WO2002056023A1

WO2002056023A1 - Optical sensor and sensor array

Info

Publication number: WO2002056023A1
Application number: PCT/EP2002/000337
Authority: WO
Inventors: Ingo Klimant; Marco Jean Pierre Leiner
Original assignee: Presens Precision Sensing Gmbh
Priority date: 2001-01-15
Filing date: 2002-01-15
Publication date: 2002-07-18
Also published as: DE10101576B4; DE10101576A1

Abstract

The invention relates to an optical sensor for determining at least one parameter in a sample. The optical sensor comprises an indicator material (in 1) responding to the parameter and having a short decay time and, a reference material (in 1) not responding to the parameter and having a long decay time. The optical sensor detects the measuring signal indicating the parameter to be detected on the basis of the luminescence responses of the indicator and the reference material that are commonly detected. The indicator and the reference material are immobilized on a common support (3). The layer facing the sample of the indicator material and of the reference material is covered by a layer (5) that allows contact between the indicator material and the sample but is substantially impermeable to the light used for exciting the indicator and the reference material. The layer prevents the sample from being influenced by the excitation light, thereby improving the measuring sensitivity of the sensor.

Description

Optical sensor and sensor field

description

The invention relates to an internally referenced optical sensor for determining at least one parameter in a sample with an indicator material having a short decay time which responds to the parameter and a reference material which does not respond to the parameter has a long decay time for detecting a measurement signal indicating the parameter to be determined on the basis of the jointly recorded Luminescence responses from indicator and reference material.

The optical sensor uses a measuring principle which determines the optical, in particular fluorometric, determination of various chemical, physical and biological parameters with the aid of e.g. B. time-resolved and phase modulation techniques. Here, for. B. the sum of the luminescence signals of the indicator material with a short decay time and the reference material with a long decay time (at least a few hundred nanoseconds) is measured. While the long-lasting luminescence is not influenced by the analyte that determines the parameter, the intensity of the indicator material co-immobilized with it changes quickly, depending on the analyte concentration. Since the phase shift between the luminescence responses of the indicator and reference material determined by phase modulation techniques is only dependent on the ratio of the intensity components of the two materials, the intensity of the luminescence response of the indicator material is directly reflected in this. An internal referencing of the signal intensity of the luminescent substances is thus obtained, so that in principle one single excitation light source and one single photodetector are sufficient. Provided that the distribution of indicator and reference material is kept constant during the manufacturing process, the phase shift depends exclusively on the concentration of the parameter to be determined, while fluctuations in the optoelectronic system, losses in light guides, which connect the sensor with the excitation light source and the photodetector connect, and the optical properties of the sample, do not affect the signal.

Details of this measurement principle and exemplary embodiments are described and shown in DE 1 97 33 341 .9, the DE 1 98 29 657.6 based thereon and the corresponding PCT / EP / 98/04779, the complete contents of which are incorporated herein by reference.

However, the light used to excite indicator and reference material could adversely affect substances in the sample, so that the measuring accuracy of the sensor decreases, e.g. B. by the fact that the substances to be measured are themselves changed by the light or the substances themselves are excited to undesired luminescence. This is of particular importance when measuring body fluids, e.g. B. blood, especially the so-called "critical care analytes", such as pH, 0 _2, CO ₂ , sodium, potassium, calcium, chloride, lithium, magnesium and the like, or culture media.

The object of the invention is therefore to increase the measurement accuracy in an optical sensor of the type mentioned.

To achieve the object, it is proposed that the indicator material and the reference material are immobilized on a common carrier and that the side of the indicator material and the reference material facing the sample is covered by a layer that allows contact between the indicator material and the sample, but for that for Excitation of the indicator and reference material light is essentially impermeable.

This layer prevents the excitation light from reaching the sample itself and the sample itself from sending light back to the detector. This increases the measuring accuracy of the sensor and thus of the entire measuring system.

The layer is preferably a pigmented polymer layer, for example by means of carbon black or metal oxide, e.g. B. iron oxide or titanium dioxide.

If the sensor is designed to measure the pH or of chloride or other ions, the layer can be a hydrogel layer permeable to substances colored in the sample, in particular an ion permeable hydrogel layer, e.g. B. hydrophilic polyurethane and / or poly-hydroxyethyl methacrylate (HEMA) and / or carbon black. For the measurement of gases, such as C0 ₂ , 0 ₂ , a gaseous substance which is permeable under normal conditions and optionally ion-permeable cover layer, e.g. B. made of silicone or Teflon. If the sensor is to be used to measure the temperature in a sample in which there are substances to which the indicator material would respond, the layer for these substances is chosen to be impermeable.

