DE102008021364A1 - Charging dry reagents into analysis unit used e.g. in biotechnology and genetic engineering, applies dry reagents to transfer reservoir to be brought into contact with analysis unit - Google Patents

Abstract

The analysis unit (103) is provided with cavities (105) to hold dry reagents (203). The dry reagents are applied initially to a transfer reservoir (201). Reservoir and analysis unit are brought into contact, such that dry reagents remain in the cavities. The analysis unit has a surface with openings through which the dry reagents are introduced into the cavities. The transfer reservoir is divided into geometric shapes by a mask. These shapes correspond with the geometric arrangement of cavities in the analysis unit. During division of the transfer reservoir it is moved over the analysis unit. Sections arising are brought into contact with the analysis unit. The preparation of the transfer reservoir and geometric arrangement of dry reagents on it, correspond with the arrangement of cavities in the analysis unit. The transfer reservoir has a surface onto which the reagents are brought. These surfaces are adhesive and fix the reagents on contact with them. The transfer reservoir is in strip form. In a corresponding kit, when the loaded transfer reservoir is brought into contact with the analysis unit, the dry reagents remain in the cavities. The analysis unit has a system of micro-channels connected to the cavities. It has two opposite surfaces, with the micro-channel system on one surface and the cavities on the other. The analysis unit comprises two separable sub-units. The micro channel system is in one sub-unit, with the cavities in the other sub-unit. An independent claim IS INCLUDED FOR a corresponding kit.

Description

The The present invention relates to a method for introducing Dry reagents in an analysis unit and a kit.

The Biotechnology and genetic engineering has become increasingly important in recent years won. A basic task in biotechnology and genetic engineering is the Detection of biological molecules like DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), Proteins, polypeptides, etc. In particular, molecules in which genetic information are coded for are many medical applications of great interest. Through her Detection, for example in a blood sample of a patient, can cause pathogens be detected, which makes a diagnosis easier for a doctor. The DNA is a double helix consisting of two cross-linked helical single chains, so-called half strands, is constructed. Each of these half strands has a lawn sequence wherein the sequence of the bases (adenine, guanine, thymine, Cytosine) the genetic information is fixed. DNA half-strands point characteristic feature, very specific only with whole certain other molecules to make a bond. Therefore, it is for the docking of a DNA half strand to another DNA half-strand requirement that the respective molecules complementary to each other are arranged.

This principle given by nature can be used for the selective detection of molecules be used in a sample to be examined. The basic idea of a biochip sensor based on this principle is that on a substrate of a suitable material first so called catcher molecules (oligonucleotides), For example, applied and immobilized by means of microdispensing be, d. H. on the surface permanently fixed to the biochip sensor. In this context it is known, bio molecules with thiol groups (SH groups) on gold surfaces to immobilize.

One corresponding biochip sensor with a substrate and bound thereto Catcher molecules that for example, to a specific DNA half-strand to be detected are sensitive, usually becomes for examining a sample, usually in the form of a liquid, be used for the presence of the DNA half strand. For this This is due to the presence of a particular DNA half-strand bring the investigating sample into operative contact with the immobilized capture molecules. are a catcher molecule and a to be examined DNA half strand complementary to each other, so hybridized the DNA half strand on the capture molecule, d. H. he is bound to it. If as a result of this binding the value a metrologically detectable physical quantity in a characteristic Way changes, so this value can be measured and in this way the presence or absence of a DNA half-strand in the sample examined be determined.

The described principle is not limited to the detection of DNA half-strands. Much more are other combinations of deposited on the substrate catcher molecules and to be detected molecules in a sample to be examined. For example, nucleic acids as Catcher molecules for peptides or proteins specific for nucleic acid bind, be used. Also known is peptides or proteins as catcher molecules for others, the capture peptide or the capture protein to use binding proteins or peptides.

