WO1995016052A1

WO1995016052A1 - Microbial optical sensors and methods

Info

Publication number: WO1995016052A1
Application number: PCT/US1994/014006
Authority: WO
Inventors: Otto S. Wolfbeis; Stanley M. Klainer
Original assignee: Fci-Fiberchem
Priority date: 1993-12-06
Filing date: 1994-12-06
Publication date: 1995-06-15

Abstract

A method and apparatus for measuring a variety of analytes is based on a biological cell culture, e.g., a yeast, bacteria or combinations thereof, and an optical chemical sensor with a species-sensitive indicator. Oxygen and carbon dioxide chemical sensors using yeast and Methylomas flagellata, respectively, are examples of sensors for measuring BOD and methane. The yeast metalobizes organic matter in a sample and consumes oxygen. The decrease in oxygen produces a measurable increase in signal from the oxygen detector by suppression of quenching of fluorescence of the oxygen sensitive indicator. The signal from the oxygen sensor can be used for quantifying BOD. The Methylomonas flagellata reacts with methane to yield CO2 which is measured by the carbon dioxide sensor. The signal from the carbon dioxide sensor can be related to methane concentration.

Description

MICROBIAL OPTICAL SENSORS AND METHODS

BACKGROUND OF THE INVENTION

The invention relates generally to optical chemical sensors, and more particularly to optical sensors for measuring species which undergo a chemical reaction with microbial materials such as yeast and bacteria which results in a measurable signal change. Biological oxygen demand (BOD) , a measure of the total amount of oxygen-demanding (i.e., biodegradable) organic matter present in water, as described in U.S. Patent Application No. 08/101,977, is a specific example of this type of sensor.

The development of compound, group or structure specific sensors, either optical or electrochemical, for small organic molecules and several inorganic compounds has proven difficult because of the lack of appropriate chemical reactions. Using target specific microbial layers, which result in a change that can be measured by an optical sensor, permits the requisite analysis to be made. For example, as shown in U.S. Patent Application No. 08/101,977, when the microbial layer is exposed to organic material, oxygen is consumed and the amount of oxygen used up can be measured by an optical oxygen sensor. The decrease in oxygen concentration can then be related to the concentration of biodegradable material present. For many other target specific microbial species, the loss of oxygen is also the measured species. Carbon dioxide and ammonia sensors are other examples of measuring devices which can be used with microbial reactions.

The use of an optical oxygen sensor is key to a device that can sensitively and accurately make many microbial measurements. Whereas it is possible to use the same approach using an electrode sensor, the results are quite different, especially at low analyte concentration ranges. Measuring oxygen consumption electrochemically is a severe problem in the case of low oxygen concentrations because the electrode itself consumes oxygen. Thus separating the oxygen used by the microbial reaction from that used by the electrode is difficult to impossible. When the analyte concentration is high, then the loss of oxygen due to the microbial reaction is much greater than that used by the electrode and measurements can be made, but an error is still introduced by the oxygen consumption by the electrode. Thus an alternative method, such as optical sensors, is desired.

A number of biosensors have been produced by using a biological transducer, e.g. an enzyme, which converts the analyte into a species for which an optrode exists. Moreno-Bondi, et al., "Oxygen Optrode for Use in a Fiber-Optic Glucose Biosensor," Anal. Chem. 1990, 62, 2377-2380, describes an oxygen sensor based on luminescent quenching of a ruthenium complex. Most importantly, optical oxygen sensors (in contrast to the electrodes cited above) do not consume oxygen. The complex is adsorbed onto silica gel and incorporated into a silicone matrix with high oxygen permeability placed on the tip of a fiber. The enzyme glucose oxidase is immobilized on the surface of the oxygen optrode. The sensor relates oxygen consumption as a result of enzymatic oxidation of glucose to glucose concentration. Similarly, an oxygen optrode with an oxygen sensitive indicator dye (decacyclene) and a C0₂ optrode with a pH sensitive indicator dye (HPTS) having the enzymes glutamate oxidase and glutamate decarboxylase, respectively, immobilized thereon are used to detect L-glutamate, Dremel, et al, "Comparison of two fibre-optic L-glutamate biosensors based on the detection of oxygen or carbon dioxide,...", Analytica Chimica Acta, 248 (1991) 351-359. U.S. Patent Application No. 08/115,843 describes an improved C0₂ sensor.