If the sensor is to be used as an enzyme optode for detecting an analyte to be determined enzymatically, the layer covering the indicator and reference material can be overlaid by an enzyme layer specific for the analyte to be measured, such as glucose oxidase for measuring glucose or lactate oxidase Lactate measurement.

In the above-mentioned sensor arrangement of DE 1 97 33 341 .9 and DE 1 98 29 657.6 or PCT / EP98 / 04779, only one is used internally referenced sensor described, with which only a single parameter can then be measured in a sample.

In many cases, however, it is desirable to measure several different parameters simultaneously in a single measurement process, such as. B. in body fluids in medical applications.

If several different parameters are to be measured at the same time, at least one second optical sensor, e.g. B. of the type mentioned above. The second optical sensor can also be a so-called decay time sensor that responds to the second parameter and whose indicator material has a decay time that varies depending on the second parameter and / or a sensor whose luminescence intensity changes depending changes from the second parameter. In both cases, the covering layer can cover all sensors of the sensor field or at least two sensors thereof together.

A preferred combination is, for example, that first internally referenced sensors for measuring the pH and the CO ₂ partial pressure and second sensors designed as a decay time sensor for measuring oxygen and possibly temperature are combined to form a sensor field.

The indicator material of the second sensor can be selected such that its decay time is in the range of the decay time of the reference material of the first sensor, these two materials preferably being identical.

An identical phase detection system can then be used to evaluate the signals from the internally referenced sensors and also the Use cooldown sensors on the base of the same phosphorescent dye. The measured phase shift, as a measurement parameter dependent on the respective analyte, describes in the case of the internally referenced sensors the intensity ratio between the indicator luminescence or fluorescence dependent on the respective analyte concentration and the constant luminescence or phosphorescence of the reference material, as described above.

In the case of the decay time sensors, the measured phase shift correlates with the decay time of the phosphorescent dye of the reference material now functioning as an indicator and also depends on the concentration of the analyte to be measured by this decay time sensor. This makes it possible to produce a field or array of internally referenced sensors and decay time sensors, all of which can be read using the identical measuring system.

The sensors of the sensor field can be combined on a common carrier. The carrier can be a film, a cassette through which the sample flows, or a planar or fiber-like light guide.

The invention further relates to a method for determining a parameter of a sample by means of an optical sensor or sensor field of the type discussed above. Here, the time or phase behavior or the temporal change in intensity of the luminescence responses of the indicator material with a short decay time and the reference material with a long decay time is used to form a reference variable for determining the parameter. A ratio of the two luminescence intensity components of the indicator material with a short decay time and the reference material with a long decay time can also be used as a reference variable, which ratio is independent of the overall intensity of the luminescence signal. There is also a possibility that the Reference quantity is determined with the aid of a time-resolved measurement, the reference quantity representing a ratio between the luminescence intensity during the excitation pulse and the luminescence intensity after the light source has been switched off.

The luminophores of the sensors of the sensor field are preferably excited jointly by a single source, and their luminescence responses can be detected by the plurality of detectors assigned to the respective sensors, or the luminophores of the sensors are excited by a plurality of respectively assigned light sources and by a single detector recorded together.

The invention will now be explained using exemplary embodiments with reference to the accompanying figure.

The internally referenced sensor, which is attached individually or as an array or array of several such sensors on a common carrier, is a sensor of the sensor types described in DE 197 33 341 .9 or DE 1 98 29 657 or sensors for Detection of pH, chloride or other ions, CO ₂ or 0 ₂ and the like, as are used in particular in the medical field. The layer containing the indicator and reference material is designated by 1 in the figure and the support on which the sensor layer is attached is designated by 3. This carrier 3 is connected to a light source and a photodetector of a fluorometric measuring device via a light guide, not shown. The light guides can be planar light guides or fiber optics. The measurement can also be carried out with conventional optics without a light guide. The opaque layer is designated 5 in the figure. The opaque layer 5 has the task of protecting the sample from interaction with the excitation light. This will falsify the measurement signal, such as z. B. by excited in the sample Background fluorescence can occur, excluded. The cover layer 5 may be a blackened polymer layer, e.g. B. with soot.

