CH711376A2 - Method and system for the determination of biological properties of samples. - Google Patents

Abstract

In a method for detecting biological or other characteristics of samples using a microplate reader, the samples are placed in wells (112) of a microtiter plate (110). The method comprises filling wells of a microtiter plate with samples containing at least one sample substance in a liquid medium, heating samples by means of a heater, placing the microtiter plate in a measurement position within a measurement chamber of the microplate reader, and examining samples during and / or after heating in at least one mode of operation of the microplate reader by measuring radiation emitted from the samples. The method is characterized in that microwave radiation is used to heat the samples. A system (100) suitable for carrying out the method has a heater (190) for heating samples located in wells (112) of a microtiter plate (110). The heating device comprises at least one microwave source (160-1, 160-2) for generating microwave radiation, which can be irradiated onto samples for heating.

Description

AREA OF APPLICATION AND PRIOR ART

The invention relates to a method for determining properties of samples by means of a microplate reader, wherein the samples are arranged in a matrix arrangement in wells of a microtiter plate, and a system for carrying out the method. A preferred field of application is the determination of biological properties of samples.

In modern biology sample multiplexing methods are widely used in which the samples to be examined in a matrix arrangement in wells of a microtiter plate (microwell plate) are arranged and examined either sequentially or in parallel with different, mostly non-contact optical analysis methods , The type of samples examined here is extremely diverse and can range from homogeneous solutions to immobilized cells. The latter in particular, but also other special sample systems ("assays"), require the most exact possible temperature for optimum reaction or growth. Modern microplate readers therefore have heating functions by means of which the samples in the microtiter plate can be brought to a defined temperature and held thereon.

In conventional systems, this temperature is achieved by electric current flowing through heating coils, which generate a temperature radiation due to line losses due to the ohmic resistance of the heating element. The heat generated by means of this electrical resistance heating is transmitted by natural air flow in the interior of a heating chamber and thus passes to the sample located there. Partly, a fan is used to homogenize the heat distribution, which distributes the heated air evenly in the heating chamber. The amount of current flowing through the heating resistor is controlled by an electrical thermal sensor, which adjusts a set target temperature with the current, detected by a thermocouple actual temperature and readjusted accordingly the current. In this way, e.g. live cells are examined at ideal 37 ° C. Furthermore, kinetic studies can be performed at different temperatures and THEN hybridizations can be controlled. Furthermore, some assay formats require working at elevated temperature for optimal performance.

US 2012/0 300 194 A1 shows universal Multidetektionssystem for Mikrotitierplatten with heaters for temperature control of samples.

TASK AND SOLUTION

It is an object of the invention to provide a method and a system for determining properties of samples, which provide improved examination possibilities by using a temperature control of samples compared to conventional methods and systems. In particular, the method and system should be suitable for determining biological properties of the samples.

This object is achieved by a method having the features of claim 1, a system having the features of claim 13 and the use of microwave radiation according to claim 26. Advantageous further developments are specified in the dependent claims. The wording of all claims is incorporated herein by reference.

In the method, microwave radiation is used for heating samples which are to be examined prior to heating, simultaneously with the heating and / or after heating by means of at least one optical examination method by measuring the radiation emitted by the sample. Preferably, the samples (some or all) are heated or heated exclusively by irradiation of microwave radiation.

A system suitable for carrying out the method has a heating device for heating samples which are located in the well of a microtiter plate. The heating device comprises at least one microwave source for generating microwave radiation, which can be irradiated onto samples for heating. In addition to the devices for generating the microwave radiation, the system requires no further heating devices, in particular no radiant heater and no heating device operating via convection or heat conduction.

The use of microwave radiation to heat samples to determine biological and / or other properties of the samples offers specific advantages over the methods and systems previously used for this purpose. The advantages are, among other things, based on the type of heat generation and the type of heat transfer. Since in the conventional method the heat reaches the sample (s) only indirectly via the ambient air, the heating of the sample (s) usually takes place relatively slowly. The environment of the sample is also heated. Conversely, later cooling to room temperature may take a very long time, since a large volume and large area interfaces (e.g., confining walls) having a correspondingly high heat capacity must be cooled by heat radiation, convection, and / or by active air transport. Due to the slow warm-up and decay times, setting an exact temperature and / or controlling the temperature can be difficult.

In the claimed invention, microwave radiation is used for sample heating and / or for temperature control of samples. This can increase the efficiency of sample heating. Ideally, virtually only the sample to be tested may be heated, while the environment may be maintained at substantially ambient temperature. This can also be referred to as direct heating and is achieved in that energy is deposited directly, without contact and essentially exclusively in the sample.

By using suitable microwave radiation can be further achieved that the biological and / or chemical integrity of the samples is not affected, in particular because no resonant excitation of individual molecular bonds is to be feared.

As e.g. biological samples are usually examined in low-viscosity liquids with permanent dipole moment (especially in water, aqueous solutions, possibly also in ethanol), it is beneficial to use for heating a radiation that interacts essentially exclusively with the liquid, which in turn as direct energy transmitter acts.

Since in this type of energy transfer on the one hand, the distances to be bridged are very low, since the heat must be transported only within the sample volume, and at the same time the shock transmission in the condensed phase substantially more efficient than in gases, one obtains a high efficiency for the Transfer of energy from the solvent (eg water) to the sample.