To the Evidence of the bond between the applied on the substrate Catcher molecule and the become in the sample to be examined, to be detected molecule often optical or electronic detection methods used. Such detection methods gain increasing importance in industrial identification and Evaluation of new drugs organic or genetic engineering Origin. These detection methods open up many applications, for example in medical diagnostics, in the pharmaceutical industry, in the chemical industry, in food analysis, as well as in the Environmental and food technology.

to execution an analysis of the type described above, for example for detection a particular DNA, are generally several treatment steps required, extracted by means of which contained in the sample DNA becomes. The following is an example of the case of the analysis of a Blood sample of a patient received, the versions are also on other analytical methods transferable.

The processing steps to be performed include a variety of biochemical processes. Thus, first of all, the components to be analyzed, for example bacteria or viruses, must be extracted from the blood sample and the remaining blood sample disposed of. By means of a so-called lysis, the surrounding envelope of the viruses or bacteria is destroyed by means of a buffer solution, thus releasing the DNA to be analyzed later. Since there are generally too few copies of the DNA in the blood sample for a detection of the DNA, a duplication of the DNA takes place by means of a known reaction, the so-called polymerase chain reaction (PCR). In the further course of the analysis, various options are known, the DNA and there to prove with the pathogens.

The used biochips are based in their simplest form on a Glass substrate on which the capture molecules immobilized are. These often synthetically produced short nucleic acid fragments are at least partially complementary to the sequence in their sequence the nucleic acid to be detected, so that the binding is highly specific. Binding of nucleic acids other than The evidence to be proved must be avoided in any case so that the Measurement result is not false positive. The actual proof the nucleic acid occurs in many procedures over Fluorescence method in which a fluorescent dye to the to be detected nucleic acid is attached, for example, over a biotin-streptavidin bond. After specific hybridization of the nucleic acid with the capture molecules the biochip rinsed, so that unbound material is removed. To be in the solution Consequently, no more fluorescent dyes. By suggestion the dyes leaves observe the fluorescence with a CCD camera, thereby creating a Proof possible becomes.

One The advantage of biochips is the multiplexing capability of the detection. So can be different on different positions of the biochip Immobilize varieties of capture molecules, which are directed against various nucleic acids to be detected. The proof let yourself then over the spatially resolved Measure the fluorescence. Thus, several detections of nucleic acids in perform a procedure.

There the number of available copies of the nucleic acids is generally insufficient to provide proof directly, become the nucleic acids duplicated before proof (Amplified). This can be done, for example, by means of a polymerase chain reaction (PCR, Polymerase Chain Reaction). The PCR is based on the Replication of nucleic acids with the help of thermostable DNA polymerases. Here is a pair of oligonucleotide primers (single-stranded oligonucleotides) with the nucleic acid to be amplified brought into contact. The primers are chosen to match the two Bind ends of a fragment to be amplified on the complementary strands. at the elongation then becomes the primers in the 3 'direction along the respective target nucleic acid strand elongated (forward and reverse primer). Forward and forward Reverse primer Alternatively, as a sense or Designated antisense primer. That way, between the complementary to the primers Spots on the target nucleic acid lying piece be amplified. For subsequent detection reactions are the PCR products geous enough of primers, nucleotides and other interfering components of the PCR approach separated.

Of the PCR procedure includes several thermocycles with three steps each: First The sample is heated (eg to 94 ° C) to the strands of the to separate in the sample contained double-stranded target DNA (Denaturation). Then the temperature is lowered (eg to 45-60 ° C), so that the primers attach themselves to the complementary regions of the now single-stranded DNA can attach (Annealing). In the last step, those tied to the single strand become Primer by the DNA polymerase extended according to the information of the DNA template strand in the 3 'direction, wherein the corresponding nucleotide triphosphates in the solution as Blocks are used (elongation, eg at 72 ° C). This Cycle is during a PCR typically about 15-50 go through it. The indicated temperatures are only for Example values and are based on the specific PCR to be performed vote.