If the sensor is used to measure the pH or chloride or other ions, a polyurethane hydrogel or poly-hydroxyethyl methacrylate (HEMA) is proposed for the cover layer 5. If C0 ₂ or 0 ₂ or another gas is to be measured, silicone or Teflon is suggested for the cover layer. If the temperature is to be measured, polyacrylonitryl, silicone or hydrogel is suggested for the covering layer.

The sensor or one of the sensors of an array can also be designed as an enzyme optode, for example for measuring glucose or lactate. In this case, the cover layer 5 is an enzyme layer 7, z. B. glucose oxidase or lactate oxidase, superimposed, which then passes on the necessary signals to the actual sensor material 1. This enzyme layer is shown in dashed lines in the figure.

The signals of the internally referenced luminescence sensors of the type mentioned at the outset can be read out using identical measuring systems, such as those optical sensors in which the change in the decay time is the measurement parameter. These measuring systems are based on phase modulation techniques.

The identical phosphorescent dyes that are used in the internally referencing sensors as long-life reference luminophores and are covered here by the cover layer 5 can also be used as a luminescence indicator in decay time sensors. An example of this is the rhutenium-tris-4,7-diphenyl-1, 0-phenanthroline complex. Built into a matrix made of polyacrylonitryl, this dye is for all potential chemical components of a sample not accessible and therefore forms an ideal reference standard for the internally referenced sensors. In this environment, its luminescence declining time is only influenced by the temperature of the sample and thus represents an ideal internally referenced decay time temperature sensor. On the other hand, if the dye is incorporated in a hydrophobic, highly gas-permeable polymer, its decay time depends on the gas partial pressure of the sample. Such a material is thus an internally referenced gas sensor, for example an oxygen sensor. An identical phase detection system can be used to trigger the signals, both from internally referenced sensors and decay time sensors based on the identical phosphorescent dyes. The measured phase shift as a measurement parameter dependent on the analyte, measured for example at a modulation frequency of 45 kHz, describes in the case of the internally referenced sensors the intensity ratio between the indicator fluorescence dependent on the respective analyte concentration and the constant phosphorescence of the reference material according to the following equation: cotφ = A _flu * cotΦ _flu + A _flu / A _ref * sinΦ _p - ₀

In the case of the decay time sensors, the measured phase shift correlates with the decay time of the phosphorescent dye now acting as an indicator and also depends on the concentration of the analyte to be measured, according to the following equation: tanφ = 2 * π * f _mod * r

In this way, the internally referenced and decay sensors complement each other in an ideal way. It can be used to produce an array of internally referenced sensors and decay time sensors, all of which can be read using the identical measuring system.

The following table shows a selection of sensors that can be combined in such an array. All of these sensors are excited with a sinusoidally modulated light-emitting diode, the modulation frequency always remaining the same, e.g. B. is 45 kHz.

The preferred array for diagnostic blood analysis consists of all the sensors listed in the following table, with the exception of temperature and glucose.

A preferred array for biotechnology and cell cultivation consists of sensors for the parameters pH, pC0 ₂ , p0 ₂ and temperature.

^'' internally referenced sensor of the type mentioned at the beginning

In the figure, arrow 9 shows the excitation light coming from the light source through a light guide and arrow 1 1 shows the light of the luminescence responses from indicator and reference material, which is passed through this or another light guide to the photodetector. In the case of several sensors, the sensors can be arranged next to one another separately from one another on the carrier 3 or they can also be mixed. The term "sample" used here includes compounds, surfaces, solutions, environmentally relevant liquids (waste water, rain water, drinking water, river water, sea water), industrial liquids and biological liquids (e.g. blood, blood plasma, blood serum, urine, cerebroscopic liquid), Emulsions, suspensions, mixtures, cell cultures, fermentation cultures, cells, tissues, secretions and / or derivatives or extracts thereof.

The term "analyte" also used herein refers to elements, ions, compounds or salts, dissociation products, polymers, aggregates or derivatives thereof.

For the internally referenced sensors, e.g. B. in question: pH optodes with fluorescein derivatives as indicators; - pH optodes with covalently bound hydroxipyrene-trisolonic acid as an indicator;

Carbon dioxide, hydrogen sulfide and ammonium sensors based on fluorescent derivatives or rhodamine dyes as

Indicator; - Optical heavy metal sensors based on

Fluorescence quenching; optical ion-sensitive sensors for the determination of calcium or magnesium with PET indicators, such as calcium green,

Calcium crimson or fur red; - Cation sensors for determining sodium, potassium, lithium,

Magnesium, calcium z. B. based on PET naphthalimide

Indicators or also zinc, lead, barium, cadmium, mercury,

lanthanum;

Anion sensors based on fluorescence quenching of acrydin and bisacridine fluorophores for the detection of chloride,

Bromide and iodide; Anion sensors for measuring chloride, bromide or nitrate based on potential-sensitive dyes (such as rhodamines or styryl fluorophores);

Cation sensors for measuring potassium or sodium based on potential sensitive dyes;

Sensors with fluorogenic reactants for measuring amines, aldehydes or alcohols;

Metabolite sensors such as glucose, lactate, urea, creatinine based on fluorogenic receptors based on boric acid derivatives.