The term "microwave radiation" refers in this application electromagnetic radiation from the frequency range of about 1 GHz to 300 GHz, which corresponds to wavelengths of about 300 mm to 1 mm. Within the scope of the claimed invention, wavelengths of the so-called 23 cm band, i. Wavelengths between 2320 and 2450 MHz, used for heating the samples to be examined. In particular, microwave radiation with a frequency of about 2.45 GHz can be used. For these frequencies, the penetration depth in aqueous or alcoholic media is sufficiently large for a largely uniform heating of a sample in the typical format for microtiter plates. Optionally, microwave radiation with frequencies up to about 900 MHz can be used.

It may be sufficient to use a single microwave radiation source and to use for heating. In some variants of the method it is provided that in a heating phase at least temporarily microwave radiation of a first microwave source and at least one separate second microwave source is irradiated simultaneously. As a result, a better control of the spatial field distribution of the microwave radiation in the region of the samples is possible. If necessary, it can be achieved, if necessary, that the same amount of energy is deposited in the time average in several or all samples, so that the respectively affected samples are tempered under comparable conditions. Selective heating of individual samples or subgroups of samples is also possible. Two separately controllable microwave sources can also be used alternatively to each other or at different times.

In some embodiments of the system, the heater has a first microwave transmitter antenna and a spatially separate second microwave transmitter antenna. As a result, among other things, a better control of the spatial field distribution and thus an adaptation to the distribution of samples in a microtiter plate are possible.

It is possible that the spatial field distribution does not change significantly during a heating phase. For this purpose, the heater may include a microwave source or multiple microwave sources that operate with fixed preset control parameters.

It is also possible that the first and the second microwave source amplitude-modulated and / or phase-modulated are controlled so coordinated that is generated by the first and the second microwave source, a time-varying field distribution of microwave radiation. For example, within a given time window, individual samples or spatially related subgroups of samples may be heated while other samples are not heated simultaneously. The spatial position of the microwave radiation detected sample (s) can be varied by means of a time-dependent control. As a result, the flexibility in the specification of temperature profiles can be increased.

This variable control of the heating effect can be realized in a system in that the heater has a first microwave source and at least one separate second microwave source. An associated microwave control device can be configured such that the first and the second microwave source can be amplitude-modulated and / or phase-modulated controlled so that a time-varying field distribution of microwave radiation can be generated by the first and the second microwave source.

In one class of systems, the heater has a microwave radiation substantially dense heating chamber with a metallic shield. The heating chamber may be dimensioned so that one or more microtiter plates within the heating chamber can be accommodated for heating. The metallic shield can ensure that the microwave radiation used for heating samples remains within the area of the shield and does not penetrate to the outside. As a result, lying outside the heating chamber components of the system can be protected against microwave radiation.

In some variants, the heating chamber is a chamber separate from the measuring chamber. In these cases, a transfer device may be provided for transferring microtiter plates between the heating chamber and the measuring chamber. The heating and the measurement are spatially and temporally separated in these cases. The heating chamber can be provided within a separate heating unit, which can be combined as an accessory to the microplate reader together with this to a system. It is also possible to accommodate a heating chamber and a separate measuring chamber in a common housing. A solution with separate chambers provides i.a. the possibility of retrofitting.

In other variants, the measuring chamber is designed as a microwave radiation substantially dense heating chamber with a metallic shield. As a result, a combined measuring and heating chamber can be created. In these cases, a microtiter plate does not have to be moved between heating up and measuring. The heating may be upstream of the actual measurement, so that the heating is completed before the measurement begins. It is also possible to carry out at least part of the heating during a measurement or to carry out a measurement during a heating phase.

In many variants, the microwave radiation is generated so that the samples (one or more) are in the range of the far field of the associated microwave generation. However, this is not mandatory. It is also possible to heat in the near field area.

In some process variants, a non-propagating microwave evanescent field is generated for heating samples, which has a spatial extent that allows selective heating of individual samples. In a corresponding system, this may be accomplished by having the heater coupled to a microwave source near-field hollow waveguide having a radiation exit aperture having an effective diameter less than half the wavelength of the microwave radiation. The diameter may for example be in the range of about 0.5 mm to about 6 mm. The geometry is chosen so that the microwave radiation can not propagate from the near-field hollow waveguide into the environment, but is reflected back into the near-field hollow waveguide. Only an evanescent portion can decouple in the region of the radiation outlet opening and falls in its field strength exponentially with increasing distance from the radiation outlet opening. As a result, the spatial resolution of the area exposed to microwave radiation in this way is determined exclusively by the size of the radiation outlet opening and is no longer diffraction-limited. The evanescent microwave field can be used for local heating of a sample.

For a very accurate setting of sample temperatures, it is expedient to measure the temperature of samples. Although a contacting measurement is possible by means of probes which may be in contact with the samples and / or the microtiter plate, preferred variants of the method are characterized by a contactless measurement of the temperature of samples for determining sample temperature values. Non-contact temperature measurement can help prevent sample degradation by measuring temperature even with small sample volumes. A determination of the temperature of individual samples or of subgroups of samples is possible here.

Preferably, the determined sample temperature values are used to control the temperature. For this purpose, the microwave radiation can be controlled as a function of the sample temperature values or values derived therefrom.

When using microwave radiation as an energy source for sample heating, the heating of samples can be done locally. The temperature can be measured without contact, for example via an infrared camera or an infrared diode or an infrared diode array and optionally controlled on the basis of the sample temperature values. Since this radiation-based temperature measurement operates in a different wavelength range (infrared range) than the microwave radiation used for heat excitation, the two types of radiation do not influence each other and the temperature measurement can be carried out continuously or intermittently if necessary even during the exposure of the sample with microwave radiation. As a result, a particularly accurate temperature control is favored.

