DE3110239A1 - Method and device for measuring the luminescence of samples - Google Patents

Abstract

The invention relates to the automatic transporting, processing and measurement of the chemiluminescence and bioluminescence of individual samples which are contained in indentations in a light-reflecting band. The band can contain either one row of indentations, intended for the samples, for measuring the luminescence by a single light detector or successive rows of in each case 2 to 10 indentations for samples or even more, in order to measure the luminescence simultaneously by means of an arrangement which has a plurality of detectors, the number of individual detectors corresponding to the number of samples provided in each row. The processing and measurement of up to 500 samples per hour is possible with a single detector, and up to several thousands of samples can be measured per hour by means of a multiple detector system.

Description

1. Seppo Kolehmainen,

Route 2, Box 66X, Riveredge Drive, Titusville, Florida 32780, USA 2 Veikko Tarkkanen, KriJgersberglaan 25, 6371 CA Schaesberg, The Netherlands Procedure and apparatus for measuring the luminescence of samples. The invention relates to this automatic transport, processing and measurement of chemiluminescence and bioluminescence of substances.

The bioluminescence and the chemiluminescence are common to many types of Measurements of substances have been used (F. Gorus and E. Schram, Clin. Chem. 25: 512-5, 1979), for measuring cell viability (P. Tarkkanen, R. Driesch and H. Greiling, Fresenius Z. Anal. Chem. 290: 180-181, 1978) and for the determination of the amount or quantification of antigen-antibody complexes (B. Velan and M. Halmann, Immonu Chemistry 15: 331-33, 1978).

Usually the measurements of bio- and chemiluminescence are carried out using carried out by hand-operated instruments, although for the purpose of automation too Flow systems have been used. A flow system divided into segments adds air bubbles to individual samples and mixes the samples with reagents (H.H. Johnston, C.J. Mitchell and G.D.W. Curtis, 210-214, in Rapid Methods and Automation in Microbiology - edited by H.H. Johnston and S.W.B. Newsom; Learned Information (Europe) Ltd., Oxford, 1976), while the so-called flow injection -Analysis (FIA) homogeneous samples are moved in capillaries.

Certain non-homogeneous samples such as cell samples and biological fluids (Blood, urine, milk) are not easy to examine using flow systems, because there are problems caused by the failure or separation of cells, proteins, fat globules and other particles in the tubes and capillaries.

Therefore, the measurement of such samples is more reliable when taken individually treated or kept in individual containers at all stages of the analysis.

An automatic luminescence measurement of individual resp.

separated samples is by means of a liquid scintillation counter possible if a special modification is made to automatically add reagents submit (compare R.H. Hammerstedt, Anal.

Biochem. 52: 449-455, 1973), or by means of an instrument that has a light-transparent filter used to hold samples and reagents.

Liquid scintillation counters (LSC) are expensive, bulky and not optimal for luminescence measurements.

The most serious drawbacks of a liquid scintillation counter are that the size of the individual samples to be examined is large (from 2-10 ml), that the dynamic range is only 2 to 3 decades and that the time delay, before the individual sample reaches the measuring position from the transport mechanism, 20 Seconds. These counters are also only cumbersome for automatic sample processing usable because an automatic injection of reagents and incubation with one certain temperature not without significant changes to the original design possible are.

The one known from US Pat. No. 3,940,250, a light-transparent filter The instrument to be used is only intended for samples to be filtered, and it is only to be used for small volume samples and reagents. Also with him can only ever one analysis can be performed at a time.

The object of the invention is to provide a method and a device for Measuring or examining samples using luminescence to provide the above Avoid the disadvantages mentioned.

To solve this problem, according to the invention, individual one another separate samples in wells of a light reflective tape or a light reflective tape Contain foil that are automatically transported and processed and where the chemiluminescence and / or bioluminescence is measured.

