DE19916430C1 - Apparatus for use in performing receptor ligand and affinity tests - Google Patents

Abstract

The invention relates to a device for use in carrying out receptor ligand and affinity tests, which may be fluorescence or enzyme immunoassays that can be used in medical diagnostics, environmental and food analysis. The invention is intended to be able to analyze various samples in a simple form and with little expenditure of time. For this purpose, a hollow piston and a hollow cylinder made of at least partially transparent material are used. In this case, a temporarily closable opening is present in the hollow cylinder, through which the sample can get into the interior of the hollow cylinder when the hollow piston and the hollow cylinder move in a corresponding relative manner. The hollow piston has a porous separating layer on its end face, which is covered with a gas and liquid sealing plate in the interior of the hollow piston. This sealing plate is designed such that it can be moved translationally inside the hollow piston and, with a corresponding relative movement of the hollow piston and sealing plate, a prepared sample from the hollow cylinder reaches a space between the porous separating layer and the sealing plate. Subsequently, for example, a fluorescence or enzyme immunoassay of the sample can then be carried out.

Description

The invention relates to a device which Performing receptor ligand and affinity tests, e.g. B. Use fluorescence or enzyme immunoassays can be detected, with possible applications in medical diagnostics, environmental and life can result in medium analysis.

Especially in environmental and food analysis So far, such tests can only be used for so-called Screening methods are used, however no concrete statement regarding a single sample can be made and only the analysis wall can be restricted accordingly in advance. The statements obtained with such tests are so far officially not yet recognized and can therefore also not be used as clear evidence.

In the known methods for such quantitative or semi-quantitative tests become so-called fest phase immunoassays performed. With such a Test, namely the company's so-called dioxin assay CAPE become analyte-specific antibodies on the wall immobilized in a test tube. With the so-called BTEX rapid assay from Coring Systems immobilization of antibodies to magnetic  Particle. In both cases, however, are complex Washing and pipetting steps required to one is badly flawed and usually also not directly at the actual place of examination, but in laboratories with a relatively high effort must be led.

However, this aspect is particularly important in environmental analysis of importance since the analyte in collected samples very easily, in a short time, with low labor and instrumental effort and ge minor error should be examined.

In US 5,077,012 there is a device for recording biological fluids and implementation of qua known literal and quantitative tests. For this becomes a cylindrical container open at both ends ter with a removable memory on the cylinder can be attached at one end. The This test memory has an open page and a cyto logical membrane is fixed in it.

A similar solution is known from US 4,960,130. It will also have a hollow cylinder on both sides is open, used. On one side is a collection storage unit attached. Furthermore, a zy Lindric hollow piston used on one side is closed with a cover and a second Cover with an opening and a connection cover used for the second side of the piston. The piston can reciprocate in the cylinder become. In addition, a can on the cylindrical container Storage tanks attached and by moving the Kol bens in the cylindrical container of the collecting container be moved into the collection and storage unit so  that liquid from the cylindrical container into the Collection container flows.

In US 4,553,553 there is a device for detection of bacterial fungi and viruses in blood fluid are described. The blood is circulating through an adsorbent material using a piston cylinder palliative unit. The piston has an opening through which the blood through the adsorbent material can get into the housing.

In the device described in US 4,057,499 Separation and collection of plasma or serum Blood samples will also be a cylinder in which blood is included, and a piston is used. Besides, is a one-way valve with a filter in the collecting cylinder which included to separate plasma or serum to reach.

With the device described in US 3,969,250 components contained in fluids are to be filtered or be isolated to post them automatically to be able to analyze chemically. Here too is a Filter element for removing particles and a Membrane valve for isolating the filtrate with a Piston cylinder unit used.

Furthermore, there is an apparatus and a method for performing fluorescence immunoassays in DE 197 47 572 C1. Building on be knew fluorescence immunoassays an optical fiber trained in a piston of a piston-cylinder unit Detete measuring chamber with the interior of the cylinder is used, so that the sample by corresponding relative movement of the piston and  Cylinder unit flowing during the measurement Is movement.

In the separating device described in DE 41 28 923 C2 device for liquid fractions becomes one in one Tubes of separating filter pistons with pistons housing, valve, filter area and socket used.

