DE102005051850A1 - Device for duplication and detection of nucleic acids - Google Patents

Abstract

A device for replicating and detecting target substances, in particular nucleic acids, from a sample is described. The device comprises at least two duplication modules and at least two detection modules, which are arranged on a component, the temperature of the duplication modules being controllable via a temperature control unit that is individually controlled.

Description

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

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

there is about to detect certain nucleic acids from which e.g. to a criminal culpability, the disposition for a hereditary disease or the genetic modification of a food shut down leaves. 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 purpose, reaction chips, Cartridges, electrophoresis chambers o. Ä., And the corresponding Devices for Reading these devices used.

There in the mentioned as well as many other cases, the nucleic acid molecules of interest are often only in very small quantities and in the immediate vicinity occur to a variety of other nucleic acid molecules, the former first targeted and specifically duplicated. These are off the prior art so-called duplication or amplification methods such as Polymerase chain reaction (PCR) or SDA known. This procedure are usually carried out in so-called thermocyclers.

Around to carry out both procedures, is therefore a big one Machinery required, which in turn is a well-equipped Laboratory required. A use outside the laboratory is therefore not readily possible. This complicates e.g. work on epidemiological investigations in the field, forensic investigations at the scene, routine examinations in the doctor's office etc.

moreover have to the nucleic acids to be detected first in the duplicating device be given, and then the duplicated ones molecules in the detection device. Each transfer step leads therefore on the one hand to material and time losses and on the other hand holds the risk of contamination with foreign nucleic acids.

Further are known from the prior art devices in which duplication function and detection function are integrated. Avoid these devices the above problems.

In some cases are the said functional units in the form of an array such miniaturized to fit on a small carrier ("Lab on a chip ") the individual reaction chambers are via miniaturized channels in conjunction, and the reaction liquids are by means of suitable Micropumps and valves as well over the capillary force is transported from one chamber to the next (microfluidics). Such chips or cartridges are thereby adapted in special Arranged equipment, which contains the necessary control and operating units, e.g. Pumps as well as heating and cooling elements etc. and control the reactions, detections etc. and evaluate.

In the scripture "flow-through polymerase chain reaction in chip thermocyclers "(Schnegaß & Köhler 2001, Rev. Mol. Biotechnol. 82; 101), such "labs presented on a chip ". As a duplication method becomes exclusive Mentioned PCR, at the success of the duplication relies on the processing of thermocycles. the authors describe a chip having a meandering channel below arranged in the ordered arrangement heating elements of different temperature are. Will the reaction liquid Pumped through the channel at a suitable speed, it passes through the for the course of the PCR required temperature cycles, and the nucleic acids reproduced.

There the duplication factor depends exponentially on the number of thermocycles, is in such a "Lab on a chip "the duplication factor already set. Such chips are therefore not very variable and consequently for different uses not to use.

Indeed These are known from the prior art devices in usually for designed the parallel examination of many samples on the same nucleic acid. So can e.g. in some devices 96 food samples of various kinds Origin at the same time parallel to the presence of a particular Gens, e.g. on an antibiotic resistance gene that indicates a genetic modification.

By contrast, the simultaneous examination of a sample for the presence of a large number of nucleic acids (as would be desirable, for example, for proteomics or genomics or the rapid diagnosis of infection) does not enable these devices. The above-mentioned document therefore proposes the serial throughput of several samples in succession. In addition to the loss of time, however, this procedure also entails the risk of contamination of a sample with products from the previous one Run.

task It is the object of the present invention to provide a duplicating device and detection of nucleic acids to provide from a biological material or a sample, which does not have the aforementioned disadvantages and with the particular means a trial order in a process a plurality of different Target substances, such as e.g. Nucleic acids, e.g. of different Infectious agents can be detected. This task is with the features of the present claim 1.

Therefore is a device in the form of an array for duplication and detection of target molecules from a biological material or a sample, which at least two duplication modules and at least two detection modules, wherein the duplicating modules each have a be controlled individually tempered Temperierungseinheit and where the reproduction and detection modules are arranged on a component. apart from a cover and any special modules available or units, the component is integrally formed.

The inventive device or chip is convenient adapted for inclusion in a control and / or operating unit (base unit), this unit being necessary for the operation of the device Control, in particular tempering, pumping, and detection elements having. These elements can be controlled by a computer.