SUMMARY OF THE INVENTION

Accordingly it is an object of the invention to provide an optical method and apparatus for determination of analytes which cannot be measured directly by chemical means or where interferences make chemical measurements unreliable.

It is also an object of the invention to provide optical method and apparatus for determination of analytes using microbial material.

The invention is microbial sensors and methods which comprise micro-organisms immobilized on optical waveguides, fiber optic chemical sensors (FOCS)/optrodes and chip chemical sensors. The oxygen and C0₂ are presented as examples. It is formed of a ruthenium complex fluorescent indicator in a PVC membrane with plasticizer; however, any optical 0₂ sensor can be used. The example is further extended by referring to the BOD measurement. A yeast cell culture (or combination of yeast and bacteria or bacteria alone) is immobilized on the 0₂ sensor, preferably in poly(vinyl alcohol) ; other microbial cell species that measure BOD could also be used. The yeast digests or metabolizes organic material in a sample, thereby consuming 0₂, which decreases the 0₂ quenching of fluorescence of the indicator. Other microbial sensors can be formed using other microorganisms immobilized on other optical sensors, e.g., C0₂ or ammonia sensors.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a cross-sectional view of a microbial sensor.

Fig. 2 is a response curve of the BOD sensor. Figs. 3-4 are response curves of the BOD sensor to glucose and glutamate, respectively, at various concentrations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As shown in Fig. 1, a microbial sensor 10 has a species sensitive layer 12 formed on an inert, optically transparent substrate 14. This layer can respond to oxygen, carbon dioxide, ammonia or any other species for which an optical chemical sensor is available. A microbial layer 16 is formed on the sensing layer 12, with an optional thin optical isolation layer 18 therebetween. A porous protective membrane 20 is formed over the microbial layer 16. The sensing layer is chosen to be compatible with the reaction of the microbial layer and the analyte.

In an illustrative embodiment, the substrate 14 is about 25-200 μm thick, the oxygen sensing layer 12 is about 2-100 μm thick, the microbial layer 16 is about 10-100 μm thick. The protective membrane 20 has a pore size of about 0.4 μm. The optical isolation layer 18 is about 10 μm thick.

In one embodiment, the oxygen sensitive layer is formed of a fluorescent 0₂ sensitive Ru complex indicator ruthenium tris(diphenylphenanthroline) perchlorate [Ru(4,7-diph)₃(C£0_A)₂ or Rudpp] in a poly(vinyl chloride) (PVC) matrix with 2-nitrophenyloctylether (NPOE) plasticizer. The indicator may also be in another matrix such as silicone, ethyl cellulose, or polystyrene; other plasticizers such as dioctylphthalate, tributyl phosphate or dioctyl sebacate may be used. The substrate is usually a polyphthalate or polyterephthalate (e.g.. Mylar™) but may be any other polyester such as poly(methyl) ethacrylate or polycarbonate. The microbial layer is formed of a micro-organism, such as the yeast trichosporon cutaneum, immobilized in poly(vinyl alcohol) (PVA) . The optical isolation layer is carbon (charcoal) . The protective membrane is porous polycarbonate.

