GB2279740A

GB2279740A - Sample preparation for luminescence measurement

Info

Publication number: GB2279740A
Application number: GB9412013A
Authority: GB
Inventors: Sergey Shnizer; Vladimir Piuk; Natan Ravin
Original assignee: Lumitest Ltd
Current assignee: Lumitest Ltd
Priority date: 1993-06-25
Filing date: 1994-06-15
Publication date: 1995-01-11
Also published as: GB9412013D0; IL106144A0; DE4421792A1

Description

2279740 METHOD AND APPARATUS FOR THE MEASUREMENT OF LUMINESCENCE OF

BIOLOGICAL FLUIDS The present invention relates to a method -and apparatus for luminiscence measurements of biological fluids. More particularly, the invention relates to a method and apparatus for the preparation of a sample to be used for the measurement of luminiscence radiation of biological fluids.

BACKGROUND OF THE INVENTION

Luminometry is a well-established method used in biochemistry and immunology. A problem which is encountered in luminiscence measurements of radiation of biological fluids, is the presence of oxygen radicals. The oxygen radicals formed during metabolic disturbances react with biomolecules such as proteins and lipids, leading to the formation of peroxides and other oxygen adducts. The is decomposition of the peroxides releases bond energy in the form of chemiluminiscence, -which of course will interfere these measurements. This decomposition which occurs mainly in the presence of water,releases an amount of energy which initiates some further reactions which are also accompanied by radiation. Moreover, the results obtained are not reproducible, since even slight changes in the humidity will strongly influence the radiation which is measured. Therefore, one of the requirements for such measurements is that the samples to be measured 2S should be substantially complete dry.

2 - In order to prevent the above undesirable reactions, the obvious solution will be to apply a prior dehydration on the sample to be measured in order to provide only one radiation source. There are a number of methods that can be used for dehydrating biological materials. among them the most encountered is the freeze drying also known as lyophilization. In this method, water is removed by sublimation from a frozen tissue; if the temperature is maintained as low as possible, substantially no changes in the physical structure of the specimen to be measured were detected. Accordingly, the free radical content remains quite constant as a result of the dehydration by this method. However, this drying operation takes a long period of time.

is In the European Patent Application Number 148,736, a new method for luminiscence measurements Of samples in transparent multi-well plates is described. According to the method, a luminiscence quenching reagent is added to the samples in sequence to provoque luminiscence reactions.

Examples for such luminiscence reagents are trichloroacetic acid, hydrochloric acid and acetic acid. The main disadvantages of the method are: (1) the formation of adducts which makes the reliability of the results obtained to be dependent on the chemical purity of the adducts used and (2) the additional chemical reactions which occur at the same time including those which generate is also radiation and thus will limit the accuracy of the results obtained.

In the recent European Patent Number 446,859 an apparatus for detecting chemical luminiscence of fluid samples is described. The apparatus is based on the use of a plurality of optical detectors, different in sensitivity, in the vicinity of a photometric cell. Nothing is mentioned in the specification on the problems of humidity and interference resulted thereof.

It is an object of the present invention to provide a novel method for the preparation of a biological fluid sample for measuring its luminiscence radiation. It is another object of the present invention, to provide a novel method for the preparation of a' biological fluid sample for measuring its luminiscence radiation, which is fast and produces accurate results." It is yet another.object of the present invention to provide a novel apparatus for the preparation of a sample of a biological fluid for measuring its luminiscence radiation.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a method for the preparation of a sample of a biological fluid to be used for the measurement of luminiscence radiation which comprises:

(a) heating under vacuum a vessel containing the sample uniformly distributed at a temperature in the range of between the freezing point of the sample and that of a - 4 peroxide possessing a ring of two carbonyl groups and two hydrocarbons, having a reaction decomposition with said fluid in the range of 300 to 650 nonometers, and (b) advancing continuously the sample in said vessel. The measurement of biochemiluminiscence radiation is carried out in an apparatus within a defined period of time after the sample has been heated at the above temperature according to the signals arriving from the luminiscence detectors. Among the advantages of the method it should be mentioned the presence of a very low concentration of peroxides decompoition products in the system fact which causes a high reproducibility of the results.