A such device allows it, a single sample simultaneously on several target substances, in particular nucleic acids to test. This is associated with a huge gain in speed over conventional Procedure that makes it possible increase the throughput of a laboratory and thus intensive screening work, as they are e.g. are required in epidemiology or cancer diagnostics, to accelerate.

By the duplication module assigned individually controlled temperature control unit can in each duplication module one for the respective target reaction or the respective target material to be detected individually adjustable sequence of duration, temperature and number the cycles performed and so on for a target to be detected and the amplification enzymes used for this purpose and to optimize denaturing conditions in each case, without disrupting parallel processes. Preferred targets are Nucleic acids. The control can be easily via a programmable PC. In each individually controlled duplicating module can also be carried out a corresponding multiplex duplication. These are typically at 3-4 Target sequences limited.

There the individual modules are arranged on a component, eliminating one Pipetting the respective sample or the duplicating product from one module to the next. Furthermore, it increases the throughput speed, and it becomes the risk of contamination reduced and minimized material loss. Since all Controls are arranged in the control and operating unit is it is possible solely by the configuration of the device according to the invention as a chip, in particular as a microchip or as a cartridge a variable and highly portable System to provide only a larger operator unit and the for the respective Use includes suitable chips. For the implementation of Analyzes is therefore not a separate laboratory required, so too a use "in Field ", e.g. at the scene of a crime or in an epidemiological study in a developing country or in a doctor's office, without further ado possible is.

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

In A preferred embodiment of the invention provides that the device as well has at least one module for the extraction of nucleic acids on the same common component is arranged.

On this way, the extraction of nucleic acids from the sample or the biological material integrated into the device, what the above mentioned advantages still increased.

Prefers is the number of duplication and detection modules equal, and the number of extraction modules is less than or equal to the number of duplicating modules.

Especially even preferred are two or more modules for the extraction of nucleic acids on the arranged common component. This way, in every extraction module an deres extraction method can be chosen to perform artifacts to exclude a less suitable method, or it may be different Samples or materials are extracted simultaneously and submitted to an analysis.

However, it may also be useful to those too Extracting amplifying nucleic acids separately and only then introduce into the device. This is the case, for example, if 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 enriched by using a large amount of material or sample must become.

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

On This creates a cost-effective, easy to manufacture, inert and easy to dispose device, usually a low heat absorption having. The latter carries to increase the speed of the process, as the while of the duplication step (in non-isothermal duplication methods) processed thermocycles can be run through faster.

As well The component can be made wholly or partly 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 can have any cross-section and, for example quadrangular, circular or U-shaped be designed.

Especially Preferably, these channels have one Diameter of 0.1-1 mm, in particular a diameter of 0.2-0.8 or 0.4-0.6 mm on. In a U-shaped or square cross-section, these values refer to the average diameter which corresponds approximately to the diameter of an equal circular cross-section.

Such Depending on the material used, structures can be injection molded, Micro-injection molding, molding, stamping, etching, photolithography, Micro sand blasting, lasers or laminates of films with and without recesses produce.

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

As well may preferably be provided that the wetting properties of the channels be specifically influenced. Thus, e.g. the surface at the exit a chamber may be made hydrophobic to leakage of reaction liquid while to prevent a reaction.

Prefers is provided that the channels after are formed closed at the top. This feature is required to excessive evaporation of the reaction liquid However, there is a risk of blistering, leading to an interruption lead the river would.

Out For this reason, it is particularly preferred that the channels after open at the top and with a liquid-impermeable, but gas permeable Structure are covered. This may be e.g. around a slide act, which is made of PTFE, for example.

In Another preferred embodiment, the control unit and / or the device according to the invention at least two temperature sensors. These are e.g. the duplicating modules assigned and allow the control and control of the temperature control units.

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

In The former case requires that a good thermally conductive Connection between the temperature control unit and the common component, especially the duplication module, so that the thermocycles can be processed quickly, without that only surrounding material has to be heated tediously. The Base unit can also e.g. 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.

These Embodiment is particularly advantageous because it is the use from inexpensive manufactured disposable components, in particular of polymer, allows, since the mentioned functionalities such as temperature control units, temperature sensors, ultrasonic generating units or drives for stirrers, but also pumps for the microfluidics or valves remain in the base unit.