As already mentioned, there are exemplary embodiments in which the measuring chamber is designed as a heating chamber substantially dense for microwave radiation with a metallic shield, so that the heating can take place within the measuring chamber. In this case, separate measures may be provided to ensure that microwave radiation-sensitive components of the measuring devices for examining the samples by the microwave radiation can not be impaired in their function and / or damaged.

Some measuring devices include, for example, a polychromatic light source, from which an optical excitation path for transmitting spectral portions of light from the light source is passed as excitation light in a measuring position in which a sample is arranged or can be arranged. Furthermore, an optical emission path is provided for the transmission of emission light emitted by the sample to a detector. With such devices, for example, fluorescence properties of samples can be determined. Some such measuring devices have at least one optical path (excitation path and / or emission path), which leads through a microwave-dense opening in the metallic shield of the heating chamber. By suitable dimensioning and / or shielding of the opening can be ensured that in the region of the aperture no microwave radiation from the heating chamber can escape to the outside. In some variants, the optical path leads through a light guide, which is guided through a microwave-dense opening in the metallic shield of the heating chamber.

In some embodiments, it is envisioned that microchemical processes, e.g. simple reaction or separation steps to perform in a multi-label device (microplate reader) with a microwave source as a central energy source. The system may have suitable facilities for this purpose.

These microchemical processes are not limited to biological systems, but may in particular include reactions in which experimental conditions such as e.g. the stoichiometry of the reactants, the temperature, the temperature gradient and / or the duration of the reaction are permuted. An embodiment from the field of non-biological samples provides for the implementation of polymerization reactions, since in this type of reactions, a relatively small number of reactants is used and the specific properties of the final polymer product can be achieved by varying the experimental parameters listed. A microplate reader with microwave heating is particularly suitable for carrying out these reactions since the microplate format allows multiple samples to be examined in parallel, and the combination with microwave heating provides the specific advantages outlined above. The possibility of directly monitoring the reaction or checking the reaction result is given in microplate readers since polymer reactions are often accompanied by changes in optical material properties such as e.g. of refractive index, absorption or autofluorescence, which can be detected and quantified with the optical measuring devices integrated in a microplate reader according to the invention.

Some variants of the method are characterized in that the heating of the microwave radiation is used to modify the sample in its molecular structure. This is preferably done indirectly by heating the solvent and by increasing the temperature initiate processes that can modify the sample substance to its molecular structure. Among other things, it is possible to use the heating by microwave radiation to separate heterogeneous sample systems into individual components, e.g. by distillation between directly or indirectly adjacent wells of a microtiter plate. The introduced via microwave radiation into the sample energy can be kept so low that an immediate modification of the sample substance by microwave radiation can be avoided.

In the system, such process variants may e.g. be made possible by an attachment over one or more wells (sample receptacles) is arranged or is such that evaporated liquid can condense on the walls of the essay and drip back into the well or the sample holder. It is also possible for an attachment to be arranged above one or more wells (sample receptacles) in such a way that vaporized liquid can condense on the walls of the attachment and be deflected in such a way that it can at least partially drip into an indirectly or immediately adjacent sample receptacle , The respective attachments should be made of a microwave non-heatable material (e.g., glass or plastic) and may be considered as accessories or as part of the system.

By appropriate dimensioning the essays can be adapted to the relatively small dimensions of wells of a microtiter plate. In particular, an outer diameter of an attachment should be slightly smaller in its cup-mounted inlet area than an inner diameter of the cup at the open end thereof so that the attachment with its inlet area can be inserted into a well.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages and aspects of the invention will become apparent from the claims and from the following description of preferred embodiments of the invention, which are explained below with reference to the figures. Showing: <Tb> FIG. 1 <SEP> schematically shows components of a system having a heating chamber separate from a measuring chamber; <Tb> FIG. 2 <SEP> schematically shows components of a system having a combined measuring and heating chamber; <Tb> FIG. 3 <SEP> schematically shows components of a system that includes a measuring device that has optical fibers for guiding excitation light into a measuring and heating chamber and emission light from the measuring and heating chamber; <Tb> FIG. 4 <SEP> schematically shows an exemplary embodiment of a near-field hollow waveguide for locally heating individual samples; <Tb> FIG. 5 <SEP> schematically shows a system with a near-field hollow waveguide for local heating of individual samples; <Tb> FIG. Fig. 6 shows another embodiment of a near field hollow waveguide system; <Tb> FIG. Figure 7 shows another embodiment of a near-field hollow waveguide system; <Tb> FIG. Figures 8A-8C show various variants of a microchemical process performed in a heating chamber using microwave radiation to selectively heat individual samples; and <Tb> FIG. Figures 9A-9C show different variants of another microchemical process performed in a heating chamber using microwave radiation to selectively heat individual samples.

DETAILED DESCRIPTION OF THE EMBODIMENTS

FIG. 1 schematically shows some components of a first embodiment of a system 100 for determining biological or other properties of samples.

The samples to be examined are arranged in a rectangular matrix arrangement in wells 112 of a rectangular microtitre plate 110 and are to be investigated sequentially or in parallel using one or more different non-contact optical analysis methods. The commercial microtiter plate may e.g. made of a plastic material, e.g. Polyvinyl chloride or polystyrene, or glass, and has a number of identically sized wells in straight rows and columns, e.g. between 6 and 1536 wells, in the example 12 * 8 = 96 wells.