In operation, the microbial surface of the sensor digests the analyte in a sample which penetrates through the pores in the protective membrane. The digestive process can consume or emit a species that can be measured with an optical sensor. The difference in the used up or increased species concentration before and after the microbial reaction is a direct measure of the amount of analyte present. For example, in the BOD sensor oxygen is consumed, thereby reducing the amount of 0₂ quenching the fluorescence of the 0₂ indicator. Thus, the increase in fluorescence correlates to the amount of organic matter which creates an 0₂ demand. Typically fluorescence or phosphorescence intensity of the oxygen-sensitive fluorescent layer is measured, but fluorescence or phosphorescence lifetime may be measured as well. Fig. 2 shows the fluorescence output of the BOD sensor; in the end regions, the sensor is exposed to a stream of buffer so there is more 0₂ to quench the fluorescence, while in the center region the sensor is exposed to a flow of an organic sample which leads to 0₂ consumption and an increase in fluorescence. Thus, when the membrane is in contact with plain water or buffer, there is no measurable oxygen consumption; if, however, the optical sensor is brought into contact with a digestible material such as glucose (Fig. 3) , glutamate (Fig. 4) or other low molecular weight organic material, metabolism begins and oxygen will be consumed (fluorescence increases) . When changing to plain water or buffer again, the signal drops to the baseline again. The fluorescence increases with the concentration of the organic material. As shown in Fig. 1, an excitation signal is provided to the sensing layer 12 from an excitation source 22 to cause the indicator in layer 12 to fluoresce. Fluorescence from layer 12 is detected by detector 24. Optical isolation layer 18 eliminates optical interferences from outside layer 12. The optical isolation can be a dispersion of a black, white, red or reflective material in an inert and analyte-per eable polymer. Examples for colored materials include carbon black, barium sulfate, titanium dioxide, red or black ferric oxide, gold particles, or glimmer pigments. Gas-permeable polymers into which the colored materials are dispersed include silicone, polystyrene or ethyl cellulose, while hydrogels are preferred polymers for use in an optical isolation when ion optrodes are used as transducers. Support substrate 14 can be a flat substrate or could also be an optical fiber core or other optical waveguide having the sensor layers formed on a lateral surface thereof (and/or on the fiber tip) . If an optical fiber is used as substrate 14, then source 22 and detector 24 are optically coupled to sensing layer 12 through the fiber. If a waveguide or chip is used then source 22 and detector 24 are optically coupled by internal reflections. The sensor can also be placed in a disposable cell or used in a flow-injection type analyzer. By using an optical chemical sensor with an immobilized microbial surface, and measuring the amount of a species consumed or generated as the result of metabolic activity, the microbial sensor provides many advantages. The advantages include: (a) the ability to use simple sensors to do complex measurements; (b) the capability of using a minimum number of sensors to do a maximum number of analytes; (c) increased specificity, (d) minimum possibility of human error, i.e, no chemicals to mix and direct readout of analyte concentration; (e) the capacity to do in-situ. real time measurements; (f) option of making it a disposable sensor, (g) remote sensing possibility via fiber optics and (h) cost advantages over electrodes and other sensor systems. Microbial sensors can be designed to detect a variety of target species, by using different microorganisms or microbes, including bacteria, yeasts and combinations thereof which produce a measurable species by acting on the target species. Table 1 gives examples, but not a complete list, of microbial sensing systems. The Rhodococcus ery./Issatchenkia combination is a mixed bacterium/yeast system while the B.substilis/Licheniformis combination is a two bacillus system.

TABLE 1

KZCSOBZJ L .rocs

MICROORGANISM TARGET FOCS/OPTRODE

Nitrosomonas europaea Ammonia Oxygen

Nitrobacter sp. Nitrite Oxygen

Chromatium sp. Sulfide Oxygen

Chlorella vulgaris Phosphate Oxygen

Trichosporon cutaneum BOD Oxygen

Clostridiums BOD Oxygen

Hansenula BOD Oxygen

Rhodococcus ery./ BOD Oxygen Issatchenkia

Baccilus substilis/ BOD Oxygen Lichenifor is

T. cutaneum Phenol Oxygen

Methylomonas flagellata Methane CO,/ Oxygen

Saccharomyces cerevisiae Glucose CO,

Streptococcus faecium pyruvate CO,

E. coli L-lysine CO,

Sarcina flava L-glutamine NH,

B. cadaveris L-Asparat NH,

Desulfovibrio desulfuricans Sulfate H,S (anaerobic)

The selection of a sensor is not only based on the measurement to be made, but also on the microbes ("bugs"), i.e., their activity, rate of reaction, extent of reaction, etc. In the case of those microbes which use the oxygen sensor, some will metabolize oxygen almost to completion and a wide dynamic range sensor is needed. In other cases small amounts of oxygen are consumed and a high sensitivity, high resolution sensor with limited dynamic range is appropriate. Three (3) possible oxygen sensors are, therefore, presented in the oxygen sensor section. Since the sensor listed under (A.) is the most versatile, its characteristics are shown in Table 2.