Thus, a small number of copies of a few available copies of a DNA (or generally of a nucleic acid) can be produced within a very short time, the concentration of which suffices for the subsequent qualitative detection of the DNA or nucleic acid in the sample. For example, after 20-fold through the PCR (typical time required 20 to 40 minutes) theoretically 2 ^20- fold, ie about 10 ^6- fold mango of the original nucleic acid used. The PCR also offers the possibility of incorporating a label into the resulting PCR product, which makes detection possible. For example, biotin-labeled PCR primers or nucleotide triphosphates can be used, resulting in the synthesized PCR products being biotinylated. The biotin is thus also immobilized on the biochip at the corresponding position after the attachment of the PCR product to the immobilized capture molecules. The biotin can then be bound in a further step with streptavidin associated fluorescent dyes, which then ultimately allow the detection of the nucleic acid or its PCR product. As an alternative to the fluorescent dye, it is possible to use other detection systems based, for example, on electrochemical detection.

When Catcher molecules can basically all in affinity chromatography used ligands are coupled. Examples for this are: Protein A, protein G, protein L, streptavidin, biotin, heparin, Antibody, Serum albumin, gelatin, lysine, concanavalin A, oligosaccharides, Oligonucleotides, polynucleotides, protein binding metal ions, lectins, Aptamers or enzymes.

Widely used z. B. in the medical diagnostics and in the research laboratory ELISA tests (enzyme-linked immunosorbent assay). For applications in the field of DNA chips, enzyme label methods are also used in a known method of redox cycling.

at the latter method, for example, the DNA to be examined a biotin molecule attached. After hybridization with the oligonucleotides of the biochip a streptavidin-linked label is added. Streptavidin and biotin Make a bond so that the label hybridizes with the label Complex of oligonucleotide and DNA to be detected is connected. After a wash, remove unbound label from the biochip surface is to be detected only in the case of a binding event DNA and oligonucleotide present a label above the biochip. By the proof of the label can thus on the present of the DNA to be detected in the original Sample be closed.

To the Proof of the label is added to a substrate through which the label of the Solved streptavidin becomes. It is hereafter in solution in front.

In certain cases, the redox behavior is diffusion-determined. As an example of a redox pair is called p-aminophenol / quinoneimine. The corresponding redox process involves 2 electrons and 2 H ⁺ ions. This system comes z. B. in enzyme-coupled detection reactions are used. The enzyme "alkaline phosphatase" is used as label or reinforcing substance. Alkaline phosphatase is capable of cleaving p-aminophenyl phosphate into p-aminophenol and phosphate. The resulting p-aminophenol is oxidized on the electrode system and the redox pair cyclized p-aminophenol / quinone imine.

The Height of finally available hanging signal in particular on the number of capture molecule-target molecule complexes formed. Therefore, it is especially important to maximize the number of immobilized capture molecules. A PCR can be enough target molecules for a Provide evidence if sufficient capture molecules are immobilized on the biochip. The quality the generated signal depends significantly from homogeneity and reproducibility of the immobilization of the capture molecules during the manufacturing process of the biochip. Therefore, quality control plays an important role the production around even in sensitive biochips.

to execution biochemical analyzes using biochips are particularly useful Point-of-care applications preferred to use integrated systems. These consist for example of a single-use card (Cartridge) and a control unit. It is desirable to design the integration so that all processing and Detection steps within the cartridge run fully automatically. For example, a drop of blood taken from a patient inserted into the cartridge be, whereupon after insertion into the control unit from the whole blood to be detected molecule (DNA, protein, ...) extracted and detected. These are in the cartridge generally provided numerous channels in which the individual process steps. To carry out the process steps are in the cartridge reagents store. This is preferably done in the form of dried reagents, as these have a longer shelf life than liquid reagents exhibit. It is also possible Reagents in the control unit to hold and guide them into the cartridge as needed. hereby but becomes the controller more complex and the flexibility of the device lower. When using cartridges with dry reagents is in control unit to hold only water, to dissolve the dried reagents and used to control microfluidics in the cartridge can.

The Introduction of Reagents into Microchannels of a Cartridge (Spotting) is with devices possible, similar, for example to work with a needle or inkjet printer. In the process, reagents are administered via a cannula in the liquid State in the microchannels the cartridge dropped. Subsequently, the reagents dried, leaving a longer Storage time possible becomes. This process is time consuming with cartridges containing a variety of microchannels requires a high number of process steps. This in turn increases the cost of Cartridge and thus the medical examination by means of Cartridge performed shall be. Especially in the case of dried reagents increases the processing time, since after each ridicule process the applied Need to dry reagents, before further reagents can be applied.