DE 35 42 331 C2 is a filter device for Liquids known, especially for Separa tion of the liquid component of the blood serum can be set. This device uses a cylindrical tube closed at the bottom, in which a piston-like sliding sample tube, which has a filter on its underside has been recorded.

It is therefore an object of the invention to provide one possibility propose using the different samples in very Simple form and quick to prepare can be tested and analyzed.

According to the invention, this object is achieved with the features of claim 1 solved. Advantageous design shapes and developments of the invention agree with those in the subordinate claims held characteristics.

The device according to the invention, for use when performing fluorescence or enzyme muntests can be used essentially of three parts. It is in egg Nem hollow cylinder received a hollow piston and ge leads, with an opening in the hollow cylinder for the Entry of a sample is present, so that the invented  device according to the invention essentially as a conventional syringe is formed. Inside the Hollow piston is one for the most diverse fluids dense sealing plate on the end face that in the  Interior of the hollow cylinder shows, arranged, the Sealing plate within the hollow piston translational is movably arranged and guided. The already he imagined face of the hollow piston is porous as Separating layer formed, a conventional Liche frit, such as that from column chromatography is known, but also a membrane can be used can.

In a room inside the hollow cylinder, between the hollow cylinder inner wall and hollow piston is formed, particles may be present. The par particles are selected so that their grain size prevents them from passing through the porous interface can penetrate.

The porous separating layer, which acts as an end face for egg NEN hollow piston is used or another porous Interface layer at the entry opening for the Sample can be arranged in the hollow cylinder, can advantageously be selected so that they are e.g. B. zel Retain lular components of a whole blood sample can and a blood plasma thus obtained, without additional preparation steps can be used as a sample can.

It is with sampling and sample preparation also important that the trained in the hollow cylinder Opening can be temporarily closed, whereby an appropriately trained stopper for this can be pushed onto the opening if necessary and this then closes, can be used. Another An alternative to this is to use a Check valve, which is in or at the opening is arranged.  

Sampling in preparation for actually tests can be carried out in such a way that a relative movement between hollow cylinder and hollow piston, with the opening of the hollow cylinder open is performed and the sample through the opening in the space in which the particles can be contained is recorded. The corresponding relative movement of hollow pistons and hollow cylinders can be fixed one of the two parts and corresponding movement of the each other or by moving both parts be enough. It is particularly beneficial if corresponding end stops on the hollow piston and / or Hollow cylinder or the relative movement manipulation unit, such as a corresponding Vending machines are available, so that in any case defined sample volume in the invention Device is included.

For performing fluorescence immunoassays in the room where the particles are located e.g. B. antigens or antibodies present, wherein either the respective antigens or antibodies a suitable fluorophore or enzyme.

Before sucking the sample into the invention Device can be either antigen or antibody reversible on the inner wall of the hollow cylinder and the other partner on the surface of the par be immobilized. If the sample is now in the sucked device according to the invention, anti genes or antibodies through the sample from the inside wall detached and bind together.

Binding the unlabeled or labeled biocom components can also be found on the porous interface,  which are arranged on the end face of the hollow piston is done, with a combination of binding the inner wall of the hollow cylinder, the porous Separating layer and on the particles may be possible. Different partners can also be contacted the inner wall, the particles or the separating layer be bound to at least two different ones Analyte a sample to perform a test.

Following this, when the opening of the Hollow cylinder an opposite relative movement performed by hollow pistons and hollow cylinders, whereby at the same time a certain part of the sample volume through the porous separating layer into the interior of the cavity piston arrives. This will now take place inside of the hollow piston from the sample volume Sealing plate covered and between porous separation layer and sealing plate in a certain sample room containing lumens.

Subsequently, the Vorrich invention with the correspondingly prepared sample analyt be evaluated quantitatively. Therefor is light with at least one fluorescent stimulating Wavelength, according to the fluoro (s) used phor (en) on the in the space between porous separation layer and sealing plate inside the hollow piston prepared sample directed and with a corresponding optic detector the intensity of the fluo Rescence light measured, with known for this Measurement methods can be used. As light sources come z. B. sending monochromatic light Laser used. But it can also be mixed light Appropriate filters can be used. Such filters can also be placed in front of the optical detector  be arranged to at least influence the scattered light to reduce.

It can also be cheap, the optical Auswer tion within an otherwise closed housing to avoid external light influences conclude.