The system 100 is set up, inter alia, for carrying out methods of fluorescence spectroscopy. In addition to fluorescence measurements, other radiation-based measurement methods, for example luminescence measurements or absorption measurements, can also be carried out. The components used for this purpose are not shown for reasons of clarity.

The system 100 has a substantially cuboid, light-tight lockable measuring chamber 120 which is dimensioned so that at least one Mikrotitierplatte can be recorded within the measuring chamber so that each one of the samples contained in the wells using a positioning system in a measuring position can be brought. For this purpose, the positioning system has a cross table which can be moved horizontally in two dimensions and has an integrated lifting device, so that the samples can be positioned in three dimensions.

The measuring chamber are associated with measuring devices, with the aid of which samples can be examined in at least one operating mode of the system by measuring radiation emitted from the samples.

Outside the measuring chamber, a primary light source 130 is arranged in the form of a xenon flash lamp with a short electrode spacing. Depending on the application, it can also be a continuous-wave lamp. The light source has a broad emission spectrum in the visible spectral range.

An optical excitation path 132 with a plane deflection mirror 133 leads from the light source to a measuring position 134 into which a sample 135 to be measured can be positioned by positioning the microtiter plate (shown in dashed lines in a measuring position) for a measurement. The sample is located in a well of the microtiter plate. The non-illustrated optical elements of the optical excitation path 132 serve to transmit spectral components of light from the primary light source as excitation light into the measurement position. The excitation light is irradiated vertically from above into a well or into the sample.

In the sample 135 is a sample substance in aqueous solution. The sample substance can be excited by means of the excitation light to emit fluorescent light, which is shifted from the excitation light to lower energies or larger wavelengths. The extent of the red spectral shift is specific to the sample substance.

The emission light (the emitted radiation) passes through an optical emission path 136 equipped with a parabolic mirror 137 to a detector 140, which generates electrical signals in response to the incident light, which are fed to an evaluation unit, not shown, to the emission light for characterizing the Spectrally evaluate the sample. In the example, the detector comprises a photomultiplier.

Furthermore, 100 absorption measurements can be performed with the system. For this purpose, the light can be guided via the optical excitation path 132 through the sample, which is located in a microtiter plate with a transparent bottom. The transmitted light, after passing through the sample, strikes a light detector, not shown, typically a wide spectral range silicon photodiode.

The system 100 is also suitable for measuring luminescence emission that is generated chemically, biologically or biochemically. For this purpose, starter substances can be injected via injectors, not shown, which initiate light emission in a sample. The emitted light is guided via the parabolic mirror 137 via the optical emission path 136 to the detector 140.

The system 100 includes a heater 190 for heating the samples held in a microtiter plate by means of microwave radiation. To the heater 190 includes a separate from the measuring chamber 120 heating chamber 150, which is arranged next to the measuring chamber at a distance from this at the same level within the housing 105 of the system. A chamber wall 155 of the heating chamber encloses a substantially cuboidal interior 152, which is dimensioned so that at least one Mikrotitierplatte completely fits. On the inside of the chamber wall, a metallic shield 154 is mounted so that the interior 152 is sealed from the environment with respect to the emission of microwave radiation. The shield 154 may be e.g. have gap-free joined metal sheets which are attached to the insides of the chamber walls.

In the interior 152 is a first microwave transmitter antenna 162-1, which is connected to a first microwave source 160-1 and can emit from this generated microwave radiation into the interior 152. An optional second microwave transmitter antenna 162-2 is spatially separated from the first microwave transmitter antenna 162-1, for example on the opposite side, and is excited by a second microwave source 160-2 for emitting microwave radiation into the interior space. The microwave sources 160-1, 160-2 are connected to a control device 170, which is configured such that the first and the second microwave source can be controlled independently of each other amplitude-modulated and / or phase-modulated.

The microwave sources are in the example to semiconductor-based microwave sources that can generate microwave radiation with a frequency of about 2.45 GHz and a rated power in the range of about 50W. Alternatively, e.g. Also suitable magnetrons suitable power and frequency.

To the heater 190 includes a temperature measuring device 180 for non-contact measurement of the temperature of samples by detecting and evaluating infrared radiation. As a result, sample temperature values can be determined on the basis of which the heating device can be controlled in order to achieve a temperature control. In the example, the temperature measuring device 180 comprises an infrared diode array 182, which is mounted above the receptacle for the microtitre plate outside the shield and connected to the control device 170. In the shield small holes are introduced in the region of the infrared diode array, through which the infrared radiation can pass, while the longer-wavelength microwave radiation is blocked.

For the transfer of a microtiter plate between the heating chamber 150 and the measuring chamber 120, a transfer unit 175 is provided which can be actuated via the control device 170 of the system. The transfer unit has a guide plate horizontally movable plate receptacle 176, which can each accommodate a single microtiter plate. The plate holder has a vertically movable frame 177, on which the microtiter plate can rest with two opposite edges. The microtiter plate can thereby be transferred to other components, e.g. by settling on a pedestal in the heating chamber. The transfer unit 175 also has drives for a fine positioning of the recorded microtiter plate, so that there is a plate manipulator which can take over both the transfer and the fine positioning. The transfer unit can thus form a common unit with the cross table for sample positioning.

The samples are heated exclusively by means of microwave radiation. Due to the dipole moment of the water molecules irradiation with electromagnetic radiation in the lower GHz range (corresponding wavelengths in the range of a few centimeters), so-called microwave radiation, a force on the water molecule, so that a torque acts on the water molecule and forces it to rotate. It is important in this consideration that the radiation frequency used does not have to correspond to a rotational resonance frequency of the water molecule.