In the drawing are exemplary embodiments of an inventive Apparatus for measuring the luminescence of samples shown schematically, namely Fig. 1 shows an overall view of a device for measuring the luminescence of an individual Samples, FIG. 2 shows a cross section through the device for producing depressions or recordings in a tape for the samples on an enlarged scale compared to FIG. 1, FIG. 3 shows a partial section through an embodiment modified from FIG. 1 the device for measuring the luminescence of a series of samples from above and Fig. 4 shows a cross section similar to that in FIG. 3 of a further modified embodiment the device.

The principles of the invention are shown in the schematic representation according to FIG. 1. For example, an upd for the transport of the samples non-transparent, reflective tape 5 made of aluminum foil is provided, in which by means of a stamp in the form of a wheel 4 (male mold) recesses 5 be pressed in by the protruding parts of the wheel 4 running over the wheel Press the foil into the recesses of another wheel 3 that are used as matrices. That Band 5 can also consist of deformable plastic such as polyvinyl chloride.

The receptacles or wells 5a intended for the samples are preferably hemispherical, but can also be conical or the shape of a have conical cylinder if they are produced with a deep-drawing punch. The diameter of the depressions 5a can vary from 2 mm to several cm such as up to 5 cm, which depends on the type of sample to be examined and the reagent system. at conical design, the depth can be between 1 and 25 mm. Small depressions are preferable because they save on reagent costs and per unit length the tape can accommodate more samples. The recordings or depressions 5a are mounted in the tape 5 so that the distance between the edges of two successive depressions is at least 1 mm and preferably smaller than 20 mm. The thickness of the band 5 can, however, be within wide limits thicknesses of 0.01 to 0.5 mm are preferred.

Two types of sample sizes may be useful.

A sample size is very small (1 to 10 / ul) for immunological Tests such as HLA tissue tests, while for more general cases the sample size is between 100 and 500 / ul. However, there are also sample sizes up to several milliliters possible.

The non-transparent tape 5 runs from a roller 1 over a guide roller 2 to the target die serving wheel 3, where by the interaction with the as patrix Serving wheel 4 recesses 5a are formed in the belt 5.

One in a container 6a such as an ampoule, tube or a Sample 6 located in the dish is transported by means of an automatic sample transport device 7a filled into the individual depressions 5a of the band 5.

The sample transport device 7a comprises a diluent 7b and a Tube 7c. The tube 7c is from that shown in Fig. 1 in full lines Position in which it is immersed in the container 6a, in the one shown in dashed lines Adjustable position in which it ends over a recess 5a. The thinner 7b contains a cleaning agent or a suitable reagent with which the from the Container 6a coming sample is dispensed from the tube 7c into the well 5a, at the same time the tube 7c is cleaned so that it can receive the next sample can without contaminating it. Details of the sample transport device 7a are not described here since they belong to the state of the art.

In addition, numbers 8 and 9 are provided for reagents. Each of these About 8 and 9 are a measured volume of a corresponding reagent the right energy to supply the sample in question and the reagent to mix with each other. In area 10 there is an incubation or chemical Reaction takes place, which is completed before another reagent (luminescent reagent) means an adder 11 is added to the sample. The intensity of the individual Samples emitted light is measured by means of a light detector 12, which located in a housing 13 and against light coming from outside by means of a diaphragm 14 is protected when the housing 13 is raised in order to transport the tape 5 further.

If the housing 13 is placed on a recess 5a containing a sample lowered, an O-ring 15 forms a light-tight cover, as FIG. 1 shows. Now the diaphragm 14 is withdrawn from the front of the light detector 12. from the light detector 12 emitted electric current or electric coming from it Pulses are supplied by an amplifier 16 and a time-counting-integrating control circuit 17 processed. Well-known printed circuit boards, microprocessors, and microcomputers can be used as control circuit 17. The intensity and amount of the measured or examined substance can via an analog output with an indicator or an oscilloscope 18 or digitally with a digital display or a digital printer 19 can be recorded.