The hollow cylinder and the hollow piston can in Entirely one transparent for the light used material. Alternatively, you can only appropriate windows for the irradiation treatment of the prepared sample and the detection of the Fluorescence light can be formed.

With the invention, the most varied can be known ten assay formats, such as competitive, titration, ver pushing and sandwich assays for the different most receptor-ligand systems, such as. B. Antibody Complement antigen, lectin carbohydrate, DNA or RNA tary nucleic acid, DNA or RNA protein, hormone Re zeptor, enzyme-enzyme cofactors, protein G or protein A-immunoglobin or avidin-biotin carried out who the. The respective analyte concentration can directly proportional and in sandwich immunoassays can be determined inversely proportional. With the he There is also the possibility of implementation competitive assays for high molecular weight analytes.

Are used for marking fluorophores in the near Infrared range can be used, can body fluids immediately, d. H. without additional preparatory steps and pre-treatment measures are examined.  

If the device according to the invention for the enzyme immunoassays are used Place the fluorophore enzymes as markers. In this case, it can be beneficial if this ent speaking enzyme-specific substrate and given if suitable buffer substances for a condition sample in advance on the underside of the Hollow piston sealing plate are deposited.

If the corresponding sample is now, as with the Performing fluorescence immunoassays, sucked into the sample according to the invention and then subsequently partly through the porous separating layer in the space between the porous interface and the seal plate inserted inside the hollow piston, can a time-resolved colorimetric evaluation be performed. On the one hand there is the possibility the intensity of one for the corresponding caused color change typical wavelength to measure time-resolved.

With the device according to the invention can also various receptor ligand or affinity tests, as fluorescence immunoassays or enzyme immunoassays be carried out again by a Relativbe movement of hollow cylinder and hollow piston a defin sample volume through the opening into the speaking of the relative movement enlarging space in is sucked inside the hollow cylinder. It can depend on at least one analyte contained in the sample of its concentration with a fluoro partner of a receptor Oust ligand systems. The fluorophore or or enzyme-labeled partners can be at the parties gel and / or the porous separation layer,  for this, in addition to covalent bonds, e.g. Belly adsorptive or affinity bonds come into question can.

The sample prepared in this way is then returned to Ver close the opening of the hollow cylinder and through opposite relative movement of the hollow cylinder and Hollow bulb through the porous interface in the accordingly enlarging space between porous Separating layer and the sealing plate within the hollow guided and the corresponding evaluation with fluorescence or colorimetric measurement then be done.

The device according to the invention can continue to do so be carried out using a hollow cylinder , which has two chambers, in each of which one appropriately trained hollow piston, which is preferred independently of each other relative to the chambers of the Hollow cylinders can be moved are introduced. This allows such a hollow cylinder in each chamber who determines at least one separate analyte the.

In such an embodiment, as in the with a simple hollow cylinder and hollow piston can also use labels with multiple fluorophores or enzymes that a Multiana lyt determination is possible. When using multiple different fluorophores should be used their immissions or excitation wavelengths do not influence each other.

The invention is described below by way of example be.  

Show:

Figure 1 shows the schematic structure of an example of a device according to the invention, before the actual sampling and sample preparation.

Fig. 2 shows part of the device shown in Figure 1 before in an enlarged view.

FIGS. 3a and 3b is a schematic representation of receiving a sample in the apparatus according to Figures 1 and 2.

Fig. 3c the transfer of part of the sample lumen into a space formed inside the hollow piston and

Fig. 3d shows a schematic representation of the actual measurement in a fluorescence immunoassay.

In Fig. 1, a possible example of an inventive device he is shown schematically. It is clearly recognizable how a hollow piston 2 is guided into the interior of a hollow cylinder 3 , the play between the hollow piston outer wall and the hollow cylinder inner wall being kept relatively small, to largely rule out tilting errors on the one hand and on the other hand to achieve a certain degree of sealing.

In the hollow cylinder 3 , an opening 8 is formed on its outer end face through which, as will be described hereinafter, the sample can get into the interior of the device.

The hollow piston 2 is designed here in such a way that its end face pointing into the interior of the hollow cylinder 3 is designed as a porous separating layer 5 . The porous separating layer 5 can be made of plastic, plastic mixtures, glass, ceramic or metal.