As biosensors for various metabolites such. B. in question: enzymatic sensors for detecting glucose or lactate based on fluorescez pH optodes as converter layer 7; - Enzymatic sensors for detecting urea or creatinine on the basis of a fluorescence ammonium, pH or ammonium optode; a microbial sensor for measuring the biological oxygen demand with a fluorescent pH sensor as a converter; - enzymatic sensors for the determination of glucose or others

Substrates based on the measurement of the intrinsic fluorescence of the enzymes or co-enzymes involved (such as in NADH).

As affinity-based biosensors come e.g. B. in question: - immunosensors with surface immobilized antigens or

Antibodies and competitive binding of fluorophore-labeled antibodies;

Biosensors for the identification and quantification of oligonucleotides or DNA strands based on competitive binding of fluorophore-labeled oligo nucleotides;

Biosensors for identifying and quantifying oligonucleotides or DNA strands with embedded dyes. Typical areas for the application of the sensors and sensor fields according to the invention are: the detection of gases, electrolytes and metabolites in

Body fluids such as blood, serum, plasma or urine; - two-dimensional mapping of chemical parameters (e.g. transcutaneous applications); diagnostic determination of antibodies, antigens and oligo-nucleotides in body fluids; fiber optic detection in tissues or entire organs of humans or animals;

Immunoassays in high throughput screening applications;

Online food analysis (determination of freshness or

Storage conditions);

Food analysis (genetic testing); - environmental analysis (fluorometric determination of humic acids,

Chlorophyll or polycyclic aromatic hydrocarbons

(PAH));

Calibration-free acquisition systems for controlling

Culture conditions in bioreactors and growth chambers; - microplates with integrated optical chemical sensors;

Immunosensors for the detection of microbiological

Contamination.

Claims

Expectations

1 . Optical sensor, for determining at least one parameter in a sample, with one that responds to the parameter

Indicator material (in 1) with a short decay time and a reference material that does not respond to the parameter (in 1) with a long decay time for detecting a measurement signal that indicates the parameter to be determined on the basis of the luminescence responses of the indicator and reference material that are recorded together, characterized in that the indicator material and the reference material is immobilized on a common carrier (3) and that the side of the indicator material and the reference material facing the sample is covered by a layer (5) which ensures contact between the

Indicator material and the sample are allowed, but are essentially opaque to the light used to excite the indicator and reference material.

2. Optical sensor according to claim 1, characterized in that it is designed for measuring parameters from biological fluids, in particular body fluids, such as blood, or culture media, in particular at least one parameter of pH, 0 ₂ , C0 ₂ , sodium, potassium, Caicium, chloride, lithium, magnesium or a combination thereof.

3. Optical sensor according to claim 1 or 2, characterized in that the layer covering the indicator and reference material (5) is permeable to the substance to be measured, which determines the parameter, in the sample.

4. Optical sensor according to claim 1, 2 or 3, characterized in that the layer covering the indicator and reference material (5) is blackened.

5. Optical sensor according to one of the preceding claims, characterized in that the layer covering the indicator and reference material (5) is a polymer layer into which pigments, in particular carbon black or metal oxide, for. B. iron oxide or titanium dioxide are embedded.

6. Optical sensor according to one of the preceding claims, characterized in that the layer covering the indicator and reference material (5) for measuring the pH or of chloride or other ions is a permeable, in particular ion-permeable, hydrogel layer for substances dissolved in the sample z. B. contains hydrophilic polyurethane and / or poly-hydroxyethyl methacrylate and / or carbon black, or for measuring gases such as C0 ₂ , 0 ₂ , one for under

Normal conditions gaseous substances permeable and optionally ion impermeable layer, e.g. B. is a silicone or Teflon layer.

