EP1780290A2 - Apparatus for amplifying nucleic acids - Google Patents

Abstract

Device for multiplying and detecting target substances, especially nucleic acids, in a biological sample comprises multiplying modules and detecting modules arranged on one component. The multiplying modules are tempered via a tempering unit which is individually controlled. An independent claim is also included for a kit comprising the above device and amplification enzymes, separating gels, ultrafiltration membranes and/or markers.

Description

The present invention relates to a device or an array for the amplification and detection of target substances, in particular of nucleic acids from a biological material or a sample.

The amplification and detection of nucleic acids plays an important role, e.g. in forensic analysis, in cancer research or in food diagnostics.

The aim is to detect certain nucleic acids that make up e.g. a criminal culpability that implies disposition for a hereditary disease or the genetic modification of a food. Conventional methods for the detection of certain nucleic acids are well known, e.g. Gel or capillary electrophoresis, Southern blot, immunoassays or hybridization with labeled nucleic acid probes. For this reaction chips, cartridges, electrophoresis chambers o. Ä., And the corresponding devices are used to read these devices.

Since in the mentioned as well as many other cases, the nucleic acid molecules of interest often occur only in very small quantities and in addition to a large number of other nucleic acid molecules, the former first have to be specifically and specifically amplified. For this purpose, what are known from the prior art are so-called duplicating or amplification methods such as the polymerase chain reaction (PCR) or SDA. These processes are usually carried out in so-called thermocyclers.

In order to carry out both methods, therefore, a large amount of machinery is required, which in turn requires a well-equipped laboratory. An application outside the laboratory is therefore not readily possible. This complicates e.g. work in epidemiological investigations in the field, forensic investigations at the crime scene, routine examinations in the doctor's office etc.

Moreover, the nucleic acids to be detected must first be added to the amplification device, and then the amplified molecules into the detection device. On the one hand, each transfer step therefore leads to material and time losses and, on the other hand, entails the risk of contamination with foreign nucleic acids.

Furthermore, devices are known from the prior art, in which duplication function and detection function are integrated. These devices avoid the above problems.

In some cases, said functional units are miniaturized in the form of an array in such a way that they can fit on a small carrier ("lab on a chip"). The individual reaction chambers are connected via miniaturized channels, and the reaction fluids are transported from one chamber to the next by means of suitable micropumps and valves as well as via the capillary force (microfluidics). Such chips or cartridges are arranged in special adapted equipment, which the necessary control and operating units, eg. As pumps and heating and cooling elements, etc., and the reactions, detections, etc. control and evaluate.

In Scripture " Flow-through polymerase chain reaction in chip thermocyclers "(Schnegaß & Köhler 2001, Rev. Mol. Biotechnol. 82; 101 ) are presented such "labs on a chip". As a duplication method only PCR is mentioned, in which the success of the duplication relies on the execution of thermocycles. The authors describe a chip having a meandering channel, under which heating elements of different temperature are arranged in an ordered arrangement. When the reaction liquid is pumped through the channel at a suitable rate, it passes through the temperature cycles required for the PCR to proceed, and the nucleic acids are amplified.

Since the multiplication factor depends exponentially on the number of thermocycles, the multiplication factor is already defined in such a "lab on a chip". Therefore, such chips are not very variable and therefore unusable for various uses.

However, these devices known from the prior art are usually designed for the parallel examination of many samples on one and the same nucleic acid. Thus, for example, in some devices, 96 food samples of various origin can be examined simultaneously in parallel for the presence of a specific gene, eg. B. on an anti-biotic resistance gene, which suggests a genetic modification.

However, the simultaneous testing of a sample for the presence of a variety of nucleic acids (as would be desirable, for example, for proteomics or rapid diagnosis of infection) does not allow these devices. The above-mentioned document therefore proposes the serial throughput of several samples in succession. However, in addition to the loss of time, this procedure also entails the risk of contamination of a sample with products from the previous run.

Object of the present invention is to provide an apparatus for amplification and detection of nucleic acids from a biological material or a sample that does not have the aforementioned disadvantages and with the particular by means of a trial order in one operation a plurality of different target substances, such. B. nucleic acids, eg. B. can be detected by different infectious agents. This object is achieved with the features of present claim 1.