With the aid of the arrangement, it can be ensured that the heating or incubation of samples takes place in a separate, metallically-conductive, enclosed space (inner space 152), which represents a cavity for the microwave excitation and thus corresponds to a hollow waveguide closed on all sides. A microtiter plate with the samples to be examined is retracted by means of the movable plate holder 176 in this cavity and can be placed there in a heating position. The transfer unit can then leave the heating chamber. The interior 152 can then be sealed by means of a likewise shielded drawer to the outside radiation-tight. At the periphery of the drawer are at most 1 mm wide and at least 1 cm long gaps, which are impermeable to the inside excited microwave radiation. The gaps can be realized for example by a rear panel on the sample drawer. With such an arrangement it is achieved that reproducible and controllable electromagnetic conditions prevail in the interior 152 and the propagation of microwave radiation from the microwave transmitting antennas to the samples does not depend on the further design of the device, since, for example, any field bends are precluded by electrically conductive components , The heater 190 may be constructed as an independent assembly that may be integrated into appropriately sized devices in addition to a measuring chamber.

A homogenous field distribution within the cavity, at least in the average time, can be achieved by controlling the amplitude and phase of the two independent microwave transmitting antennas 162-1, 162-2, which are placed in the interior, so that as far as possible no radiation hot spots are generated. Spots arise, or these can be moved over the time of the microtiter plate and thus ensure a (on average) homogeneous energy input.

After the transfer of the microtiter plate with the heated samples into the measuring chamber, radiation measurements can be carried out there in a manner known per se, e.g. Fluorescence measurements.

FIG. 2 schematically shows some components of a second embodiment of a system 200 for determining biological properties of samples. Similar to the first embodiment, fluorescence measurements and also luminescence measurements or absorption measurements on samples housed in wells of a microtiter plate 210, which may be identical or similar to the microtitre plate of the first embodiment, can be performed with the system.

A special feature of the system 200 is that the light-tight lockable measuring chamber 220 is simultaneously designed as a heating chamber of a heater 290 so that it can serve as a combined measuring and heating chamber, which allows the samples located in the measuring chamber using To heat or heat microwave radiation. As a result, heating or temperature control and measurement can be carried out simultaneously if necessary.

The substantially cuboid measuring and heating chamber may be dimensioned to be similar or the same as the heating chamber 120 of the first embodiment. In order to ensure that the measuring chamber can also serve as a heating chamber which is substantially impervious to microwave radiation, a metallic shield which is only partially shown is provided, which completely encloses the interior 252 of the measuring chamber with the measuring chamber closed in such a way that microwave radiation does not escape from the interior the measuring chamber or heating chamber can escape to the outside. For the construction of the metallic shield metal sheets may be attached to the inner walls of the measuring chamber, which are joined together at the joints gap-free or with very narrow gaps.

At least part of the metallic shield is formed by perforated plates 255-1, 255-2, which have a regular pattern of holes whose diameter is in the range of 1 mm to 30 mm, in particular in the range of 2 mm to 20 mm, is so small and that microwave radiation can not penetrate these perforated plates. However, the holes allow optical paths from measuring devices to pass through the metallic shield.

The measuring devices associated with the measuring chamber 220 include a primary light source 230 located outside the measuring chamber and emitting a continuous emission spectrum in the visible spectral range. An optical excitation path 232 leads through an opening (hole) in the lateral perforated plate 255-1 through a flat deflecting mirror arranged in the interior of the measuring chamber from the light source 230 to the measuring position 234, in which a sample to be measured can be positioned. The radiation emitted by the thus excited sample (the emission light) passes via an optical emission path 236 equipped with a parabolic mirror to a detector 240 arranged outside the measuring chamber and comprising a photomultiplier. The emission path leads through holes or openings in the perforated plate 255-2, which is mounted above the measuring position on the chamber inner wall.

Those components of the heating device 290, which are provided for the irradiation of the microwave radiation to the samples, may be identical or similar to corresponding components of the first embodiment, for which reason reference is made to the description of the first embodiment. Again, there are two independently controllable microwave transmitter antennas 262-1, 262-2, each disposed within the metallic shield to produce microwave directed radiation on the samples, which may be controlled by amplitude and phase control means. in the area of the samples, the desired field distribution results for heating the samples.

Also, the components of the non-contact temperature measuring device may correspond to those of the first embodiment, which is why reference is made to the description there. In particular, an infrared diode array 282 for contactless measurement of the temperature of the samples above the receiving position for the microtiter plate outside the shield (perforated plate 255-2) is also attached here. The measurement is made through holes in the perforated plate.

FIG. 3 schematically shows some components of a third embodiment of a system 300 for determining biological properties of samples. Here, too, the measuring chamber 320 is simultaneously designed as a heating chamber of a heating device 390, which makes it possible to heat or temper the samples in the measuring chamber by means of microwave radiation. The heating device with at least one microwave transmitter antenna 360-1 in the interior of the measuring chamber can be constructed identically or similarly to the heating devices of the previous exemplary embodiments.

Differences from the second exemplary embodiment are primarily in the way in which the optical paths of the measuring device assigned to the measuring chamber are designed. The excitation path 332, which leads from the outside of the measuring chamber arranged light source 330 to the measuring position 334, passes through a first light guide 333, which leads through a hole in the above the measuring position arranged perforated plate 355-2 of the metallic shield. The emission path 336, in which emitted radiation is guided from the measuring position to the detector 340 arranged outside the measuring chamber, passes through a second optical waveguide 337, which is likewise guided through a hole in the perforated plate 355-2 arranged above the measuring position.