Above the porous separating layer 5 , a sealing plate 4 is in the interior of the hollow piston 2 , before the samples are taken and the measurement is carried out, lying directly on the porous separating layer 5 , angeord net. The sealing plate 4 consists of a liquid and gas-tight material. It is dimensioned and designed so that it can be moved transla torically inside the hollow piston 2 along its longitudinal axis.

On the piston 2 is at least one pressure equalization opening 1 provided, which allows a gas inlet or outlet to or from the hollow piston. 2

In this example, in the hollow cylinder 3 , 2 particles 6 are present in the space between the end face of the hollow piston, the grain size of which is selected such that they cannot penetrate the porous separating layer 5 . It can e.g. B. polymers, preferably cross-linked can be used as particle materials.

In a form not shown, the opening 8 can be closed for a safe inclusion of the particles 6 in the hollow cylinder 3 by means of a further porous separating layer as a frit, a filter or a membrane, through which the respective sample can get into the hollow cylinder 3 without further notice can.

In Fig. 2 it can be clearly seen that on the outer circumferential surface of the hollow piston 2 , in the region of the porö sen separating layer 5 circumferential sealing elements 10 are present, which prevent sample liquid from entering the gap between the hollow piston 2 and the hollow cylinder 3 and with which the necessary vacuum to suck up the sample can be generated.

In addition, it is shown more clearly that the sealing plate 4 is seen with a circumferential seal 9 , which prevents sample liquid from entering the space above the sealing element 4 of the hollow piston 2 .

In order to prevent the sealing plate 4 from tilting during a transatory movement along the longitudinal axis of the hollow piston 2 , the thickness of the sealing plate 4 can be chosen accordingly, taking into account the free cross section in the interior of the hollow piston 2 .

In order to achieve the same effect, guide elements (not shown here) can also be formed on the upper side of the sealing plate 4 , which preferably rest in a radially symmetrical arrangement on the inner wall of the hollow piston 2 for guiding the sealing plate 4 with a corresponding translatory movement.

In FIG. 2 is also clearly illustrated that are attached to the various particles 6 analytspezifi specific antibody. In a form not shown, the corresponding antigens 7 are reversibly bound to the inner wall of the hollow cylinder 3 , as indicated in FIG. 3a. In this form, the device according to the invention is available for receiving and preparing a corresponding sample. Either the antibody or the antigen 7 is labeled with an appropriately suitable fluorophore for carrying out fluorescence immunoassays or with an appropriate enzyme for carrying out enzyme immunoassays.

As can be seen in Fig. 3a, opening 8 of the device according to the invention is immersed in a container containing a corresponding sample and it can by moving the hollow piston 2 , as indicated by the arrow, in the vertical direction, the volume of the space 14 inside of the hollow cylinder 3 is enlarged and the sample is taken into this space, since the sealing plate 4 forms a liquid-tight and gas-tight seal.

Subsequently, as is indicated in FIG. 3b, the bonds of the antigens 7 on the inner wall of the hollow cylinder 3 are released and the fluorophor-labeled antigens 7 can move freely within the liquid sample.

As already mentioned in the general part of the description, by adhering to a certain pre-given path, which, in the example shown here, the hollow piston 2 travels, a precisely defined sample volume from the container into the device according to the invention, i.e. into the room 14 be sucked.

Following this, the opening 8 of the Hohlzylin ders 3 can be closed with a stopper 13 and after the end of an analyte-specific predetermined time, the starting position can be restored by moving the hollow piston 2 alone or by corresponding relative movement of the hollow piston 2 and hollow cylinder 3 , a Part of the sample volume passes through the porous separating layer 5 into the interior of the hollow piston 2 , that is to say into a space 11 between the porous separating layer 5 and the sealing plate 4 and is held there enclosed.

In this form, the actual evaluation of the fluorescence immunoassay, as shown schematically in FIG. 3d, can be carried out.

For this purpose, the sample is irradiated from one side within the space 11 with a suitable light source and fluorescence is excited. The intensity of the fluorescent light can be measured with an optical detector 12 arranged diametrically to the light source and the respective analyte quantity can be deduced from the corresponding measurement signals.

Since the sample is enclosed on all sides within the device according to the invention by the stopper 13 and by means of the sealing plate 4 as well as the hollow piston 2 and hollow cylinder 3 , the entire device can be disposed of without risk without further ado after the measurement has been carried out.