The control circuit 17 synchronizes the step-by-step movement of the Tape 5, the input of the samples 6 into the wells 5a by means of the sample transport device 7a and the drawers 8, 9 and 11 and also the movements of the housing 13 and the cover 14. A wheel 21 moves the non-transparent tape 5 step by step and in a synchronized manner with wheels 3 and 4 forward. The tape 5 is collected on a roll 22, while the samples 6 drop into a container 23 as waste 24. Energy becomes the whole Device fed through a line 25.

The band 5 with the recesses 5a is made of a metal Block 27 (Fig. 3) guided and supported, the block 27 extending in the longitudinal direction Contains grooves 28 which have a slightly larger diameter than the depressions 5a, however, correspond in shape to the depressions 5a. This block 27 allows that a light-tight seal between the light detector 12 and a recess 5a is achieved and the samples 6 during the incubation at a constant temperature be heated. For this purpose, the O-ring 15 between the housing 13 and the band 5 resting on the block 27 is pressed together.

Fig. 2 shows a modified embodiment of the device for Generating the intended for receiving the samples 6 wells 5a of the not transparent tape 5. This device has a punch 26 with a convex Head end 26a and an anvil 30 which includes a concave recess 30a. The Stamp 26 replaces the wheel 4 of the embodiment from FIG. 1 and is controlled by the control circuit 17 controlled so that it is synchronous with the other movements of the device goes up and down. The head end 26a of the punch 26 can be spherical, conical or be designed as a slightly truncated conical rod, while the anvil 30 is a mirror image is designed so that certain wells 5a with hemispherical shape for receiving samples, conical or cylindrical shape can be generated.

Fig. 3 shows an embodiment for one with a plurality of detectors working device, which is made of aluminum with a band 5 and grooves 28 cooperates, which the wells intended for receiving samples 5a and the band 5 wear. The non-transparent tape 5 with the in its depressions 5a located samples 31 is from the block made of metal or plastic 27 supported, the grooves 28 of which are slightly wider than the depressions 5a. At this A device having multiple detectors will measure the light intensity of each one Sample located in a row of parallel samples with its own detector 32 measured. The signal delivered by each detector 32 or the corresponding pulses are amplified by means of an amplifier 33 and are used for data control and Data processing specific control circuit 17 fed. In Fig. 3 are as a solid trained detectors 32 (silicon photodiodes) shown, each in a Housing 13 are housed, the housings 13 are lined up next to one another Fig. 3 also shows the principle of a multi-channel instrument. The band 5 can be 2 to 20 have parallel depressions 5a for receiving samples, each of these Samples 31 is measured by means of its own light detector 32, which is a silicon photodiode or a photomultiplier. Each detector 32 has its own amplifier 33, where the measured light intensity (as an electric current or as pulses) processed and recorded separately for each detector through individual lines 34 will.

Fig. 4 shows an embodiment equipped with several detectors, which uses optical fibers to collect light from a number of photomultiplier detectors to feed. Of the in the depressions 5a of the re- inflected Band 5 located samples 31 emitted light is through bundled optical Fibers 36 photomultiplier tubes 37 supplied. The ends 36a of the optical fibers 36 are sealed to the outside by means of an O-ring 38, which is secured against the on the Block 27 lying band 5 is pressed. The optical fibers 36 lie in one non-transparent sheath 39, the other ends 36b being attached to a photocathode 40 of a photomultiplier tube 41 are located and by means of individual serving as a shutter Apertures 42 are covered between the individual measurements. These screens 42 protect the photomultiplier tubes 41 against light incident from outside when the measuring head 43 is loaded to advance the belt 5 to allow the next series of samples 31 arrives at the measuring point.

The diaphragms 42 are automatically removed from the photocathodes before each measurement process 40 withdrawn and automatically in the closed position after each measurement moved back. The electrical current (s) output by each detector 72 electrical pulses are generated by means of individual lines 44, not shown Amplifier and a circuit intended for data processing. The photomultiplier tubes 41 are accommodated in housings 45 lined up next to one another.

