GB2056670A - Method and apparatus for luminescent measurement - Google Patents

Abstract

The invention relates to automatic transportation, processing and measurement of chemiluminescence or bioluminescence in samples contained in depressions 5 in non-transparent light-reflecting tape. The depressions are made by indenter 4. The tape can have one cue of sample well is for measurement of luminescence with single light detector 12, or it can have consecutive rows of 2 - 10 samples for simultaneous measurement of luminescence with a multi-detector array having an equal number of individual detectors as there are samples in one row. Transport mechanism 3, 4, 21 moves the tape past reagent injectors 8, 9, 11 and the detector. <IMAGE>

Description

SPECIFICATION Method and apparatus for luminescent measurement The invention relates to automatic transportation, processing and measurement of chemiluminescence and bioluminescence in discrete samples contained in depressions on a light-reflecting-tape. The tape can have either one cue of sample wells for measurement of luminescence with a single light detector, or it can have consecutive rows of 2-20 samples for simultaneous measurement of luminescence with a multidetector array having an equal number of individual detectors as there are samples in one row. The invention allows the processing and measurement of upto 500 samples per hour and upto several thousands of samples per hour with the multi-detector system. With the multidetector system it is possible to measure several parameters simultaneous in different aliquotes of the same sample.

The mechanics of the system is simple and inexpensive. Furthermore, the use of such apparatus is economical because there is no need for measuring cuvettes or vials and the consumption of reagents can be reduced considerably from that of manual systems by application of small volumes for sample and reagents. Miniaturizing with this apparatus is possible due to the application of accurate, automatic dispensers and the high measuring sensitivity resulting from optimal optical sample geometry. Small sample size and the simple principle ofthe apparatus enables a large number of samples being processed in a small space. The system can be applied for various types of analysis requiring multiple reagent dispensing, time delays and in cubation at constant temperatures as well as rapid heating of sample.

Bioluminescence and chemiluminescence have been applied for various types of measurements of substances (Gorus, F. and E. Schram. Clin. Chem. 25: 512-5, 1979), viability of cells (Tarkkanen, P. R.

Driesch and H. Greiling. Fresenius Z. Anal. Chem.

290:180-181, 1978) and as a means ofquantitation of antigen - antibody complexes (Velan, B. and M.

Halmann. Immonu Chemistry 15:331-33, 1978).

Normally the bio- and chemiluminescence measurements are carried out in manually operated instruments although fiow-through systems have been applied for automation. Segmented flowthrough system applies air bubbles to separate samples and to mix samples with reagents (Johnston, H.H., C.J. Mitchell and G.D.W. Curtis, pp.

210-214. In Rapid Methods and Automation in Microbiology (H.H. Johnston and S.W.B. Newsom, Eds.); Learned Information (Europe) Ltd., Oxford, 1976), while in so-called Flow Injection Analysis (FIA) the homogeneous samples move in capillaries.

Certain non-homogeneous samples, such as cell samples, biological fluids (blood, urine, milk) are not easily applicable to a flow-through system because of problems associated with precipitation of cells, proteins, fat globules and other particles in the tubes and capillaries. Therefore, the measurement of such samples is more reliable when the samples are handled discretely in individual containers through all steps in the analysing procedure.

Automatic luminescence measurement of discrete samples has been possible with a iiquid scintillation counter by applying special modifications to provide automatic reagent dispensing (see Hammerstedt, R.H. Anal, Biochem. 52:449-455, 1973) or in an instrument applying a light-transparent filter for carrying samples and reagents.

Liquid scintillation counters (LSC) are expensive, voluminous and non-optimal for luminescence measurements. The most important drawbacks of a liquid scintillation counter are that the sample size is large (from 2-10 ml), dynamic range is only 2-3 decades and there is a 20 second delay before sample goes from the transport mechanism to the measuring position. Furthermore, these counters are difficult to apply for automatic sample processing as automatic injection of reagents and incubation at a given temperature cannot be accomplished without significant changes in the original construction.

The instrument using a light-transparent filter (U.S. Pat. No.3,940,250) is meant only for samples to be filtered and its use is limited to small sample and reagent volumes only. Furthermore, it can be used for one analysis at the time.

The invention offers several advantages over the manual and the automatic instruments mentioned above. These include simplicity, versatility in sample size and sample processing, high sample output, high measuring efficiency and possibility to use single and multiple detectors, and the possibility to perform several different analysis in aliquotes of the same sample simultaneously. Conventional, commercially available sample changers can be coupled with this instrument. The principles and details of the present invention are given in the following paragraphs and illustrations.

The principles of the present invention are schematically described in Figure 1. Sample container and sample transport is based on an aluminium of other non-transparent, reflecting tape (1) of the thickness of 0.01-0.5 mm such as aluminium foil which allows forming of constant shape depressions (5) with a punch of wheel (male part) that presses the tape against an anvil of wheel (3) having corresponding depressions (female part). The shape of the sample wells is preferably that of a half sphere, but can be conical or slightly cone-shaped cylinder when made with a punch. The diameter of such wells can vary from 2 mm to several centimeters depending on the sample type and reagent system. Small wells are preferable as they save in reagent costs and allow more samples to be contained per unit length of tape.The wells are positioned in the tape in such a way that the distance between the edges of two consecutive wells is a minimum of 1 mm and preferably less than 2 mm.

Two types of sample volumes for this instrument can be anticipated, one being very small (1-10 yl) for immunological test (such as HLA tissue typing) and a more generally applicable sample size between 100 and 500 Ftl. Sample size upto several millitres are possible.

An alternative method of making sample wells is a punch, illustrated in Figure 2. The head of the punch (1) presses the foil (3) into a depression in the anvil part (2) forming a constant shape well into the foil.

The head of the punch can be sperical, conical or a slightly conical blunt rod and the anvil part a mirror image of the above to form sample wells of the shape of a half sphere, conical orcylindrical cup, respectively.

The tape with sample wells is guided and supported with a metal block (1 in Figure 3.) that has grooves slightly larger than the size of the crosssection of the sample well, but having the same shape as the cross-section of the wells (2). This guiding block allows the forming of a light-tight seal between the light detector and the sample well with an O-ring (4) as well as to accomplish heating of samples to a constant temperature during incubation. Figure 3. also illustrates the principle of a multichannel instrument. The tape (5) has 2-20 parallel sample wells (3) and each one of them is measured with a separate light detector (8) which can be either a silicon photodiode or a photc multiplier.

The detectors are sealed light-tight against outside light and light from surrounding samples with an O-ring (4) which is pressed between the detector housing (9) and the tape (5) which is supported by the grooved block (1). The detectors have individual amplifiers (6) and the measured light intensity (as electrical current or pulses) is processed and reconded individually for each detector through individual leads (7).

When photomultipliertubes are used in the multidetector system, the detector head consists of an array of optical fibre terminals to allow the measure mentofsmall samples (Figure 4). The photomultipliers are protected from exposure to daylight by a shutter that makes a light-tight seal when detector head is lifted up for moving of the tape. It is also possible to use one bundle of optical fibres (3) and a single photomultiplier detector (4) to measure a whole row of samples (11. In this system the fibre bundle (3) will move from one sample position to another until all wells in the row are measured sequentially. After the scanning of one row, the tape (2) will move to place the next row of samples to the measuring position.The bundle of fibres will again move from the first well through all positions in the row to scan all samples. The tape will move and the measuring cycle starts again, etc.

The invention will now be described, by way of examples, with references to the accompanying drawings, wherein Figure 1. is a schematic illustration of the invention wherein (1) is a roll of non-transparent tape, (2) a guiding roller, (3) a counter wheel with identations forforming sample wells in the tape with wheel (4) having corresponding protrusions, (5) sample wells in the aluminium tape, (6) a sample in a container (vial, tube, cup) in an automatic sample transport unit, (7) a dispensing tube for aspirating samples with pump (20) to wells in the tape, (8) a dispenser for reagent 1, (9) a dispenserfor reagent 11(10) a region for incubation or time delay for a chemical reaction to proceed to completion before introducing the last reagent (luminescence reagent) with dispenser (11); the light intensity emitted by the sample is measured with a light detector (12) that is closed in a housing (13) and protected from outside light with a shutter (14) when housing is lifted up for moving the tape. When the housing is lowered on the sample, O-ring (15) makes a light-tight seal as shown in Figure 1. In this position shutter is moved from the front of the detector (12). The electrical current or pulses from the detector are processed by an amplifier (16) and timer/counter/integratoricon- trol unit (17). Light intensity and quantity of measured substance can be recorded through an analogue output with a recorder or oscilloscope (18), or digitally with a digital panei meter or printer (19).

Control unit (17) synchronizes the stepwise movement if tape, aspirating samples to sample wells (5), dispensers (8, 9, 11) as well as the movement of detector housing (13) and shutter (14). Wheel (21) moves non-transparenttape forward stepwise together with wheels (3) and (4). The tape is collected on roll (22) and the samples drop to waste (24) in the container (23).

Figure 2. shows a device for making sample wells in the non-transparent foil (3) with a punch having a convex head (1) and an anvil having a concave depression (2). The punch moves up and down in a synchronous manner controlled by the unit controlling thewhole instrument (see 17 in Figure 1.).

Figure 3. schematically illustrates an example of a multidetector system applied with aluminium tape and the principle of grooves supporting the sample wells and the tape. Non-transparent tape (5) having the samples (3) in the wells, is supported with a metal or plastic block (1) having grooves slightly widerthan the sample wells (2). In the multi-detector system the light intensity of each sample in a row of parallel samples is measured with a separate detector (8). The signal or pulses of the detector are intensified with an amplifier (6) and are forwarded to a date control and processing unit (see 17 in Figure 1.). The illustration shows solid state (silicon photodiode) detectors (8) enclosed as an array in a housing (9). Amplified signal or pulses are carried through a lead (7) to data output.

Figure 4. is a schematic illustration of a multidetector applying optical fibres to conduct light to an array of photomultiplier detectors. Emitted light from samples (1) in the wells of reflecting tape (2) isb conducted through bundles of optical fibres (3) to photomultiplier tubes (4). The ends of fibres (5) are isolated from outside light by O-rings (16) which are pressed against the tape that is supported by the grooved block (12). The optical fibres are isolated with a non-transparent sheath (7) and the other end at photocathode of the photomultiplier tube (8) is covered with a shutter (9) between the measurements. This shutter protects the tube from ambient light when the measuring head (10) is lifted upto move the tape to the next row of samples. The shutters are removed from the front of the photocathodes automatically prior to measurement and returned back after the measurement. The electrical current or pulses from each detector are led to an amplifier and data processing with individual wires (11). The photomultiplier tubes are enclosed in an array of housings (13).

Claims (18)

1. A method of measuring the intensity of bio- or chemiluminescent light emitted from a sample which comprises depositing the sample in well formed in or on a non-transparent light-reflecting foil and arranging the foil in relation to light detector means so that light emitted from the sample imping es on the detector means.

2. A method according to claim 1 in which the well comprises an indentation formed in the foil.

3. A method according to claim 1 or claim 2 in which a plurality of wells are formed at respective spaced locations on the foil, respective samples are deposited in the wells and the intensity of light emitted by the samples is measured by sequentially arranging the wells in relation to the detector means so that light emitted from the samples impinges thereon.

4. A method according to any preceding claim wherein the foil is a sheet of thin, 0.01-0.5 mm thick, non-transparent, light-reflecting metal or synthetic material.

5. A method according to any preceding claim wherein the sample wells are of the shape of a half sphere, conical or cone-shaped cylinder having an inside diameter of 2-50 mm and being 1-25 mm deep.

6. A method according to any preceding claim wherein sample wells are arranged in a line, there being a minimum distance of 1 mm and a maximum distance of 200 mm between the inside edges of the consecutive wells.

7. A method according to claim 6 wherein the light reflecting foil has a single line of sample wells for the measurement of bio- or chemiluminescence intensity with a single light detector.

8. A method according to claim 6 or claim 7 wherein the light reflecting foil has a line of rows of 2-20 parallel samples wells.

9. A method according to claim 8 wherein each well in the row contains a different sample for the measurement of one type of luminescent reaction.

10. A method according to claim 8 wherein each well in the row contains an aliquot of the same sample for the measurement of different lumines cent reaction.

11. A method according to any preceding claim wherein the detector means comprises a detector assembly consisting of an array of 2-20 parallel detectors for a simultaneous measurement of re spective samples.

12. A method according to claim 11 wherein the light detector array consists of silicon photodiodes.

13. A method according to claim 11 wherein the light detector array consists of photomultiplier tubes and the light emitted by the sample is conducted through bundies of optical fibres to the photomulti plier tubes.

14. A method according to any one of claims 8 to 10 wherein the individual sample wells of a row are scanned sequentially with a laterally moving bundle of optical fibres conducting the emitted light to a single light detector.

15. Apparatus for measuring the intensity of bioor chemiluminescent light emitted from a sample, comprising indenting meansforforming a well in a non-transparent light reflecting foil in which a sample may be deposited, light detector means for detecting light emitted from the sample and transport means for moving the foil between the locations at which well formation, depositing and detecting are effected.

16. Apparatus according to claim 15 wherein the transport means is adapted to move a continuous band of foil between said locations, the indenting means is adapted to form a series of wells spaced longitudinally along the band and the transport means is adapted to move the band so that the wells are presented sequentially to the detecting means.

17. Apparatus according to claim 15 or claim 16 including automatic means for depositing a sample or samples in said wells.

18. Apparatus according to any one of claims 15 to 17 including: A. a wheel or punch for pressing the sample wells on the foil, B. an aspirator system for delivering a measured volume ofthe sample in each well, C. several dispensers for delivering measured volumes of a plurality of reagents into each well at proper force to provide the mixing of the sample and reagents, D. a single or multiple system of light detectors placed on the top of the foil carrying the samples, E. an amplifier for the electrical current or pulses registered by the light detector, F. a data output and data processing unit, G. a sample transport mechanism, H. a control circuitry for the synchronizing and controlling of the aspirator, dispensers, sample transport mechanism, light detectors and data processing systems.