When carrying out fluorescence immunoassays, an internal standardization can also be carried out. For this purpose, a first measurement of a fluorescence intensity is carried out when the sample has been sucked up via the opening 8 into the space 14 within the hollow cylinder 3 , as shown in FIG. 3b. For this purpose, light with at least one wavelength with which the fluorescence of the fluorophore or the fluorophores used can be excited is directed and the fluorescence intensity is detected with a suitable optical detector.

The fluorescence intensity measured in this way can then subsequently be compared with and / or related to the fluorescence intensities which, as has been shown and described in FIG. 3d, have been recorded. As a result, the measurement accuracy can be increased in a simple manner and the measurement effort required can be increased only insignificantly.

Claims (18)

1. Device for use in carrying out receptor ligand and affinity tests, in which a hollow piston ( 2 ) from at least partially transparent to light material in a with a temporarily closable opening ( 8 ) provided hollow cylinder ( 3 ), which also at least partially consists of a transparent material for light, recorded and guided;
the end face of the hollow piston ( 2 ), which faces into the interior of the hollow cylinder ( 3 ), consists of a porous separating layer ( 5 ); and
In the hollow piston ( 2 ) parallel to the porous separating layer ( 5 ), a gas and liquid-tight sealing plate ( 4 ) which is translationally movable within the hollow piston ( 2 ) is arranged. 2. Device according to claim 1, characterized in that within the hollow cylinder ( 3 ) in a space ( 14 ) between the hollow cylinder ( 3 ) and hollow piston ( 2 ) particles ( 6 ) are present, the grain size of which is selected so that it is porous Interface ( 5 ) can not penetrate. 3. Apparatus according to claim 1 or 2, characterized in that the opening ( 8 ) of the hollow cylinder ( 3 ) by means of a closure ( 13 ) or a return check valve can be closed. 4. Apparatus according to claim 1 or 2, characterized in that the sealing plate ( 4 ) has an outer circumferential seal ( 9 ). 5. Device according to one of claims 1 to 4, characterized in that the sealing plate ( 4 ) with respect to the inside width within the hollow piston ( 2 ) has a thickness that a cant th during a translational movement within half of the hollow piston ( 2nd ) prevented. 6. Device according to one of claims 1 to 5, characterized in that on the sealing plate ( 4 ), tilting of the sealing plate ( 4 ) during a translational movement within the hollow piston ( 2 ) preventing guide elements are angeord net. 7. Device according to one of claims 1 to 6, characterized in that the hollow piston ( 2 ) has on its outer lateral surface in the region of its in the interior of the hollow cylinder ( 3 ) facing end face circumferential sealing elements ( 10 ). 8. Device according to one of claims 1 to 7, characterized in that on the hollow piston ( 2 ) at least one pressure compensation opening ( 1 ) is present. 9. Device according to one of claims 1 to 8, characterized in that the particles ( 6 ) consist of polymers. 10. Device according to one of claims 1 to 9, characterized in that on the particles ( 6 ) is a partner of a receptor-ligand system is the. 11. Device according to one of claims 1 to 10, characterized in that the porous frit ( 5 ) consists of plastic, plastic alloys, glass, ceramic or metal. 12. The device according to one of claims 1 to 11, characterized in that on the hollow cylinder ( 3 ) and / or on the hollow piston ( 2 ) there are limit stops. 13. Device according to one of claims 1 to 12, characterized in that the opening ( 8 ) is closed by means of a further frit, a membrane or a filter. 14. A method for carrying out receptor ligand or affinity tests using a device according to one of claims 1 to 13, characterized in that
that a defined sample volume is sucked through the opening ( 8 ) into a space ( 14 ) which increases in accordance with the relative movement, by moving the hollow cylinder ( 3 ) and the hollow piston ( 2 ) relative to one another,
at least one fluorophore-labeled partner of a receptor-ligand system, which is reversibly immobilized on the inner wall of the hollow cylinder ( 3 ) and / or the porous separation layer ( 5 ), is dissolved in the sample;
depending on the concentration of the analyte in the sample and according to the selected test format, the respective partner partially reacts with the other partner of the receptor-ligand system, which is bound to particles ( 6 ) and / or the porous separating layer ( 5 ) ,