It is also possible to have a bundle of optical fibers 36 and a single one Detector in the form of a photomultiplier tube 37 to use a complete Series of samples 31 to measure. In this case, the bundle of optical fibers 36 is then from one sample point to the next until all samples are in a row one after the other measured. have been. After scanning a row, the belt 5 is advanced, to bring the next row of samples to the measuring point. The bundle of optical fibers 36 is then again from the first recess 5a through all positions moved in the row to scan all samples 31. Then the belt 5 is moved forward again and the next measuring process is initiated, etc.

This device can be used to measure quantities of substances or measure a number of living cells using bio- and chemiluminescence reactions.

The following is a specific example of measuring somatic Cells in milk indicated: an automatic sample changer became a milk sample 6 is placed in a well 5a in a volume of 10 to 100 / µl. After this When this sample was moved under the drawer 8, 10 to 500 / µl of an extraction reagent were added entered into this sample. This reagent consisted of 0.02 to 0.5% aqueous solution an ethoxylated alkylphenol type nonionic surfactant.

During the passage of the sample through the incubation section 10 became ATP (adenosine triphosphate) from the somatic cells of milk (leukocytis) and epitelic cells extracted by the extraction reagent. As the sample under the label 11 was a firefly luciferin luciferase reagent added to the pretreated sample in an amount of 10 to 100 / µl. The Luciferin Luciferase Reagent consisted of 0.01 µg / ml luciferase enzymes, 10-100 / µg / ml luciferin, 0.5-5 mM EDTA, 5 to 20 mM magnesium salt in a 10-100 mM biochemical buffer (tris, HEPES, MOPS, TES) with a pH value between 7 and 8.2, but preferably 7.75.

After the luciferin luciferase was added to the sample, generated they emit light, which is the concentration of ATP and the amount of somatic Cells in the sample was proportional. After the sample under the light detector 12 has been brought, the light intensity was measured and converted into the number of somatic cells converted by the readable sample value with known standard values was compared by means of the control circuit 17. The results were either analog or digital shown by the oscilloscope 18 or the printer 19.

The invention offers numerous advantages over by hand or also automatically operated known instruments. One of these benefits is the Simplicity, versatility in sample size and sample processing, a high speed in the sample examination, high measurement accuracy and the Possibility to use a single or multiple detectors. In the end This also includes the possibility of various different analyzes on fractions the same sample at the same time. Well-known and commercially available sample changers can be connected to this device.

The mechanisms of the device are simple and inexpensive. Also is the use of this device is economical because it is unnecessary to use measuring cuvettes or measuring ampoules to use and because the consumption of the reagents is significant manually operated devices can be lowered because you only have small Volume of the samples and thus only small amounts of reagents are required. The device can be built extremely small because you work very precisely automatic Zugber can use and you have a high measurement sensitivity because of optimal optical Sample geometry received. Because of the small size of each sample and how easy it is The construction of the device can handle large amounts of samples in a small space or to be examined. The device can be used for numerous types of analysis multiple reagent additions, time delays and incubation at constant temperatures as well as requiring rapid heating of the samples.

As mentioned above, the belt 5 can either be a number of Wells 5a for samples 6 for measuring the luminescence with a single light detector or consecutive rows of 2 to 20 samples or more for simultaneous Measurement of the luminescence with one of several detectors lined up next to each other in have a number corresponding to the number of samples in a series. Here you can more samples can also be provided in a row. The invention allows up to up to 500 samples per hour with a single row of wells and up to several to process a thousand samples per hour with a multi-detector system and to eat. With the system having multiple detectors, it is possible to do so simultaneously measure different parameters in different fractions of the same sample.