EXAMPLE 1 Preparation of 0₂ Indicator - Ruthenium(II)-tris(4,7-diphenyl-l,10-phenanthroline) (C£0₄)₂ ("Rudpp") .

Dissolve 225.9 mg. ruthenium(III) chloride trihydrate (Alfa Products) in 5 m£ of ethylene glycol and 0.5 m£ of water. Heat to 160°C upon which the color changes from orange to blue-green. Then cool to 120°C and add 862.6 mg of 4 , 7 -d ipheny1 - 1 , 10 -phenanthro 1 ine ("bathophenanthroline") (Alfa Products) and heat to 160°C for 45 minutes. Let cool to room temperature, add 50 rat of acetone and filter. Add drop-wise 10 m£ of 1 M aqueous perchloric acid to the filtrate and finally 50 m£ of water. A precipitate is formed which, after several hours, is collected by suction and washed with ether. It is recrystallized from hot acetone and again washed with ether. Rudpp forms an orange powder, insoluble in water but with good solubility in acetone, methanol and tetrahydrofuran (THF) . The yield is 83%. Preparation of 0₂ Sensor Membrane

A. PVC Sensor. Dissolve 1.0 g of polyvinyl chloride (PVC) (FLUKA, Switzerland, high molecular weight material)) and 1.0 g nitrophenyloctyl ether (NPOE) (FLUKA) in 20 m£ of a solution of 120 mg Rudpp in 100 m£ THF. The membrane is made by spreading this solution onto a 175 μm layer of polyester (Mylar™, Dupont) using a home-made coating device such that the thickness of the sensing layer is approximately 50 μm after solvent evaporation. By additionally spreading, a thin layer (« 1 μm) of carbon black on top of the oxygen membrane while still wet, a black optical isolation layer is provided.

Table 2 summarizes the sensor properties of the 0₂ membrane.

TABLE 2 0 PROPERTIES OF OXYGEN SENSOR MADE OF PLASTICIZED PVC

Stern-Vol er constant (K. 7.3 m Torr "'

Quenching from nitrogen to 10% -35% Oxygen

Quenching from nitrogen to air -52%

Oxygen sensitivity range 0-200 Torr

Fluorescence intensity very high

Response time to oxygen in less than 1 minute Water

Photostability no bleaching detectable with blue LED

Storage stability 6+ months

Operational lifetime 1 week minimum

Leaching of the dye Not detectable after 1 week in water

Leaching of the plasticizer Negligible in water but mea¬ surable when detergent added |

Reproducibility of the ± 2% | quenching constant (K.

No interference by CO, (1000 ppm), H,S (100 ppm) |

Interference observed by SO, (20 ppm) | B. Polystyrene (PS) Sensor. Make a 10% solution of PS in MEK by dissolving 1.0 g. PS in 9.0 g methyl ethyl ketone (MEK) , and make a 10"³ M solution or Ru(dpp) in MEK (3 mg dye in 2.5 m£ MEK) . Mix 1.0 g of the PS solution and 0.5 m£ of the Ru(dpp) solution. An oxygen sensitive coating is obtained by spreading this solution onto a polyester support using a 50 μm spacer. The thickness of the resulting membrane is 5 μm after complete drying. Then, a layer of black silicone (using a 20 μm spacer) is placed on top of the PS layer. Its thickness is 8 μm after drying. This sensor has a lower sensitivity, but a wider dynamic range than PVC sensor A.