It is therefore an object of the present invention, a method and to provide a kit that provides a time-efficient introduction of reagents into an analysis unit.

These Task is by method according to claim 1 and a kit according to claim 8 solved.

• – Bereitstellen einer Analyseeinheit, die mehrere Kavitäten zur Aufnahme der Trockenreagenzien aufweist,
• – Bereitstellen wenigstens eines Transferreservoirs auf dem die Trockenreagenzien aufgebracht sind,
• – Inkontaktbringen des Transferreservoirs mit der Analyseeinheit derart, dass die Trockenreagenzien in den Kavitäten verbleiben.

A method is provided for introducing dry reagents into an analysis unit, comprising the following method steps:

• Providing an analysis unit which has a plurality of cavities for receiving the dry reagents,
• Providing at least one transfer reservoir on which the dry reagents are applied,
• - Contacting the transfer reservoir with the analysis unit such that the dry reagents remain in the cavities.

Advantageous across from known method for the introduction of reagents is that the Reagents already in dry form on the transfer reservoir, so that at the assembly the analysis unit with the dry reagents only one transfer is required. additional Drying times of the reagents during the assembly of the analysis unit can thereby omitted.

In an advantageous embodiment According to the invention, the analysis unit has a surface with openings through which the dry reagents can be introduced into the cavities are. This simplifies the corresponding contacting of the transfer reservoir with the analysis unit, since the dry reagents are directly in the cavities introduced can be.

Advantageous is a method such that the transfer reservoir by means of a Mask is divided into geometric shapes, which is a geometric arrangement the cavities correspond to the analysis unit. In this way, even more complex Arrangements of cavities in a simple manner with dry reagents from the transfer reservoir equip. Splitting the transfer reservoir increases the flexibility of the process, because standardized transfer reservoirs for equipping analysis unit with different arranged cavities can be used.

In An advantageous embodiment of the invention is the transfer reservoir while moved and created its division above the analysis unit sections are brought into contact with the analysis unit. That's the way to go the drying reagents in sections into the cavities of Introduce analysis unit. The division process and contacting the transfer reservoir with the analysis unit is carried out by means of each other nested procedures.

Advantageous is a method such that in providing the transfer reservoir the dry reagents geometrically on the transfer reservoir be arranged so that their arrangement of the arrangement of the cavities of the Analysis unit corresponds. This has the advantage that the transfer reservoir brought in contact with the analysis unit in a single step can be, with all dry reagents introduced into the appropriate cavities become. Thus, even complex geometric arrangements can be a variety of cavities easily time-efficiently fill with dry reagents.

In an advantageous embodiment of the invention, the transfer reservoir a surface on, on which the dry reagents are applied, with adhesives on the surface are arranged, through which the transfer reservoir after contacting can be fixed on the analysis unit. This offers the advantage that the openings the cavities after filling the dry reagents are closed by the transfer reservoir can. The adhesive serves to fix the transfer reservoir on the analysis unit.

According to the invention is a Kit provided with an analysis unit that holds multiple wells of dry reagents and a transfer reservoir on the the dry reagents are applied, the transfer reservoir being so is formed by contacting with the analysis unit the dry reagents remain in the wells.

Further Advantages and embodiments of the invention will become apparent from the hereinafter described embodiments in conjunction with the attached Drawings:

It demonstrate:

1 a cartridge,

2 an adhesive film with dry reagents,

3 Contacting of adhesive film and cartridge,

4 Assembly of a cartridge with dry reagents,

5 to 10 different mix options for liquids in the cartridge.

In 1 is schematically a cartridge 101 shown. The cartridge 101 includes a smart card-shaped plastic card 103 in the multiple microchannels 105 are formed. The microchannels 105 are just shown for the sake of clarity. In general, however, they will have a variety of curves and turns. In the microchannels 105 are to store reagents at predefined positions for carrying out various analysis and preparation steps. This is done in the embodiments illustrated here by means of an adhesive film, the in 2 is shown.