7. Optical sensor according to one of the preceding claims, characterized in that the layer covering the indicator and reference material (5) is overlaid with the formation of an enzyme optode from an enzyme layer (7) specific for the substance to be measured, e.g. B. glucose oxidase for measuring glucose or lactate oxidase for measuring lactate.

8. Optical sensor according to one of the preceding claims, characterized in that the carrier (3) is an at least partially transparent carrier.

9. Optical sensor according to one of the preceding claims, characterized in that, with formation of a sensor field on the carrier (3), at least one second optical sensor (in FIG. 1) responsive to a second parameter is attached, the second optical sensor (in FIG. 1) can be carried out according to the preamble of claim 1.

1 0. Optical sensor according to claim 9, characterized in that the cover layer (5) covers at least two of the sensors together.

1 1. Optical sensor field for determining a plurality of parameters in a sample, with at least one first sensor (in FIG. 1) which has an indicator material with a short decay time which responds to a first parameter and an associated reference material with a long decay time which does not respond to the parameter for detecting a parameter to be determined Has parameter-indicating measurement signal on the basis of the jointly recorded luminescence responses of the indicator and reference material, and with at least one second optical sensor responsive to a second parameter (in FIG. 1).

1 2. Optical sensor field according to claim 1 1, characterized in that the indicator material of the second sensor has a decay time which is variable as a function of the second parameter and / or a luminescence intensity which is variable as a function of the second parameter.

1 3. Optical sensor field according to claim 1 1 or 1 2, characterized in that the second sensor (in 1) an indicator material responding to the second parameter short decay time and an associated reference material not responding to the parameter long decay time for detecting a to determining parameter indicating measurement signal on the basis of the luminescence responses of the indicator and

Has reference material.

14. Optical sensor field according to one of claims 1 1 to 1 3, characterized in that the first sensor, and in the case of claim 1 3, optionally also the second sensor, is designed according to one of claims 1 to 8.

1 5. Optical sensor field according to one of claims 1 1 to 1 4, characterized in that the indicator material of the second optical sensor is a luminescent dye, preferably from the group of luminescent transition metal complexes with Ru, Re, Os, Rh, Ir or Pt as the central atom and σ-diimine ligand is selected.

1 6. Optical sensor field according to one of claims 1 1 to 1 5, characterized in that in the case of a decay time sensor, the decay time and / or the spectral properties of the indicator material of the second sensor in the range of the decay times or the spectral properties of the

Reference material of the first sensor lies or lie.

1 7. Optical sensor field according to one of claims 1 1 to 1 6, characterized in that the first sensor responds to pH or C0 ₂ or chloride or salinity.

1 8. Optical sensor field according to one of claims 1 1 to 1 7, characterized in that the second optical sensor is a decay time sensor for measuring the temperature.

1 9. Optical sensor field according to one of claims 1 1 to 1 8, characterized in that the second optical sensor is a decay time sensor for measuring a gas, preferably 0 ₂ .

20. Optical sensor field according to one of claims 1 1 to 1 9, characterized in that it is designed for measuring parameters from biological liquids, in particular body fluids, such as blood, or culture media, in particular at least one parameter of pH, 0 ₂ , C0 ₂ , sodium, potassium, calcium, chloride, lithium, magnesium or a combination thereof.

21. Optical sensor field according to one of claims 1 1 to 20, characterized in that the first sensor comprises a pH sensor and a C0 ₂ sensor, and that the second sensor comprises a 0 ₂ decay time sensor and optionally a temperature decay time sensor.

22. Optical sensor field according to one of claims 1 1 to 21, characterized in that the sensors of the sensor field are arranged on a common carrier (3).

23. A method for determining at least one parameter of a sample, characterized in that an optical sensor or a sensor field according to one of the preceding claims is used.

24. The method according to claim 23, characterized in that the indicator and reference materials of the sensor or sensors are excited together by a single light source.

25. The method according to claim 23 or 24, characterized in that only a single light source, but several detectors assigned to the respective sensors are used for the measurement.

26. The method according to claim 23, characterized in that the luminescence responses of the indicator and reference materials of the sensor or sensors are detected together by a single detector.

27. The method according to claim 23 or 26, characterized in that light sources assigned to the respective sensors, but only a single detector, are used for the measurement.

28. The method according to claim 24 and 26, characterized in that the plurality of sensors of the sensor field are arranged separately from one another and that the light source and the detector are moved relative to the sensor field between the individual sensors.

29. The method according to any one of claims 23 to 28, characterized in that the excitation light for the sensor or sensors is modulated with a single constant frequency, for. B. 45 KHz.