In this embodiment, therefore, a common chamber for incubation and measurement is combined with measuring devices which have optical fibers from the light source to the sample position and from the sample position to the detector. Illustrated is a variant in which the optical fibers are designed as a y-split, so that the excitation path and emission path are guided in sections next to each other. It is also possible to coaxially conduct optical fibers for the excitation path on the emission path such that both paths can be passed through the same aperture within the metallic shield. The detail in FIG. 3 shows a cross-section through a coaxial fiber bundle 350 in which a central optical fiber guides the light of the emission path and the outer optical fibers arranged around the central optical fiber are provided for guiding the excitation light. It is also possible to use the coaxial fiber bundle in reverse order so that a central optical fiber guides the excitation light while the emission light is guided through the outer optical fibers arranged around the central optical fiber. The division of optical fibers, which are exposed to excitation or emission light, may also be arbitrary, so that a homogeneous mixing of excitation and emission optical fibers is achieved.

When a light pipe terminates inside the heating chamber, the ferrule of the light pipe should ideally be made of plastic or other electrically non-conductive material so as not to disturb the microwave field distribution. Metallic ferrules are also conceivable, but should then be included in the design of the heating chamber and the calculations to optimize the field distribution of the microwave radiation.

In the exemplary embodiments illustrated so far, the heating device has in each case a heating chamber equipped with a metallic shield, into which at least one microtiter plate with samples has to be introduced in order to heat the samples by means of microwave radiation. The samples are located in the far field area of the microwave transmitting antennas.

Chamber-free systems are also possible, that is to say systems without a separate heating chamber for accommodating a microtiter plate. The systems described below use a non-propagating evanescent field of the microwave radiation for sample heating. 4 shows schematically an exemplary embodiment of a near-field hollow waveguide 400 which can be used for this purpose, and FIG. 5 shows schematically a system 500 which is equipped with such a near-field hollow waveguide for heating samples.

In the case of the evanescent microwave excitation, the microwave radiation is initially generated similarly to the other exemplary embodiments by means of a microwave source 460 and emitted via an antenna 410 into the interior of the metallic near-field hollow waveguide 400. In the exemplary embodiment, the near-field hollow waveguide tapers exponentially at its end opposite the antenna for continuous impedance matching and ends in a round or angular radiation exit opening 420 whose effective diameter D is significantly smaller than the wavelength of the microwave radiation. The diameter may for example be in the range of 0.5 to 3 cm. The microwaves can not propagate into the far field here, but an evanescent field exponentially decaying in its field strength forms immediately in front of the radiation exit opening and can be used to locally heat a sample.

In a non-illustrated embodiment, the near-field hollow waveguide has a geometry by which the reflected microwave radiation is not reflected back to the source, but passes it. By suitable deflection of the microwave radiation, the above-described process can be repeated several times, so that a large number of samples in the well of a microtiter plate can be heated simultaneously. By lateral movement of the microtiter plate relative to the radiation outlet openings, all cells of the microtiter plate can thus be successively subjected to microwave radiation.

FIG. 5 schematically shows an example of application of a chamber-free system 500 with microwave evanescent radiation and beam guidance of the measuring system via optical fibers. The microtiter plate 510 is in this case within a measuring chamber, not shown, of the system, which does not have or require any internal metallic shielding against the emission of microwave radiation. It can thus be the measuring chamber of a conventional system. The excitation light reaching the measurement position from the light source 530 via the excitation path 532 is irradiated from above into the sample, which emits emitted radiation upwards, which passes through the emission path 536 to the detector 540.

The heating device 590 of the system 500 comprises at least one near-field hollow waveguide 400, which in the example below the receiving plane for the microtiter plate is arranged so that the upwardly directed outlet opening 420 is located in a plane immediately below the level in which the floors the wells 512 of the microtiter plate are arranged. The distance between the radiation outlet opening and the bottom of a well is dimensioned such that the microwave evanescent field emerging from the radiation outlet opening can penetrate from below through the material of the microtiter plate into the aqueous sample and heat it up.

Advantageously, the radiation outlet opening is so small in diameter that in typical microtiter plates in this way only a single sample can be warmed up by means of microwave radiation, while the other samples are practically unaffected by microwave radiation at ambient temperature (or another previously set temperature). stay. Selective heating of individual samples is thus possible in a targeted manner. The heating can be monitored and controlled under observation by the non-contact temperature measuring device. This has, similar to the other embodiments, an above the receiving position for the microtiter plate arranged infrared sensor 582. Using a three-dimensionally movable sample manipulation device 575 for moving the microtiter plate 510 relative to the radiation outlet opening 420 individual samples can be successively positioned in front of the outlet opening.

In the systems 600 in FIGS. 6 and 700 in FIG. 7, the means for positioning the microtiter plate, for heating samples by means of near-field hollow waveguides and for temperature control are identical to the embodiment of FIGS. 4 and 5. Differences exist only with regard to the structure of the measuring equipment.

In the system 600 of Fig. 6, the light of the light source 630 is irradiated to the sample from above without using optical fibers via an inclined reflecting mirror, while the emission light passes through a parabolic mirror to the detector (see, for example, first embodiment).

In the variant of FIG. 7, the excitation path 732 and the emission path 736 are separated via a geometric beam splitter 770 in the form of a hole mirror, which is arranged above the measurement position and has a centric opening through which the excitation light directly from above Measuring position arranged light source 730 can be irradiated in a sample. The emission light passes over a reflecting surface of the geometric beam splitter to the detector 740.