Accordingly, a device in the form of an array for duplicating and detecting target molecules from a biological material or a sample is provided which has at least two duplicating modules and at least two detection modules, wherein the duplicating modules are respectively tempered via a temperature control unit to be controlled individually,
and wherein the duplicating and detection modules are arranged on a component. Apart from a cover and optionally present special modules or units, the component is integrally formed.

The device or chip according to the invention is expediently adapted to be accommodated in a control and / or operating unit (base unit), this unit having the control, in particular temperature control, pumping and detection elements necessary for the operation of the device. These elements can be controlled by means of a computer.

Such a device makes it possible to test a single sample simultaneously on a plurality of target substances, in particular nucleic acids. Associated with this is an enormous gain in speed over conventional methods, which makes it possible to increase the throughput of a laboratory and thus intensive screening work, such as. in epidemiology or cancer diagnostics are required to accelerate.

By each amplification module associated with each individually controlled tempering can be performed in each duplicating module for the respective target reaction or the respective target material to be detected individually adjustable sequence of duration, temperature and number of cycles, and so for a target to be detected and each for this purpose Optimize the amplification enzymes and denaturing conditions used in each case without interfering with parallel processes. Preferred targets are nucleic acids. The control can be done in a simple manner via a programmable PC. In each individually controlled reproduction module, a corresponding multiplex reproduction can also be performed. These are typically limited to 3-4 target sequences.

Since the individual modules are arranged on a component, pipetting of the respective sample or of the duplicating product from one module to the next is no longer necessary. Furthermore, the throughput rate increases, and the risk of contamination is reduced and material loss is minimized. Since all control elements are arranged in the control and operating unit, it is possible to provide a variable and highly portable system solely by the configuration of the device according to the invention as a chip, especially as a microchip or as a cartridge, which only a larger operator unit and the respective Use includes suitable chips. It is therefore not necessary to have an in-house laboratory to carry out the analyzes, so that use "in the field", e.g. at the site of a crime or in an epidemiological study in a developing country or in a doctor's office, is readily possible.

In an alternative embodiment, the device may be designed to detect bacteria.

In a preferred embodiment of the invention it is provided that the device also has at least one module for extracting nucleic acids, which is arranged on the same common component.

In this way, the extraction of the nucleic acids from the sample or the biological material is integrated into the device, which further increases the advantages mentioned above.

Preferably, the number of replication and detection modules is the same, and the number of extraction modules is less than or equal to the number of duplicating modules.

Even more preferably, two or more modules for the extraction of nucleic acids are arranged on the common component. In this way, a different extraction method can be chosen in each extraction module to exclude artifacts by a less suitable method, or different samples or materials can be simultaneously extracted and analyzed.

However, it may also be useful to separately extract the nucleic acids to be amplified and only then to introduce them into the device. This is e.g. the case when it is a very solid sample, for the extraction of mechanical force is required, or if the material to be examined contains very few nucleic acids, so that the extracted nucleic acid must first be enriched by using a large amount of material or sample.

In a particularly preferred embodiment of the invention it is provided that the common component, the extraction module, the duplicating module and / or the detection module are predominantly or entirely made of a polymer material. Suitable polymer materials are polyamides, polycarbonates, PMMA, polysulfones, polyether ketones, polyesters, polyethylene, PET, a cycloolefinic polymer (COC) such as. B. TOPAS, polyacetates, polymethanes. In a further preferred embodiment, the detection unit may consist of a second material which is particularly suitable for a detection radiation used.

In this way, a cost-produced, lightweight, inert and easy to dispose of device, which usually has a low heat absorption. The latter contributes to increasing the speed of the process, since the thermocycles worked off during the amplification step (in non-isothermal amplification processes) can be passed through more quickly.

Likewise, the component may be wholly or partially made of an inorganic material such. As metal, silicon, ceramic and / or glass.