2 schematically shows an adhesive film 201 , the dimensions of the plastic card 103 out 1 is adjusted. At the positions where in the microchannels 105 the plastic card 103 Reagents to be introduced are dry reagents 203 on the adhesive film 201 appropriate. For better illustration, here are the microchannels 105 indicated.

In 3 is a side view of the contacting process between the adhesive film 201 and the plastic card 103 shown. On the adhesive film 201 are the dry reagents 203 applied, their geometric arrangement on the adhesive film 201 corresponds exactly to the microchannels 105 in the plastic card 103 , The adhesive film 201 has a surface 205 on which a suitable adhesive is applied. The adhesive film 201 gets on the plastic card 103 applied, what by the arrows 207 is indicated. As a result, on the one hand the dry reagents 203 into the corresponding microchannels 105 introduced, with the microchannels 105 on the other hand through the adhesive film 201 be closed. A microchannel 105 ' in the plastic card 103 is not equipped with dry reagents and only sealed. For example, it could be used for the microchannels 105 to connect with a liquid reservoir, not shown here.

alternative Is it possible, by means of a mask of a broader strip with applied Dry reagents to part out in such a way that the geometric Arrangement of the dry reagents of the desired arrangement within the microchannels equivalent.

In 4 an alternative embodiment of the invention is shown. It is the same plastic card 103 in turn, micro-channels 105 are formed. The plastic card 103 is two-dimensionally movable in the horizontal plane, which is indicated by the double arrow 251 is shown. A stripe 253 is on its bottom with dry reagents 255 stocked. He is across the plastic card 103 movable, what by the double arrow 257 is shown. Through the two-dimensional coordinate movement of the plastic card 103 and the strip 259 it is possible to use any of the dry reagents 255 above the plastic card 103 to position at any location. The vertical distance of the strip 253 from the plastic card 103 is preferred to keep small. After the foremost of the dry reagents 255 on the strip 253 placed at the desired position is by means of a vertically movable punch 259 (Double arrow 260 ) part of the strip 253 punched out so that the corresponding dry reagents 255 at the appropriate position in one of the microchannels 105 remains. This is how the microchannels gradually become 105 with dry reagents 255 equip. In the figure four are already illustrative four dry reagents 255 ' in microchannels 105 brought in. Above are pieces separated by the punching process 261 of the strip 253 remained.

In an alternative embodiment of the invention, it is also possible that the punch 259 the strip 253 not cut, but only on the plastic card 103 to push that by re-moving the strip 253 the corresponding dry reagent 255 into one of the microchannels 105 can be stripped off. In this case, no pieces remain 261 of the strip 253 on the plastic card 103 , In either alternative, the plastic card may 103 be sealed with an adhesive film, not shown here.

alternative Is it possible, to provide the plastic card with a photolithographic mask, the only at the places where reagents are introduced into the microchannels be permeable is. In a next step, reagents can be passed through the passages the mask into the microchannels of the Transfer the plastic card and then remove the mask.

In 5 is a microchannel 301 represented, through which a liquid is passed. This is by the arrow 303 shown. Along the micro channel 301 are reservoirs 305 arranged for reagents. The reagents should be mixed with the liquid in the microchannel 301 be mixed. When passing the liquid through the microchannel 301 they will be in the reservoir 305 stored reagents dissolved, what by the arrow 307 is shown. As a result, a mixing takes place, after which a mixture of the liquid with the reagents further in the microchannel 101 what flows through the arrow 309 is clarified.

In 6 is also the microchannel 301 shown at the two other reservoirs 311 are arranged for other reagents. The other reagents should turn with the mixture of the liquid with the reagent from the reservoir 305 be mixed. Mixing the liquid with the reagent from reservoir 305 (Arrow 309 ) dissolves in passing by the reservoir 311 the there stored further reagent, what by arrow 313 is shown. In the further course of the microchannel 301 Consequently, a mixture of the liquid flows with the reagents of the reservoirs 305 and 311 what's by arrow 315 is shown.