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Claims (23)

Claims: 1. Method for automatically measuring the bio and Chemiluminescence of a wide variety of samples, which is not indicated n e t that in a non-transparent, light-reflecting tape recordings like depressions be generated that this tape is transported along a path that on a First place this way a measured volume in each recess of the tape a liquid is entered that you enter at a second point along the way measured volume of at least one reagent in each of the individual receptacles of the tape located liquid enters that one of the mixture of the liquid and the at least one reagent in each well measures and emits light generates a corresponding electrical signal that this signal is processed and that the tape transport, the addition of liquid, the introduction of the reagents, illuminating and measuring the emitted light and processing it obtained signals synchronized. 2. The method according to claim 1, characterized in that one in the band A multitude of parallel rows of VOIs create wells and different fluids in each row of wells. 3. The method according to claim 1, characterized in that one Multiple parallel rows of depressions are created in the tape and in each row of Wells parts of the same liquid. 4. Device for the automatic measurement of bio- and chemiluminescence a plurality of samples for performing the method of any one of the claims 1 to 3, characterized in that they have a device 3, 4; 26, 30) to generate of recordings (5a) in a non-transparent, light-reflecting tape (5) a Means (21) for transporting the tape along a path, a device (7a) for filling a measured volume of liquid into each receptacle at a first point along the path, means (8, 9, 11) for dispensing one measured volume of at least one reagent in each receptacle of the tape liquid located at a second point along the path, a light detector (12; 32; 37) above the path of the belt and at a distance from the dispensing devices (8, 9, 11) to get from the mixture of the liquid and the at least one reagent Detect light reflected in each recording (5a) and a corresponding one to form an electrical signal, means 16, 17; 33, 17) to process this electrical signal and with a control device (17) for synchronizing the Operation of the transport device, the filling device, the dispensing device, of the light detector and the processing device. 5. Apparatus according to claim 4, characterized in that the device to create depressions (5a) or recordings a wheel (4) and one with this having cooperating mating gear (3) which have projections and depressions. 6. Apparatus according to claim 4, characterized in that for generating a stamp (26) is provided for recesses in the band (5) serving as receptacles which cooperates with an anvil (30) containing at least one recess (30a). 7. Device according to one of claims 4 to 6, characterized in that that the device (3, 4; 26, 30) for generating recordings (5a) in the band (5) at least a series of consecutive recordings is produced in the tape, moving in the direction of movement string together. 8. Device according to one of claims 4 to 7, characterized in that that the device (3, 4; 26, 30) for generating recordings parallel rows of Recordings (5a) in the band t5) are generated, each row being perpendicular to the direction of movement of the band (5) extends. 9. Device according to one of claims 4 to 8, characterized in that that the band (5) is a metal foil with a thickness of about 0.01 to about 0.5 mm is. 10. Device according to one of claims 4 to 9, characterized in that that the band (5) consists of aluminum. 11. Device according to one of claims 4 to 8, characterized in that that the band (5) consists of plastic. 12. Device according to one of claims 1 to 8, characterized in that that each receptacle (5a) has approximately the shape of a hemisphere. 13. The apparatus according to claim 12, characterized in that the Inside diameter of each hemispherical receptacle (5a) between about 2 and about 50 mm. 14. Device according to one of claims 4 to 11, characterized in that that each receptacle (5a) has the shape of a cone or a conical cylinder. 15. The device according to claim 14, characterized in that the individual recordings (5a) are between about 1 and about 25 mm deep. 16. Device according to one of claims 4 to 15, characterized in that that the smallest distance between the edges of consecutive recordings (5a) the same row about 1 mm and the largest distance is about 20 mm. 17. Device according to one of claims 4 to 16, characterized in that that a single light detector (12) is provided. 18. Device according to one of claims 4 to 17, characterized in that that the belt (5) extends transversely to the direction of movement of the same extending rows of Contains 2 to 20 recordings (5a). 19. The device according to claim 18, characterized in that a A plurality of light detectors (32; 37) corresponding to the number of those in each row located recordings (5a) of the tape (5) is provided. 20. The device according to claim 19, characterized in that the Light detectors (32) are silicon photodiodes. 21. The device according to claim 19, characterized in that the Light detectors (37) are photomultiplier tubes and that optical fibers (36) light transferred from the relevant receptacle (5a) of the tape (5) to the associated tube. 22. The apparatus according to claim 17, characterized in that the Light detector (12) is a silicon photodiode. 23. The device according to claim 17, characterized in that the Light detector (12) is a photomultiplier tube.