C. Ethylcellulose (EtCell) Sensor. 0.5 g EtCell was dissolved in 10 m£ of a solution of 20 mg. Ru(dpp) in 10 m£ chloroform. The coating was made as described above using a 50μm spacer which resulted in a final thickness of the membrane (after drying) of approximately 5 μ . Growth of Cell Culture

For the BOD sensor, yeast Trichosporon cutaneum (from DSM, Brunswick, Germany) was grown under aerobic conditions in a rotating shaker at 30°C for 36 hours in a medium containing 0.25% malt extract, 0.25% peptone, 0.25% yeast extract and 1% glucose. After completion of cell growth, the broth was centrifuged at 5000 rpm for 10 minutes and the resulting cell mass washed .twice with 0.1 M phosphate buffer of pH 6.8. For other microbial species, standard growth methods are used. Immobilization of Microbial Cells on the 0₂ Sensor

100 mg of the above cell mass were mixed with 100 μ£ of a 10% solution of poly(vinyl alcohol) in water. The viscous mix was spread onto the oxygen membrane (same method as was used to spread oxygen chemistry) . The thin film was left to dry at 4°C for 24 hours. A cross-section of the resulting multilayer sensor is shown in Fig. 1.

EXAMPLE 2

Preparation pf C0₂ Indicator - l-hydroxypyrene-3,6,8-trisulfonic acid hexadecyl trimethyl ammonium ion pair [HPTS(CTA)₃]

264 mg of l-hydroxypyrene-3,6,8-trisulfonic acid trisodium salt (HPTS) (Lambda, Austria) were dissolved in 20 m£ of distilled water. 546.5 mg of hexadecyl trimethyl ammonium bromide (CTA-Br) (Aldrich, Germany) were dissolved in 50 ml of distilled water at 50°C and 1 ml of 0.5 M HC£ was added to the solution. The HPTS solution was added dropwise to the stirred CTA-Br solution, and stirred 10 minutes more and then cooled to room temperature (ca. 20°C) . The resulting yellow precipitate was centrifuged (10 min at 2000 rpm) , decanted and then dissolved in 50 m£ methyl-ethyl ketone. The solution was dried over 10.0 g of Na₂S0₄ for 3 hours. Finally, the solution was decanted, the solvent evaporated and the product recrystallized from methyl-ethyl ketone. The HPTS(CTA)₃ ion pair (M=1309.05) forms a yellow powder, only slightly soluble in water, very well soluble in acetone, ethanol, methanol, toluene, THF, and methyl-ethyl ketone. 1000 mg of HPTS(CTA)₃ were mixed with 1.53 ml of a freshly prepared 0.5 M tetraoctyl ammonium hydroxide solution [TOA-OH, prepared from tetraoctyl ammonium bromide (TOA-Br) (FLUKA, Switzerland) according to the procedure of Mills, A.; Chang, Q. ; and McMurray, N. , Anal. Chem. 1992, 64. 1383 and Raemer, D.B.; Walt, D.R. ; and Munkholm, C. , U.S. Pat. 5,005,572, 1991.] in methanol and diluted with 3.47 ml of 100% methanol. This ion pair solution (IPS) can be stored in a refrigerator without changes in the fluorescence properties over a long time period (>5 month tested) . Preparation of C0₂ sensor Membrane 20 mg of IPS is mixed with 50 mg of plasticizer

[trioctyl phosphate (TOP) , trisbutylphosphate (TBP) or dioCtyl-phthalate (DOP) , all from FLUXA, Switzerland], 1,000 mg of toluene, 150 mg of ethyl cellulose derivatives [amino-ethyl-cellulose (AE) , diethylamino-ethyl cellulose (DEAE-32) or triethyl-amino ethyl cellulose (TEAE-23) (all from Serva, Germany) or quarternary-am no-ethyl cellulose (QA-52) (Whatman, England)] and titanium dioxide (Ti0₂) Aldrich, Germany) . The solution was kept in an ultrasonic bath for 5 minutes, then 1,000 mg of RTV silicone A-07 (Burghausen, Germany) was added and mixed. The reason for adding the plasticizer is to decrease response time. The reason for adding Ti0₂ is to provide scattering centers for more efficient excitation and to optically isolate the sensor from the sample and ambient light.