Fig. 8A shows an embodiment of a configuration for heating a sample in a microtiter plate to a maximum of the boiling temperature of the solvent used for a longer period. On a well 812 of a microtiter plate 810, a cap 820 is made substantially gas-tight, consisting essentially of an upwardly open tube of a non-metallic material, e.g. carbon fiber-reinforced plastic or glass.

The outer diameter of the attachment is in its placed on a well 812 inlet portion slightly smaller than the inner diameter of the cup at its upper open end, so that the essay can be introduced with its inlet portion a small piece in the well. Subsequently, at the inlet region, an outwardly projecting shoulder is formed, whose outer diameter is slightly larger than the diameter of the well opening, so that the attachment seals the well substantially gas-tight.

By heating the sample 835 by means of microwave radiation from a lower temperature Ti to a higher temperature T2> T1 according to one of the methods described above, the solvent begins to boil and rises as a vapor up into the interior of the tower. Since the patch tube is not heated substantially by the use of microwave radiation as an energy source, the steam cools on the tube walls and condenses. The liquid condensate may drip back into the well 812.

In a further embodiment according to FIG. 8B, the tube is such that it tapers upwards. This arrangement achieves a more efficient recondensation of the solvent vapor.

In another embodiment, the tube has an upwardly increasing wall thickness, as shown in FIG. 8C. As a result, due to the high heat capacity of the tube, a greater thermal gradient of T2 to T1 and thus more efficient gas condensation at a given tube length is achieved.

9A shows an exemplary embodiment for the distillative separation of liquid substance mixtures in microtiter plates. In this case, two adjacent wells 812-1, 812-2 are connected to one another via a bridge 830 sloping down to the second well, such that the bridge can be referred to as the distillation well on the first well 812-1, in which the substance mixture is located , Essentially gas-tight, while on the second well 812-2, which can be referred to as a suppository, rests so that a gas exchange with the environment can take place. By heating the sample with microwave radiation according to one of the methods described above, the solvent begins to boil and ascends as steam. Since the patch bridge is essentially not heated by the use of microwave radiation as an energy source, the vapor cools on the bridge walls and condenses. The condensate flows off the sloping side of the bridge and thus enters the master cup. By utilizing different boiling points of a substance mixture, two fractions can be separated from one another in this way. The separation efficiency can be increased by having webs, not shown in the bridge, which protrude above the distillation well into the gas space. At these webs, the vapor can condense, drip and re-enter the gas phase.

This increases the number of effective distillation steps and the separation efficiency is increased. In classical preparative chemistry, this process is referred to as increasing the number of theoretical plates by introducing a distillation column (column distillation).

In an exemplary embodiment illustrated in FIG. 9B, the bridge has a wall thickness increasing in the direction of the suppository cups. As a result, due to the high heat capacity of the bridge, a greater thermal gradient of T2 to T1 is achieved, and thus more efficient gas condensation for a given bridge length.

Another embodiment shown in Fig. 9C uses as a bridge a curved tube having on the side of the distillation well a substantially horizontal opening to the gas space and having on the side of the task suppository a substantially vertical opening, so that in the tube condensed gas can drip into the master cup.

In both methods described above for heating samples in microtiter plates with microwave radiation to modify the molecular structure or to separate a homogeneous substance mixture, the microwave radiation is not used so that the molecular integrity of the sample is directly influenced by the exposure to the microwave radiation. Rather, the microwave radiation serves only to heat the solvent by orientation polarization of the dipoles contained therein. It is therefore guaranteed that the findings gained scale for larger amounts of substance.

Current research investigates to what extent intensive microwave radiation can directly influence the chemical properties of substances. This influence is not provided in the examples described, but may optionally be exploited if powerful microwave sources are used.

Embodiments of the claimed inventions may provide one or more of the following advantages.

An exclusively local heating of the sample: As a result, much shorter heating times in the range of a few seconds can be achieved. At the same time, the device interior is not heated and therefore does not have to cool down accordingly. Therefore, measurements at different temperatures can be made in direct sequence.

An exact temperature control: If the temperature of the sample is tracked in real time via an IR diode or another IR sensor, this can be controlled very precisely via feedback by adjusting the microwave power (in cw or pulse mode) control). Thus one achieves a high-precision sample thermostatization, which is not possible in a comparable manner with conventional methods.

A generation of temperature profiles: An active temperature control loop, in conjunction with the short reaction times, can ensure that user-specific temperature profiles can be run. This is on the one hand e.g. for two-dimensional kinetic studies of advantage and on the other hand opens the field of DNA hybridization: by an accurate determination of the heating and Abkühlprofils hybridization errors can be almost avoided.

An extension of the application spectrum of the devices. According to the inventors, it is possible to carry out simple reaction and separation steps in a multilabel device with a microwave source as a central energy source in the context of u-chemical processes. In combination with injectors or dispersing units, it is possible to reflux and stir with appropriate attachments and to distil from one well into a neighboring well. The prerequisite for this is a large temperature gradient, which is achieved by preventing the distillation bridge from heating up (for example, as a simple bent pipe). Local heating with the help of microwave radiation meets this requirement.

In this way, a multilabel reader or microplate reader can develop from the pure measuring device to the integrated synthesis robot with analysis unit. Such an approach is useful for systemic approaches e.g. This is extremely helpful in polymer research, since reaction conditions and concentrations are permuted with a limited number of starting materials in order to achieve desired product properties.