BRIEF DESCRIPTION OF THE DRAWINGS.

Figure 1, correlates the influence of a reduced pressure on the heating time, on an empty vessel (1) and a vessel with a sample.

Figures 2,3, and 4 illustrate schematically an apparatus for the luminiscence measurement according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The drying operation may be carried out in a vessel,generally being a disposable one, possessing a shape corresponding to that of a miniature Petry cap. The sample to be measured is distributed uniformly over the vessel surface, most preferably up to a film thickness of 0.5 nm. In principle the measurements of biochemiluminiscence is - E.

radiation may be carried out also for a film possessing a larger thickness but in.this case the accuracy of the measurement might be affected. The continuous movement of the sample into the vessel, provides only a small amount of bubbles to appear during the drying and thus will maintain a uniform distribution of the dry matter on the surface.

According to a most preferred embodiment, the peroxide used has a reaction decomposition temperature with the fluid to be tested in the range of between 400 to 600 nanometers. As known, the intensity of biochemiluminiscence radiation of heated samples changes with the time after reaching the reaction initiation temperature. At the beginning. the luminiscence radiation intensity- is constantly increased until will reach a stable value, which should be measured by means of photo-electron converter and is compared to standard signal.

During the measurement of biochemiluminiscence radiation of the heated samples, the registered signals will also include noises from different origins, such as noises of the photo-electric itself. In order to obtain an accurate result of the radiation weasurement of the sample, it is required to isolate the signal which is proportional to the luminiscence of the beam flux.

During the measurement of biochemiluminiscence radiation of the heated samples, the registered signals will also include noises from different origins, such as noises of the photo-electric itself. In order to obtain an accurate result of the radiation measurement of the sample, it is required to isolate the signal which is proportional to the luminiscence of the beam flux.

The correlation of the reduced pressure of the sample which prevails during the drying versus the time of heating, is illustrated in Figure 1. Graph 1, represents this correlation for an empty vessel and graph 2, for the vessel which contains a sample of 200 pl of blood plasma.

As can be noticed from graph 2, a constant vacuum value of 1 mbar, as obtained with the empty vessel, is reached already only after about 16 minutes of heating, which indicates that the drying operation has been completed.

is In fact, the preparation is completely dried when it is obtained in vacuum camera at pressure not exceeding mbar.

According to another aspect of the present invention, an apparatus for luminiscence measurements of biological fluids, using the above method is described. As illustra ted in Figures 2, 3 and 4, the apparatus consists of the following main parts:

A - Analysis unit, which consists of the following main components:

(1) a photo-electronic converter; (2) a photo-electronic converter cooler; (3) an obturator, and (4) a sample signal source.

B - Signal control and processing unit, which consists of the following main components:

is c - (1) a photoelectronic converter signal amplifier (5) which amplifies the electric signals at the photo-elec- tronic converter output and serves as, a filter fcr dis- turbances. (2) a programmable controller (6); (3) a control unit (7), and (4) an automatic temperature control unit (8). Drying and heating unit, which consists of the following main components: (1) an opaque loading cap (9); a device for blocking the opaque cap removal (10); a vessel (11) and a vessel positioning unit (12); a vacuum cap (13); a flask (14); - a vacuum pump (15); - a beater (16); a device for liquid distribution in the vessel (17); - a pressure sensor (18); a controlled valve (10, a device to enable the transfer of an exact amount of liquid to the vessel with the sample (20).

8 - is The flask (14) can be located underneath vacuum cap (13) and they form a vacuum camera.