Especially It is preferably provided that the tempering unit of the duplicating module includes a Peltier element. Peltier elements are easy to control and allow a fast and accurate generation of a desired temperature. moreover they are able to heat both also cold to create. Due to the arrangement of Peltier elements directly on one of the liquid facing away surface the devices according to the invention, especially on the surfaces of the Reproduction units can extremely fast tempering, d. H. Heating and / or cooling achieved become. In this way, a further acceleration of the respective Examination possible. In an expedient embodiment the Peltier elements are not in the device or polymer cartridge integrated.

In alternative embodiments However, the use of air blowing devices, microwaves, IR lamps, heating resistors, Heating or cooling liquid or conductive and resistive polymers. The latter can be directly in the material be incorporated in the component. In addition, all are Combinations of said temperature control conceivable.

In A particularly preferred embodiment of the invention is provided, that in the duplicating module a nucleic acid amplification reaction feasible is. 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 (Strand Displacement Amplification) act.

The The above methods are roughly in isothermal and non-isothermal Divide the amplification process. It should be noted that no thermocycles are processed in isothermal amplification processes become. Nevertheless, the device according to the invention is also in these Method usable with the advantages mentioned.

In In any case, it is necessary that during the duplication step the one to be reproduced nucleic acids Primers, mononucleotides and one or more polymerase can be added. Prefers is therefore intended that the device according to the invention a device for delivery of primers, mononucleotides and / or one or more Having polymerases. This can also be found both on the common Component be arranged as well as on the base unit. The invention therefore, also relates to a reaction kit combining these substances with inventive device contains.

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

As well For example, the temperature sensors may be elements of chromogens Polymers whose color changes as a function of temperature.

So can e.g. the bottom of a chamber is made of chromogenic polymer, while whose lid is transparent. The temperature measurement then takes place with a photodetector through the lid of the chamber. A photodetector may include a photodiode or a camera. Such measurements are also used as a reflection measurement or as a transmission measurement feasible.

As well For example, the temperature sensors may be particulate elements act of the reaction liquid and their optical properties, e.g. their transmission, color, fluorescence or phosphorescence depending change from the temperature.

advantage All of these methods is that the sensors mentioned in the immediate Close to Sample and have only a small own heat input, so that very accurate measurements are possible. Furthermore diminishes the fact that all these measurements are contactless are the danger of contamination.

In a further preferred embodiment of the invention is provided that the extraction in the extraction module by chemical means he follows. This can e.g. by enzymatic digestion and / or by customary precipitation methods happen.

As well can be provided that the extraction in the extraction module done by ultrasound. When using a suitable frequency and Performance become cell walls and open-minded membranes, allowing the nucleic acids out can escape the cell interior.

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

As well but may be the extraction in the extraction module and / or mixing in one of the modules by stirring respectively. Decisive here are the shear forces that occur by selecting a suitable speed or a suitable stirrer accordingly can be adjusted.

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

It It goes without saying that the said extraction methods and / or for mixing with each other can be combined with each other to the Efficiency of extraction and / or mixing.

Prefers is provided in this embodiment, in particular, that the outlet channel of the extraction module has a cross-section which is smaller than that of the magnetic Particles or magnetic particles are used, the Cross section is larger as that of the outlet channel. This will prevent them from being stirred or extracted used particles are transported.

In a further preferred embodiment of the invention is provided that the chambers of the extraction and / or duplication modules of Recesses are surrounded in the material of the component.

These Recesses can formed in particular in the form of upwardly closed channels on the one hand serve to thermally isolate the chambers and so accelerate the processing of the thermocycles. On the other hand Due to the thermal insulation stresses in the material due reduced temperatures. Finally, these recesses can also serve the acoustic insulation of the chambers. This increases e.g. the efficiency of using ultrasound for mixing or for extracting the nucleic acids.

in principle can the recesses e.g. be closed by means of a cover to channels. They are usually with air, gas or a liquid filled or have a vacuum. But it can also be provided that the temperature of the chambers over these channels he follows. In this case, e.g. cold or warm liquids in the channels directed. In one embodiment is the basic device designed to provide the cold and warm liquid.

With The advantage is thus a rapid temperature change of the duplicating modules achievable.

In a further preferred embodiment of the device according to the invention is provided that the detection module in the duplicating module is integrated. In this way, it allows the progress of the duplication reaction track in real time. The corresponding procedures are as Online PCR or Real-Time PCR.

The Detection of the duplication products takes place while e.g. by means of FRET (fluorescence resonance energy transfer) or by quenching fluorescence molecules. In principle, the detection can also by measuring an electric current or voltage, which is generated by a suitable electrochemical reaction.