The support substrate is Mylar™ (GA-10) (Dupont, USA) . A dust free sheet was coated with a primer consisting of 1,000 mg of A-07 silicone and 1,500 mg of toluene. No spacer was used, but the thickness of this film was consistently 1 μm thick after drying at room temperature for 2 hours. The purpose of the primer is to assure that the sensing layer does not detach from the substrate when placed in water. The sensing chemistry is then spread on the primed substrate with a 100 μm using the coating device discussed in the oxygen section. Other methods such as spin coating or sieve printing may be used as well. After allowing the solvent to evaporate at room temperature for 5 minutes, the membrane was first cured at 100% relative humidity (RH) in a desiccator for 4 hours. The final membrane thickness is very close to 55μm. After curing has occurred, a black silicone (N189) (Burghausen, Germany) optical isolation layer was spread on the membrane using a 10 μm spacer. The membranes were, placed into the desiccator again for total curing (24 - 36 hours). The membranes are stored at 100% RH.

The C0₂ sensor is further described in U.S. Patent Application Ser.No. 08/115,843 which is herein incorporated by reference.

Table 3 summarizes the properties of the C0₂ membrane. TABLE 3 PROPERTIES OF A CARBON DIOXIDE SENSOR MADE FROM A SOLUTION OF AN INDICATOR ION PAIR IN SILICONE

Growth of Cell Culture

For a methane sensor, the bacterium Methylomonas flagellata (German Collection of Micro-organisms, Germany) is used. They were prepared to the specific instructions of the supplier.

Immobilization of Microbial Cells on the C0₂ Sensor 100 mg of the above cell mass were mixed with 100 μ£ of a 10% solution of poly(vinyl alcohol) in water. The viscous mix was spread onto the carbon dioxide membrane (same method as was used to spread carbon dioxide chemistry) . The thin film was left to dry at 4°C for 24 hours. A cross-section of the resulting multilayer sensor is similar to that for oxygen and is, again, represented by Fig. 1. Since the methylomonas flagellata consumes oxygen, it could also be immobilized on an 0₂ sensor.

EXAMPLE 3 Preparation of NH₃ Sensor

Dissolve, in 1.6 m£ chloroform, 4 mg of bromothymol blue (BTB) octyltrimethylammonium salt (made by analogy to J. Reichert, et al; Sensors & Actuators, 1991, 25A. 481.) and 100 mg silicone (Elastosil E4, Wacker, Germany). The solution is cast on a 175-μm thick polyester film (Mylar, type GA-10, from General Electric) and the solvent slowly evaporated at room temperature. The silicone cures and the resulting rubber forms a thin yellow film. In order to complete polymerization and to remove the acetic acid released during polymerization, the membrane was dried at 90°C for two days. The resulting coating was estimated to be 2-4 μm thick. Before actual measurements, the membrane was conditioned for 1 hour in a 100 mM phosphate buffer of pH 7.38. The membranes, when in contact with ammonia, assume the blue color of the BTB anion and this can be monitored photometrically at around 580 nm.

If bromothymol blue (BTB) is replaced by a fluorescent dye of a pK similar to that of BTB (7.2), a fluorescent sensor is obtained. One example for a fluorescent dye of similar pKa is _l hydroxypyrene-3,6,8-trisulfonate with a pK of 7.3. It can excited at around 460 nm and fluoresces at above 500 nm, with a maximum at 512 nm.

Immobilization of Microbial Cells on the NH₃ Sensor The resulting membranes can be covered with a layer of a bacterium producing ammonia during its biological action. The bacteria preferentially are immobilized in a hydrophilic polymer such as poly(vinyl alcohol) or hydrogel. Sarcina flava and used on an ammonia such as B.cadaveris are two bacteria which can be used on an ammonia sensor. Changes and modifications in the specifically described embodiments can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims.

Claims

1. An optical sensor for detecting a first species, comprising: a support; an optical sensing means formed on the support which produces an optical signal which varies as a function of concentration of a second species; a microbial material immobilized on the optical sensing means which acts on the first species to produce the second species.