when the opening ( 8 ) of the hollow cylinder ( 3 ) is closed by opposing relative movement of the hollow cylinder ( 3 ) and hollow piston ( 2 ), part of the sample prepared in this way from the space ( 14 ) through the porous separating layer ( 5 ) into one corresponding to the respective one Sample volume increasing space ( 11 ) within the hollow piston ( 2 ) between the porous separating layer ( 5 ) and sealing plate ( 4 );
and the sample in the room ( 11 ) is irradiated from the outside with light of a fluorescence-stimulating wavelength of the fluorophore used and the fluorescence intensity is measured with a detector ( 12 ). 15. A method for carrying out receptor ligand or affinity tests using an on device according to one of claims 1 to 13, characterized in that by relative movement of the hollow cylinder ( 3 ) and hollow piston ( 2 ) a defined sample volume through the opening ( 8 ) sucked into a space ( 14 ) that increases in size according to the relative movement,
at least one analyte contained in the sample, depending on its concentration, the fluoro-labeled partner of a receptor-ligand system, which due to its affinity for the other partner of this system, the particle ( 6 ) and / or the porous separation layer ( 5 ) bound, ousted;
and when the opening ( 8 ) of the hollow cylinder ( 3 ) is closed by opposing relative movement of the hollow cylinder ( 3 ) and hollow piston ( 2 ), part of the sample prepared in this way from the space ( 14 ) through the porous separating layer ( 5 ) in a corresponding manner the space ( 11 ) increasing the respective sample volume within the hollow piston ( 2 ) between the porous separating layer ( 5 ) and the sealing plate ( 4 );
and the sample in the room ( 11 ) is irradiated from the outside with light of a fluorescence-stimulating wavelength of the fluorophore used and the fluorescence intensity is measured with a detector ( 12 ). 16. The method according to claim 14 or 15, characterized in that the sample recorded in the room ( 14 ) is irradiated from the outside with light of a fluorescence-stimulating wavelength of the fluorophore used, the fluorescence intensity measured and the fluorescence intensity measured in this way with the fluorescence measured in the aftermath Resence intensity of the affected sample, which was measured in the room ( 11 ), compared and / or related. 17. A method for performing receptor ligand or affinity immunoassays using a device according to one of claims 1 to 13, characterized in that a relative sample volume through the opening ( 8 ) by relative movement of the hollow cylinder ( 3 ) and hollow piston ( 2 ) sucked into a space ( 14 ) which increases in accordance with the relative movement,
at least one enzyme-labeled partner of a receptor-ligand system, which is reversibly immobilized on the inner wall of the hollow cylinder ( 3 ) and / or the porous separating layer ( 5 ), is dissolved in the sample;
depending on the concentration of the analyte in the sample and according to the selected test format, this partner partially reacts with the other partner of the receptor-ligand system, which is bound to particles ( 6 ) and / or the porous separating layer ( 5 ) ,
when the opening ( 8 ) of the hollow cylinder ( 3 ) is closed by opposing relative movement of the hollow cylinder ( 3 ) and hollow piston ( 2 ), part of the sample prepared in this way from the space ( 14 ) through the porous separating layer ( 5 ) into one corresponding to the respective one Sample volume increasing space ( 11 ) within the hollow piston ( 2 ) between the porous separating layer ( 5 ) and sealing plate ( 4 )
and then the sample is subjected to a time-resolved colorimetric measurement within the space ( 11 ). 18. A method for carrying out receptor ligand or affinity tests using a device according to one of claims 1 to 13, characterized in that a relative sample volume through the opening ( 8. ) By relative movement of the hollow cylinder ( 3 ) and hollow piston ( 2 ) ) sucked into a space ( 14 ) that increases in size according to the relative movement,
at least one analyte contained in the sample, depending on its concentration, the enzyme-labeled partner of a receptor-ligand system, which due to its affinity to the other, to the particles ( 6 ) and / or the porous separation layer ( 5 ) bound partner of this system bound, ousted,
when the opening ( 8 ) of the hollow cylinder ( 3 ) is closed by opposing relative movement of the hollow cylinder ( 3 ) and hollow piston ( 2 ), part of the sample prepared in this way from the space ( 14 ) through the porous separating layer ( 5 ) into one corresponding to the respective one Sample volume increasing space ( 11 ) within the hollow piston ( 2 ) between the porous separating layer ( 5 ) and sealing plate ( 4 )
and then the sample is subjected to a time-resolved colorimetric measurement within the space ( 11 ).