Claims (26)

1. A method for determining properties of samples by means of a microplate reader, wherein the samples are arranged in wells of a microtiter plate, comprising the following steps: Filling wells of a microtiter plate with samples containing at least one sample substance in a liquid medium; Heating samples by means of a heating device; Placing the microtiter plate in a measuring position within a measuring chamber of the microplate reader; Examine samples before, during, and / or after heating in at least one mode of operation of the microplate reader by measuring radiation emitted from the samples; characterized in that For heating the samples microwave radiation is used. 2. The method according to claim 1, characterized in that for heating microwave radiation from a frequency range of 2320 MHz to 2450 MHz is irradiated. 3. The method according to claim 1 or 2, characterized in that in a heating phase at least temporarily microwave radiation of a first microwave source and at least one separate second microwave source is irradiated simultaneously. 4. The method according to any one of the preceding claims, characterized in that a first microwave source and a second microwave source amplitude-modulated and / or phase-modulated controlled in such a coordinated that is generated by the first and the second microwave source, a time-varying field distribution of microwave radiation. 5. The method according to any one of the preceding claims, characterized in that during heating in the time average in several or all samples substantially the same amount of energy is deposited. 6. The method according to any one of the preceding claims, characterized in that at least a portion of the heating is carried out during a measurement. 7. The method according to any one of the preceding claims, characterized in that a non-propagating microwave Evaneszenzfeld is generated, which has a spatial extent, which allows selective heating of individual samples. 8. The method according to any one of the preceding claims, characterized by a non-contact measurement of the temperature of samples for determining sample temperature values, preferably for measuring the temperature of the samples emitted infrared radiation is measured. 9. The method according to claim 8, characterized by controlling the microwave radiation as a function of the sample temperature values or values derived therefrom. 10. The method according to any one of claims 8 or 9, characterized in that a measurement of the temperature of samples is carried out at least in phases during the heating. 11. The method according to any one of the preceding claims, characterized in that the heating by microwave radiation is used to modify the sample in its molecular structure. 12. The method according to any one of the preceding claims, characterized in that the heating by microwave radiation is used to separate heterogeneous sample systems into individual components, in particular by way of distillation between directly or indirectly adjacent wells of a Mikrotitierplatte. 13. System (100, 200, 300, 500, 600, 700) for determining properties of samples (135) which are arranged in wells (112) of a microtiter plate (110, 210, 510), comprising: a measuring chamber (120) for receiving at least one Mikrotitierplatte in a measuring position; a heater (190, 290, 390, 590) for heating samples located in wells of the microtiter plate; Measuring devices for examining samples arranged in the measuring chamber during and / or after heating in at least one operating mode by measuring radiation emitted from the samples; characterized in that the heating device (190, 290, 390, 590) has at least one microwave source (160-1, 160-2) for generating microwave radiation, which can be irradiated onto samples for heating. 14. System according to claim 13, characterized in that the heating device (190) has a microwave radiation substantially dense heating chamber (150) with a metallic shield (154). 15. System according to claim 13 or 14, characterized in that the heating chamber (150) is a separate chamber from the measuring chamber (120) and that a transfer device (175) for transferring microtiter plates (110) between the heating chamber (150) and the Measuring chamber (120) is provided. 16. System according to claim 13 or 14, characterized in that the measuring chamber (220) is designed as a microwave radiation substantially dense heating chamber with a metallic shield. 17. System according to one of claims 13 to 16, characterized in that the heating device (190, 290) comprises a first microwave transmitting antenna (162-1, 262-1) and a spatially separated second microwave transmitting antenna (162-2, 262-2). 18. System according to any one of claims 13 to 17, characterized in that the heating device (190) has a first microwave source (160-1) and at least one separate second microwave source (160-2). 19. System according to claim 18, characterized by a control device that is configured such that the first microwave source and the second microwave source are amplitude-modulated and / or phase-modulated in a coordinated manner such that a time-varying field distribution of microwave radiation can be generated by the first and the second microwave source. The system of claim 13, characterized in that the heater comprises a near-field hollow waveguide (400) coupled to a microwave source (460) having a radiation exit aperture (420) having an effective diameter (D) less than that Half the wavelength of the microwave radiation, wherein the diameter is preferably in the range of 0.5 mm to 6 mm. The system of claim 20, characterized by sample manipulation means (575) for moving the microtiter plate (520) relative to the radiation exit port (420). 22. System according to any one of claims 13 to 20, characterized by a temperature measuring device (180) for non-contact measurement of the temperature of samples for determining sample temperature values, wherein preferably the temperature measuring device has at least one temperature sensor (182, 282, 582) which is selected from the group infrared diode, infrared diode array and infrared camera. A system according to claim 22, characterized by temperature control means configured to control the microwave radiation in dependence on the sample temperature values or values derived therefrom. 24. System according to any one of claims 14 to 19 or 22 to 23, characterized in that the measuring means comprise at least one optical path (232, 332, 336), which by a microwave-dense aperture in the metallic shield (255-1, 355-2) of the heating chamber (290, 390), wherein preferably the optical path through an optical fiber (333, 337) leads. 25. System according to any one of claims 14 to 24, characterized by at least one attachment (820) which can be arranged over one or more wells such that evaporated liquid of the sample can condense on walls of the essay and drip back into the one or more wells and / or at least one attachment (830) that can be arranged above one or more wells such that vaporized liquid can condense on the walls of the attachment and be deflected so that the liquid can at least partially drip into an indirectly or immediately adjacent well. 26. Use of microwave radiation to heat samples placed in wells of a microtiter plate in a method of detecting biological or other properties of the samples.