2. The sensor of Claim 1 wherein the microbial material is a bacterium, a yeast, or a combination thereof.

3. The sensor of Claim 2 wherein the microbial material is selected from the group consisting of Nitrosomonas europaea, Nitrobacter sp. , Chromatium sp. , Chlorella vulgaris, Trichosporon cutaneum, Clostridiums, Hansenula, Rhodococcus ery./Issatchenkia, Baccilus substilis/Licheniformis, T. cutaneum, Methylomonas flagellata, Saccharomyces cerevisiae, Streptococcus faecium, E. coli, Sarcina flava, B. cadaveris, Desulfovibrio desulfuricans.

4. The sensor of Claim 1 wherein the microbial material is immobilized on the optical sensing means in a polymer matrix.

5. The sensor of Claim 4 wherein the polymer matrix is poly(vinyl alcohol) or hydrogel.

6. The sensor of Claim 1 wherein the optical sensing means is an 0₂ sensor.

7. The sensor of Claim 6 wherein the 0₂ sensor comprises an 0₂ sensitive indicator in a polymer matrix.

8. The sensor of Claim 7 wherein the indicator is a fluorescent indicator.

9. The sensor of Claim 8 wherein the indicator is a ruthenium complex.

10. The sensor of Claim 9 wherein the ruthenium complex is Ru(4,7 diphenyl-1,10-phenanthroline)₃(C£0₄)₂.

11. The sensor of Claim 7 wherein the polymer matrix is polyvinyl chloride, silicone, ethyl cellulose, or polystyrene.

12. The sensor of Claim 7 further comprising a plasticizer in the matrix.

13. The sensor of Claim 1 where the support is a flat transparent support.

14. The sensor of Claim 13 wherein the support is a polyphthalate or polyterephthalate film.

15. The sensor of Claim 1 wherein the support is an optical waveguide.

16. The sensor of Claim 15 wherein the waveguide is an optical fiber.

17. The sensor of Claim 1 further comprising a porous protective layer on the microbial material.

18. The sensor of Claim 1 further comprising an optical isolation layer formed between the optical sensing means and microbial material.

19. The sensor of Claim 18 wherein the optical isolation layer comprises a colored or reflective material in an inert polymer which is permeable to the second species.

20. The sensor of Claim 1 wherein the optical sensing means is a C0₂ sensor.

21. The sensor of Claim 20 wherein the C0₂ sensor comprises a C0₂ sensing chemistry in a polymer matrix.

22. The sensor of Claim 21 wherein the C0₂ sensing chemistry comprises a pH sensitive indicator.

23. The sensor of Claim 22 wherein the C0₂ sensing chemistry comprises: a pH sensitive dye with a hydroxy group; a base which converts the dye into its conjugate base form and forms a soluble ion pair therewith; an amino-alkyl cellulose derivative.

24. The sensor of Claim 23 wherein the dye is HPTS(CTA)₃ and the base is tetraoctylammonium hydroxide.

25. The sensor of Claim 21 wherein the polymer matrix is silicone.

26. The sensor of Claim 20 wherein the optical sensing means is an ammonia sensor.

27. An optical method for detecting a first species, comprising: exposing the first species to an optical sensor having an optical sensing means which produces an optical signal which varies as a function of concentration of a second species and a microbial material immobilized on the optical sensing means which acts on the first species to produce the second species; detecting a change in the optical sensing means; determining changes in the first species from detected changes in the optical sensing means.

28. The method of Claim 27 further comprising selecting the microbial material from a bacterium, a yeast, or a combination thereof.

29. The method of Claim 28 further comprising selecting the microbial material from the group consisting of Nitrosomonas europaea, Nitrobacter sp., Chromatium sp., Chlorelia vulgaris, Trichosporon cutaneum, Clostridiums, Hansenula, Rhodococcus ery./Issatchenkia, Baccilus substilis/Licheniformis, T. cutaneum, Methylomonas flagellata, Saccharomyces cerevisiae, Streptococcus faecium, E. coli, Sarcina flava, B. cadaveris, Desulfovibrio desulfuricans.

30. The method of Claim 27 further comprising optical sensing means from an 0₂ sensor, a C0₂ sensor or an ammonia sensor.