Due to its surface structure, cells generally do not grow on the sensor, but around it. However, the sensor does not disturb the cells in any way: Alamar Blue staining of chondrocytes under the fluorescence microscope. The yellow part with the red edge is the sensor spot. No dead cells (purple color) are visible, and no difference of the cell growth near the sensor compared to cells further away can be detected.
Why do I see peaks in the graphs of the SDR software (especially in the oxygen signal) when I open the incubator door / take the plates out of the incubator?
There are different reasons:
- Temperature effect: The signal of the optical sensor, especially the oxygen sensor, depends on temperature. If you open the incubator, the temperature changes, but the software uses the constant, user-defined temperature typed into the software for calculation. A temperature drop leads to decreasing oxygen / pH signals and vice versa.
- OxoDish & hypoxic conditions: Opening of the incubator door leads to an upward peak in oxygen because ambient air enters the hypoxic sample.
- HydroDish & CO2 atmosphere: Opening the incubator door leads to an upward peak because CO2 leaves the incubator. As the pH of the buffer for cell cultivation is regulated by the CO2 atmosphere in the incubator, this leads to a higher pH within the sample.
Sensor Plates
Is my microplate reader compatible to the SensorPlates?
Your microplate reader must be able to read out fluorescence instensity from the bottom side and must have the required filter pairs (HydroPlate: 485/540 nm and 485/620 nm; OxoPlate: 540/650 nm and 540/590 nm). For kinetics, it must be capable of measuring in the dual kinetic mode, changing the filter pairs for each measurement point.
The filters of my reader do not exactly match the combination in the disposable brochure. Nevertheless, can I use them?
SFR
Which types and sizes of shake flasks with integrated sensors are offered by PreSens?
PreSens offers 3 types of shake flasks with integrated sensors:
- Plastic flasks made of poly carbonate (PC):
PreSens offers plastic shake flasks with integrated oxygen and pH sensors. PreSens uses Corning flasks made of polycarbonate to manufacture the flasks with sensors. Sizes of 125 ml, 250 ml, 500 ml, 1000 ml and 2000 ml are offered. All offered sizes are available with and without baffles. - Glass flasks with oxygen sensor:
PreSens offers glass flasks with oxygen sensors only, as there is no autoclavable pH sensor available. These glass flasks can be autoclaved for about 50 cultivations. - Integration of oxygen sensors into your glass flasks:
PreSens also offers the integration of oxygen sensors into specific glass flasks. The customer sends his / her flasks to PreSens and PreSens integrates autoclavable oxygen sensors into these flasks. These flasks are autoclavable for about 50 times.
Can the customer integrate the sensors by himself/herself?
The customer can integrate the oxygen sensors by himself. This procedure is complex. PreSens only recommends the integration if it is not possible to send the glassware to PreSens. PreSens can not assure that sensors integrated by a third party are working properly.
Can I re-use the shake flasks with integrated sensors?
Plastic flasks with integrated sensors can only be used for one cultivation. They are not autoclavable.
Glass flasks with integrated oxygen sensor are autoclavable. They can be used for about 50 cultivations.
Does the SFR fit into my incubator/shaker?
The SFR is designed to fit into most incubators/shakers. Currently, drillings are integrated so that the system fits on most trays from Infors HT, Kühner, New Brunswick Scientific, Sartorius Stedim Biotech and Thermo Scientific. PreSens can supply attachment sets for other shakers on request.