In a further preferred embodiment of the invention, it is provided that the individual modules have reaction chambers, which via microchannels with a cross-sectional area of particularly <1 mm ² or with cross sections of about 0.005 or 0.007 mm ² to about 1 mm ² or 0.8 mm ² , through which the biological material, the sample, the extracted nucleic acids or the amplification products are transported.
The chambers consist for example of depressions in the common component.
The channels may have any cross-section and be configured, for example, quadrangular, circular or U-shaped.

Particularly preferably, these channels have a diameter of 0.1 to 1 mm, in particular a diameter of 0.2 to 0.8 or 0.4 to 0.6 mm. In the case of a U-shaped or angular cross-section, these values relate to the average diameter which corresponds approximately to the diameter of an equally large circular cross-section.

Such structures can be produced depending on the material used by injection molding, micro injection molding, molding, embossing, etching, photolithography, micro sandblasting, lasers or laminate of films with and without recesses.

The liquid is transported by suitable pumps and valves as well as by capillary forces from one module to the next or from one reaction chamber to the next.

Likewise, it can preferably be provided that the wetting properties of the channels are specifically influenced. Thus, e.g. the surface at the exit of a chamber may be made hydrophobic in order to prevent leakage of reaction liquid during a reaction.

It is preferably provided that the channels are formed closed at the top. This feature is required to prevent excessive evaporation of the reaction liquid, but carries the risk of blistering, which would lead to interruption of the flow.

For this reason, it is particularly preferred that the channels are designed to be open at the top and covered with a liquid-impermeable, but gas-permeable structure. This may be e.g. to act a film that is made of PTFE, for example.

In a further preferred embodiment, the control unit and / or the device according to the invention has at least two temperature sensors. These are for example the Associated with duplicating modules and allow the control and control of the temperature control units.

In a likewise preferred embodiment, the common component can be inserted into the control / operator unit (base unit), in which the temperature control units and / or temperature sensors are also arranged.

In the former case, it is necessary that there is a good thermally conductive connection between the tempering unit and the common component, in particular the duplicating module, so that the thermocycles can be processed quickly, without having to heat the surrounding material for a long time. The base unit may moreover be e.g. have one or more ultrasonic sources, pumps for microfluidics, valves or optical detectors such as cameras or photomultipliers and photodiodes as a single detector or in array form.

This embodiment is particularly advantageous because it allows the use of inexpensive manufactured disposable components, in particular of polymer, since the mentioned functionalities such Temperierungseinheiten, temperature sensors, ultrasonic generating units or drives for stirrers, but also pumps for microfluidics or valves in the Base device remain.

Particularly preferably, it is provided that the temperature control unit of the duplication module comprises a Peltier element. Peltier elements are easy to control and allow fast and accurate generation of a desired temperature. Moreover, they are able to generate both heat and cold. By the arrangement of Peltier elements Directly on a surface of the devices according to the invention facing away from the liquid, in particular on the surfaces of the duplicating units, an extremely rapid tempering, ie heating and / or cooling, can be achieved. In this way, a further acceleration of the respective investigation is made possible. In an expedient embodiment, the Peltier elements are not integrated in the device or polymer cartridge.

In alternative embodiments, however, the use of air blowing devices, microwaves, IR radiators, heating resistors, heating or cooling liquid or conductive and resistive polymers is also provided. The latter can be incorporated directly into the material of the component. In addition, all combinations of said temperature control units are conceivable.

In a particularly preferred embodiment of the invention, it is provided that a nucleic acid amplification reaction can be carried out in the amplification module. This may be e.g. PCR (polymerase chain reaction), rtPCR (reverse transcription PCR), real-time PCR (real-time PCR), NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), RCA (rolling circle amplification), or SDA Act (beach displacement amplification).

The processes mentioned can be roughly subdivided into isothermal and non-isothermal amplification processes. It should be noted that in isothermal amplification processes no thermocycles are processed. Nevertheless, the device according to the invention can also be used in these methods with the stated advantages.

In any event, it is necessary that primers, mononucleotides, and one or more polymerases be added to the nucleic acids to be amplified during the amplification step. It is therefore preferably provided that the device according to the invention has a device for dispensing primers, mononucleotides and / or one or more polymerases. This can also be arranged both on the common component as well as on the base unit. The invention therefore also relates to a reaction kit containing these Subs-dance together with inventive device.