In another embodiment of the invention It is envisaged that the detection of the amplified nucleic acids in the detection module by electrophoresis, Southern blot, immunoassay or Hybridization with labeled nucleic acid probes takes place.

In an alternative embodiment are for the detection of the duplicated nucleic acids electrochemical or mass-sensitive detectors such as a quartz crystal or a surface acoustic wave device provided on its surface have a capture nucleic acid.

at the biological material is preferably material selected from the group containing blood, serum, cerebrospinal fluid, urine, sputum, stool, Plant material and tissue samples.

application areas here are e.g. forensics, medical diagnostics or the Genetic analysis.

at the sample, however, is preferably a sample of the Group containing water, liquid Food, solid foods, baby food, soil samples, and Air samples. Areas of application are here e.g. the environmental, water or food analysis, in particular analytics genetically modified Food.

Furthermore, a method for extraction, amplification and detection of nucleic acids from a biological material or a sample vorgese hen, in which a device according to one of the preceding claims is used.

The The invention will be described below with reference to the attached figures exemplified.

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

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

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

As in 1 1, the device according to the invention has one or more inlets or application devices ( 1 ) for various liquids obtained from a sample with already extracted targets to be detected, in particular nucleic acids. These are then transferred to 4 different nucleic acid amplification units ( 4 ) in which they are multiplied under different conditions in terms of enzymes, temperature and cycle length and then in the detector ( 5 ) are detected. 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 by 2 is based on a sample order ( 1 ) the sample to each of 2 different extraction units ( 2 ) and subjected there to various extraction methods. The extraction process itself can be carried out by means of stirring and / or ultrasound or by application by sudden release of pressure. Also chemically triggered processes, eg by pH change are possible. By means of the device according to the invention, at the same time, minute amounts of a single sample can be subjected to completely different conditions in the extraction units ( 2 ) and the extracts thus obtained are then applied to various amplification units ( 4 ), where they are individually and specifically reproduced in each unit at different temperature, cycle duration and enzymes.

As in 3 the device according to the invention comprises a polymer basic component ( 9 ), which is one or more multi-storey. In the polymer base ( 9 ) are microchannels ( 8th ) are arranged, in which the respective extraction or duplication reactions are carried out. The device is replaced by a cover ( 11 ) locked. On the opposite side of the canal ( 8th ) is a temperature sensor ( 7 ) 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 else another component such as, for example, a stirrer, can be arranged, which holds in the reaction space or channel ( 8th ) is mixed and / or digested. Next to the reaction space ( 8th ) an insulating space ( 10 ), which isolates lateral propagation 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 ) may be formed as a liquid-tight and gas-permeable film.

Claims (15)

Device for duplication and detection of target substances, in particular of nucleic acids, from a sample comprising at least two duplication modules and at least two detection modules, where the reproduction and detection modules are arranged on a component, and the Duplication modules via a individually tempered Temperierungseinheit tempered are. Device according to claim 1, characterized in that it comprises at least one module for extraction of nucleic acids contains which is arranged on the component. Device according to a the previous claims, characterized in that the component, the extraction module, the duplication module and / or the detection module consist 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. Device according to claim 4, characterized in that the microchannels have different wetting properties exhibit. Apparatus according to claim 4 or 5, characterized in that the channels are closed by means of a cover which is a liquid impermeable, but gas-permeable structure. Device according to a the previous claims, characterized in that the device also comprises at least two temperature sensors having. Device according to a the previous claims, characterized in that the common component for receiving is adapted in an operating unit in which the temperature control units and / or Temperature sensors are arranged. Device according to a the previous claims, characterized in that the temperature control unit has a Peltier element. Device according to a the previous claims, characterized in that the temperature sensors thermocouples, Platinum sensors, thermoresistors, Silicon sensors or PTC or NTC elements and / or elements from chromogenic polymers whose color depends on the temperature changes, are. Device according to a the previous claims, characterized in that the chambers of the extraction and / or the reproduction modules surrounded by recesses in the material of the component. Device according to a the previous 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 a the previous claims, characterized in that one of the at least two detection modules one of the at least two duplicating modules downstream is. Kit of Parts comprising a device according to one the previous claims together with amplification enzymes, separating gels, ultrafiltration membranes and / or markers. Use of a device according to one of the preceding claims Extraction, duplication and detection of target substances, in particular of nucleic acids a biological material or a sample.