The heater (16) and the device for liquid distribution in the vessel (17) are located inside the flask (14). In the device for the vessel (17) shown in Figure 3 there is a motor (21) with eccentric 22, located on its axis and going into the washer opening (23), which -is connected rigidly to the heater (16). The washer (23) is connected to the flask (14) by an elastic element (24). The flask (14) is located on a vertical motion screw (25) of the transportation unit D (Figure 2). The transportation unit D includes a movable platform (26), its drive (271), a vertical motion screw (25) with a nut (28) and a nut drive (29). The feeding unit (E) provides voltage to all the device units. The device is operated by signal processing and control unit. According to the program stored in the controller, it supplies signals to the control unit (7). From the output of the control unit (7) the control signals are conveyed to all the parts and units. As a result, the following sequence of operations is automatically performed in a closed lcop (Figure 2): - Vessel location (position I); - Loading of the original material for analysis and the stage of preparing: drying and heating (position II); is - Preparation analysis (position III) and resetting the entire device to the initial position.

In the initial position I the movable platform (26) is set to enable fixing of flask (14) under the removable opaque cap (9), while the site for vessel positioning (12) is in its upper position.

When the vessel site is so located, the cup (9) is unblocked and it is shifted to enable the vessel location or removal.

The empty vessel (11) is located in the place (12), while the cap (9) is'returned to its original position and is blocked. Signal arriving from the control device (7) will operate the drive (29).

The flask (14) with the device inside is fixed in the lower position. Upon operating the drive (27), the platform (26) is shifted horizontallyand fixed in the position II where flask (14) will be located coaxially with the vacuum cap 13. Then the flask (14) will be raised vertically until it will become tightly connected with the vacuum cap (15) thus generating a vacuum camera.

The control device operates the vacuum pump (15),controls the vaLlve unit (19),'-for the liquid distribution (17) and the heater (16). The tested liquid is introduced into the vessel (11) by means of proportioner (20). The pressure in the vacuum camera will decrease, and thus will cause an intensive drying of the material to be measured and as S is a result, the desired preparation is formed for its further analysis. The sample under study is- heated during the drying process to prevent its freezing when an intensive liquid evaporation takes place.

A uniform distribution of the material under study at the bottom of vessel (11) is provided by an horizontal movement of the vessel (11) along a clo.sed contour during the rotation of an eccentric unit (22). The drying process is terminated when a signal arrives through the sensor (18) from the drying and heating unit output to the control device. At the end of the drying process, the following operations should be done:

- The vacuum pump (15) in unit C is switched off. - Air is supplied to the vacuum camera.

- The flask (14) with the vessel is lowered to the lower position.

Then the platform (26) is moved to the position Ill, in which the vessel 11 with the sample inside is located underneath a Photo-ElectronicConverter (PEC) cathode (1). The flask (14) with the sample inside is then shifted to the upper position, in which the sample analysis is performed.

In order to carry out the analysis, the sample is heated to the required temperature by means of the heater (16). The heater (16) consists of a heating element (30) and a temperature sensor (31) which together with an automatic S is temperature regulator form the beating scheme. A particular feature of the heating scheme is that a powerful transistor has to be used as heating element, being at the same time used as a functioning element of the ATC unit (8). The power dissipated by the transistor, and accordingly the released energy. are defined by a control device voltage supplied to the transistor base from the ATC (Automatic Temperature Control) unit.

The ATC unit which maintains a stable temperature regime operates as follows:

Temperature sensor 31, which is located directly ort the transistor body, senses the temperature changes occurring in the transistor body. The sensor output is connected to one of the ATC unit comparators, while a sample signal is supplied at its other output from the dc source. The comparator output is connected to the heating elementtransistor base, whose emitter and collector are connected directly to the DC source. When the temperature of the transistor body changes, the electric signal magnitude at the temperature sensor output will be varied and will accordingly cause a change of the electric signal voltage at the comparator output (transistor base). The stable temperature regime will be defined by temperature sensor parameters. The sample is being heated during both the analysis and the preparation drying, however the heating temperatures in the two cases are much different. The is temperature gradient is effected by changing the reference signal magnitude in the ATC unit according to the signal arriving from the control device.

The analysis of the sample is then carried out by a known instrument, for example a PEC. In order to reduce the heat currents in the PEC, it is suggested to place the sample inside a cooler (2). in which a co nstant temperature is maintained. The cooler switching on and off, is effected by the signals arriving from control scheme.