Furthermore, it is preferably provided that the temperature sensors are thermocouples, thermoresistors, silicon sensors, PTC or NTC elements or platinum sensors (PT 100, PT 1000).

Likewise, the temperature sensors may be elements of chromogenic polymers whose color changes as a function of the temperature.

Thus, e.g. the bottom of a chamber made of chromogenic polymer, while the lid is transparent. The temperature measurement is then carried out with a photodetector through the lid of the chamber. A photodetector may include a photodiode or a camera. Such measurements can also be carried out as a reflection measurement or as a transmission measurement.

Likewise, the temperature sensors may be particulate elements which are added to the reaction liquid and their optical properties, such as change their transmission, color, fluorescence or phosphorescence as a function of the temperature.

The advantage of all these methods is that the said sensors are in close proximity to the sample and have only a small own heat input, so that very accurate measurements are possible. In addition, the fact that all these measurements are non-contact reduces the risk of contamination.

In a further preferred embodiment of the invention it is provided that the extraction takes place in the extraction module by chemical means. This can e.g. done by enzymatic digestion and / or by conventional precipitation methods.

Likewise it can be provided that the extraction in the extraction module takes place by means of ultrasound. Using a suitable frequency and power cell walls and membranes are disrupted so that the nucleic acids can escape from the cell interior.

Ultrasound can also be used for mixing the reaction liquid in one of the modules. However, other frequencies and benefits may be considered.

Likewise, however, the extraction in the extraction module and / or the mixing in one of the modules can be effected by stirring. Decisive here are the shear forces that can be adjusted accordingly by selecting a suitable speed or a suitable stirrer.

A variant of this embodiment is the extraction or mixing by means of magnetic particles which are set in motion by an externally applied rotating magnetic field. Here it can be e.g. to act the well-known stir-fry, but also to magnetic microparticles. It is particularly preferred, as already mentioned, provided that the magnetic drive is arranged in the base unit.

It goes without saying that the said extraction and / or mixing methods can be combined as desired to improve the efficiency of extraction and / or mixing.

In this embodiment, it is preferably provided that the outlet channel of the extraction module has a cross-section which is smaller than that of the magnetic particles, or the magnetic particles are used whose cross-section is greater than that of the outlet channel. In this way, the particles used for stirring or extraction are prevented from being transported further.

In a further preferred embodiment of the invention it is provided that the chambers of the extraction and / or the duplicating modules are surrounded by recesses in the material of the component.

These recesses may be formed in particular in the form of channels closed at the top and on the one hand serve to thermally insulate the chambers and thus to accelerate the processing of the thermocycles. On the other hand, the thermal insulation stresses in Material reduced due to different temperatures. Finally, these recesses can also serve the acoustic insulation of the chambers. This, for example, increases the efficiency of using ultrasound to mix or extract the nucleic acids.

In principle, the recesses z. B. be closed by means of a cover to channels. They are usually filled with air, gas or a liquid or have a vacuum. But it can also be provided that the temperature of the chambers takes place via these channels. In this case, e.g. cold or warm liquids are led into the channels. In one embodiment, the base unit is configured to provide the cold and warm fluids. Advantageously, therefore, a rapid temperature change of the duplicating modules can be achieved.

In a further preferred embodiment of the device according to the invention it is provided that the detection module is integrated in the duplicating module. In this way it is possible to follow the progress of the duplication reaction in real time. The corresponding methods are known as online PCR or real-time PCR.

The detection of the duplication products takes place, for example. by means of FRET (fluorescence resonance energy transfer) or by quenching of fluorescence molecules. In principle, the detection can also be carried out by measuring an electrical current or voltage, which is generated by a suitable electrochemical reaction.

In another embodiment of the invention it is provided that the detection of the amplified nucleic acids in the detection module by means of electrophoresis, Southern blot, immunoassay or hybridization with labeled nucleic acid probes.

In an alternative embodiment, electrochemical or mass-sensitive detectors such as a quartz crystal or a surface acoustic wave device are provided for detecting the amplified nucleic acids, which have a capture nucleic acid on their surface.