In 7 is shown that the mixing of the liquid with the reagent of the reservoir 305 can be improved when the liquid passes through the reservoir 305 is directed (arrow 317 ).

In 8th is a microchannel 301 shown, the further mixing, such as the Liquid with one of the reagents from the reservoir 305 or 311 , improved. These are constrictions 319 arranged on the channel walls. The constrictions cause turbulence in the liquid transport, so that the mixing is improved.

In 9 is in the reservoir 305 ' a partition 321 arranged so that here a microchannel is formed. There are constrictions on the walls in this microchannel 319 arranged so that already arise when dissolving the stored there reagents through the liquid turbulence and thereby the mixing is improved.

In 10 is a reservoir 323 for reagents on the microchannel 301 arranged. In the reservoir 323 is a piston 325 provided by an actuator 327 is movable. The reagents are in reservoir 323 on the microchannel 301 facing side of the piston 325 stored. By moving the piston 325 let the stored reagents in the microchannels 301 and transfer the liquid moving there. This is particularly advantageous in the case of liquid reagents. In the case of dry reagents, by moving the piston away 325 from the microchannel 301 a part of the liquid moving there targeted into the reservoir 323 to dissolve the dry reagents there. Subsequently, the liquid mixed with the dry reagents can be moved by oppositely directed movement of the piston 325 back into the microchannel 301 transfer back.

The embodiments can be preferably applied to a cartridge which is a microchannel system for receiving and transporting liquids. The microchannel system has microchannels on that with the cavities connected to receive the dry reagents. The microchannel system and the cavities can on the same surface the cartridge can be arranged. Alternatively, they can be opposite Surfaces (upper and bottom) of the cartridge or on separable parts of the cartridge be arranged. This increases the contamination safety when introducing the reagents and the flexibility of the system. A fluidic connection between the microchannel system and the cavities is to produce.

Claims (13)

Method for introducing dry reagents into an analysis unit, comprising the following method steps: providing an analysis unit which has a plurality of cavities for receiving the dry reagents ( 255 . 255 ' ), - providing at least one transfer reservoir on which the dry reagents ( 255 . 255 ' ), - contacting the transfer reservoir with the analysis unit in such a way that the dry reagents ( 255 . 255 ' ) remain in the cavities. The method of claim 1, wherein the analysis unit has a surface with openings through which the dry reagents ( 255 . 255 ' ) can be introduced into the cavities. The method of claim 1 or 2, wherein the transfer reservoir is divided into geometric shapes by means of a mask, the one geometric arrangement of the cavities correspond to the analysis unit. The method of claim 1 or 2, wherein the transfer reservoir while its division is moved above the analysis unit and resulting sections be brought into contact with the analysis unit. Method according to claim 1 or 2, wherein in the provision of the transfer reservoir the dry reagents ( 255 . 255 ' ) are arranged geometrically on the transfer reservoir such that their arrangement corresponds to the arrangement of the cavities of the analysis unit. Method according to one of the preceding claims, in which the transfer reservoir has a surface on which the dry reagents ( 255 . 255 ' ) are applied, wherein on the surface adhesives are arranged, can be fixed by the transfer reservoir after contacting on the analysis unit. Method according to one of the above claims, wherein the transfer reservoir is strip-shaped. Kit with an analysis unit containing several wells for holding dry reagents ( 255 . 255 ' ) and a transfer reservoir on which the dry reagents are applied, wherein the transfer reservoir is designed such that by contacting with the analysis unit the dry reagents ( 255 . 255 ' ) remain in the cavities. Kit according to claim 8, wherein the transfer reservoir has a surface on which the dry reagents ( 255 . 255 ' ) are applied, wherein on the surface adhesives are arranged, can be fixed by the transfer reservoir after contacting on the analysis unit. Kit according to claim 8 or 9, wherein the analysis unit has a surface with openings through which the dry reagents ( 255 . 255 ' ) can be introduced into the cavities. Kit according to any one of claims 8 to 10, at the analysis unit has a microchannel system connected to the cavities. Kit according to claim 11, wherein the analysis unit two opposite ones surfaces having the microchannel system on one surface and the cavities arranged on the other surface are. