Figure 1. Absorption spectra of the pH-sensitive dye.
* Mr. Brechignac is now with C.E.A. (France).
Résumé
La détermination de concentrations en solutés de
fluides biologiques par des sondes opto-chimiques fait appel à des
développements récents dans l'utilisation de fluorophores
immobilisés de manière stable dans ou sur des matrices
polymériques en contact avec le fluide et à la mesure optique de
l'intensité de luminescence. Des prototypes de senseurs pour
l'oxygène, le pH et le dioxide de carbone en solution ont été
developpés. Ces senseurs présentent la particularité
nouvelle de ne pas contaminer le milieu biologique, ne comportant
pas d'électrolytes, ni d'en modifier la composition car ne
consommant pas les espèces mesurées.
Contractors: Joanneum Research (A), DBA (D) and AEA (UK)
Funding: Funded jointly by ESA's Basic Technology Research Programme and the Esprit Programme of the European Communities
The control of proton activity and of major metabolic chemicals like oxygen and carbon dioxide is a classical and fundamental method for optimising growth conditions of biological organisms. The changes in their concentration are also commonly used to indirectly assess the biological activity correlated to the consumption or production of these analytes.
For life sciences experimentation in space, an additional constraint to the selection of a suitable measuring instrument for these analytes is the very small volume of fluid available for measurement, which may be either a sample or even the content of the miniature bioreactor itself. It is therefore essential that the measurement induce negligible perturbation to prevent modification of the medium and to avoid introducing a bias into the calculations of the corresponding rates and yields. For these reasons, classical instruments in biotechnology, like the Clark electrode for oxygen and the glass-bulb pH electrode are usually not optimum for life science experiments in space.
On the other hand, the simple infra-red absorption method for gaseous CO(2) (carbon dioxide) is not practical for determining dissolved CO(2) in small volumes of liquid because it presupposes the existence of a chemical equilibrium between the phases, which is not necessarily achieved, and it requires difficult calculations for correcting the effects of partial degassing. Opto- chemical sensors have the potential to overcome these major limitations.
The oxygen sensor, on one hand, and the pH and CO(2) sensors on the other are governed by different concepts. Measurements made by optical oxygen sensors depend on the principle of fluorescence quenching. Because of energy transfer at molecular level, through contact between the excited fluorophore and oxygen, the excited dye molecule returns to the ground state without the emission of light. The observed intensity of the fluorescence is a function of the oxygen concentration. The concept selected for the oxygen optrode is based on the formation, on a transparent rigid optical window, of a film of gas-permeable polystyrene/silicone co-polymer containing the fluorophore. The film is optically protected by a black silicone layer to prevent interference by ambient light and intrinsic sample fluorescence. The biological fluid is maintained in contact with the black, gas-permeable, silicone layer and fluorescence is measured on the opposite side, through the optical window.
For the pH optrode, a pH-sensitive dye is covalently immobilised on a cellulose triacetate hydrophillic polymer supported by a 100 micron polyester foil. In contact with a sample solution, the colour of the dye-layer changes from blue to yellow when the pH value is increased from 5 to 10. This change is reversible and the pH can be determined by measuring transmittance at a suitable wavelength. Figure 1 shows the absorption spectra of the dye selected for the pH sensor for various pH values. It can be seen that the optimal wavelength for detection is 597 nm, where the variation of absorption with change in pH is strongest.
Figure 1. Absorption spectra of the pH-sensitive dye.
The optical CO(2) sensor is also based on changes in absorptivity of a pH-sensitive dye. The indicator is dissolved in a thin layer of ethyl cellulose as an ion pair with a quaternary ammonium which acts as a phase-transfer catalyst. The proposed reaction mechanism in response to a change in concentration of CO(2) involves carbonation/decarbonation of the quaternary ammonium coupled with protonation/deprotonation of the dye inducing a shift in the absorption spectra. In order to prevent leaching of the dye and interference by pH, the indicator layer is protected by a gas-permeable silicone coating.
Prototype optrodes have been developed for all three analytes and tested under real laboratory conditions while connected to a bioreactor during the Bioprocessing Space Technology study.
The oxygen optrode (Figure 2) comprises a ruthenium phenantroline-based oxygen-sensitive film excited at 480 nm by a pulsed blue light-emitting diode (LED). The high intensity fluorescence, whose maximum is at 600 nm, is filtered by a longpass glass filter and detected by a silicon photodiode. The optrode contains a flow-through cell for the biological fluid and is temperature-controlled to prevent signal shift due to the thermal dependence of both the maximum fluorescence intensity and oxygen sensitivity.
Figure 2. An optrode for sensing oxygen gas.
The general optical design for the pH and CO(2) sensors are similar; a flow-through cell is covered with the analyte sensitive film and illuminated by a LED. The light from the source passes through the liquid sample and two distinct zones of the film, not-stained and dye-containing respectively. The light signals pass respectively through the measurement path and the reference path, which are similar and symmetrical. The intensities of both signals are measured by matched silicon photodiodes.
For example, in the case of the pH sensor (Figure 3) an amber LED with peak emission at 592 nm and spectral half-width of 15 nm, is used and the signal is measured through interference filters, whose transmission is maximum at 589 nm and whose half-width is 10 nm. The housing itself is made from Teflon and the complete head can be wet heat-sterilised by conventional means.
Figure 3. A complete optical pH sensor. The indicator film shows
a yellow to blue shift.
The performance of this prototype has been compared, under real laboratory conditions, against standard techniques and found to be superior in general, and certainly in the case of the oxygen optrode. An example of this was an independent trial in which the oxygen optrode was used for breath analysis under clinical conditions. The simplicity and excellent characteristics of these sensors made possible a direct measurement of the primary gas flow, whose results are given in Figure 4.
Figure 4. Instantaneous oxygen concentration measured by a
clinical breath analyser equipped with an oxygen optrode, using
CO(2) as a reference.
Opto-chemical sensors for oxygen, carbon dioxide and pH have been developed and tested. Their use brings advantages over classical methods, particularly because of their non-invasive sensing of the composition of the medium and their suitability for measurements of oxygen and carbon dioxide in either the gaseous or liquid phase. The rapid on-going advances in the development of sensing materials has brought significant improvements to the sensors' characteristics, even during the time frame of this particular study, suggesting potentially rapid progress in practical applications.
Preparing for the Future Vol. 6 No. 1