The biological material is preferably material selected from the group consisting of blood, serum, cerebrospinal fluid, urine, sputum, stool, plant material and tissue samples.

Areas of application are here e.g. forensics, medical diagnostics or genetic analysis.

The sample, on the other hand, is preferably a sample from the group consisting of water, liquid foods, solid foods, baby food, soil samples, and air samples. Areas of application are here e.g. the environmental, the water or the food analysis, in particular the analysis of genetically modified food.

Further provided is a method of extracting, amplifying and detecting nucleic acids from a biological material or a sample using a device according to any one of the preceding claims.

The invention will now be described by way of example with reference to the accompanying drawings.

Fig. 1 shows a schematic representation of the device according to the invention with order, duplication and detection units.

Fig. 2 shows a schematic representation of an analysis chip according to the invention with extraction and duplication unit.

Fig. 3 shows a cross section through such a device according to the invention.

4 shows an exemplary representation of a device according to the invention.

As shown in FIG. 1, the device according to the invention has one or more inlets or application devices (1) for different liquids obtained from a sample with already extracted targets to be detected, in particular nucleic acids. These are then directed to 4 different Nukleinvielvielfältigungseinheiten (4) in which they are multiplied under different conditions with respect to enzymes, temperature and cycle length and then detected in the detector (5). In this case, the nucleic acid extraction is performed outside the device according to the invention.

According to a preferred embodiment of the invention, the chip polymer component additionally contains a disruption device (2), in particular nucleic acid extraction unit. In the example shown in FIG. 2, starting from a sample application (1), the sample is conveyed to in each case 2 different extraction units (2) and subjected to different extraction processes there. The extraction process itself can be carried out by means of stirring and / or ultrasound or by application by means of sudden pressure relief. Also chemically induced processes, eg. B. by pH change are possible. By means of the device according to the invention, at the same time tiny amounts of a single sample completely different conditions in the extraction units (2) treated and the extracts thus obtained on different duplicating units (4) are passed, where they individually and specifically in each unit at different temperature, cycle time and Enzyme be duplicated.

As shown in Fig. 3, the device according to the invention comprises a polymer base member (9), which is one or more multi-storey. In the polymer base member (9) microchannels (8) are arranged, in which the respective extraction or amplification reactions are carried out. The device is closed by means of a cover (11). On the opposite side of the channel (8), a temperature sensor (7) is arranged, on the opposite side of a temperature control unit (6), in particular Peltier element opposite. In a further embodiment, instead of the Peltier element (6) or in addition to this, an ultrasound head (not shown) or another component such as a stirrer may be arranged which mixes and / or dissolves the reaction medium present in the reaction space or channel (8) , In addition to the reaction space (8), an insulating space (10) can be arranged, which isolates a lateral spread of the temperature to further reaction units (not shown). In principle, the space can also be formed as a channel and serve by applying by means of a gas or fluid of the temperature. Alternatively, the cover (11) as Liquid-tight and gas-permeable film may be formed.

A further exemplary representation of a device according to the invention which serves for the duplication and detection of target substances is shown in FIG. 4. The device comprises the polymer base member (9) and the cover (11). The polymer basic component (9) has two inlets (1), three further inlets (3), three duplication units or modules (4), two separate detection modules (5) and three outlets (14). The inlets (1) and the further inlets (3) are connected to the three replication modules (4) by means of microchannels (8). Likewise, the three duplicating modules (4) are connected to the three outlets (14) by means of microchannels (8). In this case, two of the three replication modules (4) are each coupled to two of the three outlets (14) via a respective detection module (5). Insulating spaces (10), which are formed as recesses, are arranged around one of the three replication modules (4). A third of the three duplicating modules (4) already comprises the associated detection module (5). Each of the three replication modules (4) is associated with a temperature control unit (6), each comprising a Peltier element. For reasons of clarity, only one temperature control unit (6) is shown. In order to be able to control the temperature cycles, each of the three replication modules (4) is also assigned a temperature sensor. A not shown operating unit comprises the temperature sensors and the temperature control units (6). Both are arranged in an operating phase on the polymer base member (9) and the cover (11), in particular pressed.