During the analysis, the light flux from the preparation is periodicaly interruped by means of obturator (3),which provides the signal measurement in case of back-ground disturbances.

The present scheme of the chemiluminescence signal measurement is achieved by a comparison with a sample signal. For this purpose, a high frequency light diode is used as a sample signal source. The PEC will analyze the sample signal when the vessel will be in position II.

The sample signal magnitude value is loaded to a controller (6) memory. When an error signal is present, an automatic correction of the magnitude of the signal arriving from the sample under study is performed.

The processing of the analysis results in unit (B) is performed using a generally known method of signal processing. The controller (6) fixes and reffeirbers the instantaneous voltage values at the output of PEC during the r - 13 - time period T with equal time intervals I'V1. Signal is processed by the minimum of the absolute value of the derivative, in an ideal case at the point where the deri vative is equal to zero,which corresponds to the straight section of the characteristic or to the bending point.

The analysis results can be presented either. in a digital form at the image device screen, or in the form of a printout when a printing device is connected. Signal control and processing unit can be connected to a PEC via a standard interface RS-232.

At the end of the analysis, the f lask (14) is lowered to its lower position,the platform (26) is returned to position 1, the flask (14) with vessel (11) are raised to the upper position. Then. the vessel (11) witt the sample is under study is removed from the device.

Of course,.a person skilled in the art may also conceive to use other versions of the device implementation which contain the blocks included in Figure 2.

- 14 C 1 A I M S:- 1. A method for the preparation of a sample of a biological fluid to be used for the measurement of its luminiscence radiation by a detector which comprises the steps of:

(a) heating under vacuum a vessel containing the sample, uniformly distributed therein, at a temperature in the range of between the freezing point of said sample and that of a peroxide possessing a ring of two carbonyl and two radicals having a decomposition reaction with said fluid in the range of 300 to 600 nanometers; (b) advancing continuously the sample in said vessel.

Claims

2. The method according to Claim 1, wherein the drying operation is

carried out in a vessel possessing a shape corresponding to that of a miniature Petry shape cap.

3. The method according to Claims 1 or 2, wherein said sample distributed uniformly over the vessel surface has as a thin film with a thickness in the range of between 0.1 to 0.5 mm.

4. The method according to Claims 1 to 3, wherein said has a decomposition reaction with said fluid in the range off 400 to 600 nahometers.

5. The method according to Claims 1 to 4, wherein said measurement is carried out continuously in a closed loop.

6. An apparatus for the measurement of luminiscence of a biological sample which comprises:

(a) an analysis unit; (b) a signal control and processing unit; (c) a drying and heating unit; (d) a transportation unit, and (e) a feeding unit. 7. The apparatus accerding to Claim 6, wherein said analysis unit comprises: (a) a photo-electronic converter; (b) a photo-electronic converter cooler; -(c) an obturator, and (d) a sample signal source. 8. -The apparatus acccrding to Claim 6.. wherein-said signal control and processing unit comprises:

(a) a photo-electronic converter signal amplifier; (b) a programmable controller; (c) a control unit, and (d) an automatic temperature control. 9. The apparatus according tc Claim 6, wherein said drying and heating unit comprises: (a) an opaque loading cap; (b) an element for blocking the opaque cap removal; (c) a vessel with its positioning element; 16 - (d) a vacuum cap; (e) a flask; (f) a vacuum pump; (g) a beater element acting also as a functioning element of the Automatic Temperature Control unit; (h) a device for the sample distribution in the vessel a pressure sensor; (k) a controlled valve, and (1) a proportioner.

10. The apparatus according to Claim 9, wherein said heater is implemented in a single element which provides an automatic regulation and constant temperature.

11. A method for the preparation of a sample of a biological fluid to be used fcr the measurement-of luminiscence radiation, substantially as described in the specification and claimed in any one of Claims 1 to 5.

11.. An apparatus for the preparation of a sample of a biological fluid to be used for the measurement of luminiscence radiation, substantially as described in the specification and claimed in any one of Claims 6-to 10.