The cover (11) is shown separately from the polymer base member (9) for ease of illustration. It is fixedly and durably arranged in the manufacture of the device on the polymer base member (9). The cover (11) at least partially contains a polymer material that is transparent at the wavelength of the optical detection method used. If, for example, a fluorescence method is used as the detection method, the coverage at the excitation wavelength and at the wavelength of the fluorescence radiation emitted by the detection modules (5) is permeable. The polymer base member (9) also comprises a polymer material.

The liquid extracted from a sample with extracted targets is supplied via the inlets (1) to the polymer base member (9). At the further inlets (3), the substances necessary for the duplication are supplied to the polymer basic component (9). Via microchannels (8) and by applying a pressure difference between the inlets (1) and the further inlets (3) and the outlets (14), the liquids with the targets and the substances used for the duplication are fed to the three replication modules (4). The nucleic acid to be amplified is multiplied by means of temperature cycles generated by the three temperature control units (6). In two of the three amplification modules (4), the nucleic acid produced is fed to two detection modules (5) and detected there by means of fluorescence methods. In the third of the duplicating modules (4), the detection already takes place in the combined unit, comprising the duplicating module (4) and the detection module (5). The cover (11) serves to guide the liquids and prevents evaporation of the liquids. Next prevented it penetration of particles and other interfering substances during transport into the recesses of the polymer base member (9). The cover (11) is designed so that it can be easily opened at the inlets (1), the other inlets (3) and the outlets (14). The opening can be done for example by piercing the cover (11).

Thus, advantageously, a plurality of nucleic acids can be duplicated and detected simultaneously and in parallel from one sample. Advantageously, the temperature cycles for the duplication in the three duplicating modules (4) may be different, since each duplicating module (4) is assigned its own tempering unit (6).

In an alternative embodiment, not shown, extraction modules are connected between the inlets 1 and the duplicating modules (4).

In a further alternative embodiment, the detection modules (5) comprise electrochemical detectors. The electrochemical detectors comprise an array of electrodes.

Claims (15)

Device for duplicating and detecting target substances, in particular nucleic acids, from a sample, comprising
at least two duplicating modules and at least two detection modules,
wherein the duplication and detection modules are arranged on a component, and the duplicating modules can be temperature-controlled by means of a temperature control unit which is to be controlled individually. Apparatus according to claim 1, characterized in that it contains at least one module for the extraction of nucleic acids, which is arranged on the component. Device according to one of the preceding claims, characterized in that the component, the extraction module, the duplicating module and / or the detection module made of a polymer material. Device according to one of the preceding claims, characterized in that the individual modules are connected via microchannels with a cross-sectional area of less than or equal to 1 mm ² , via which the biological material, the sample, the extracted nucleic acids or the amplification products are transported. Apparatus according to claim 4, characterized in that the microchannels have different wetting properties. Apparatus according to claim 4 or 5, characterized in that the channels are closed by means of a cover which has a liquid-impermeable, but gas-permeable structure. Device according to one of the preceding claims, characterized in that the device also comprises at least two temperature sensors. Device according to one of the preceding claims, characterized in that the common component is adapted for receiving in an operating unit in which the temperature control units and / or temperature sensors are arranged. Device according to one of the preceding claims, characterized in that the temperature control unit has a Peltier element. Device according to one of the preceding claims, characterized in that the temperature sensors thermocouples, platinum sensors, thermoresistors, silicon sensors or PTC or NTC elements and / or elements of chromogenic polymers whose color changes as a function of the temperature, are. Device according to one of the preceding claims, characterized in that the chambers of the extraction and / or the duplicating modules are surrounded by recesses in the material of the component. Device according to one of the preceding claims, characterized in that one of the at least two detection modules is integrated in one of the at least two duplicating modules. Device according to one of the preceding claims, characterized in that one of the at least two detection modules is connected downstream of one of the at least two duplicating modules. Kit of parts comprising a device according to one of the preceding claims together with amplification enzymes, separating gels, ultrafiltration membranes and / or markers. Use of a device according to one of the preceding claims for the extraction, duplication and detection of target substances, in particular of nucleic acids, from a biological material or a sample.

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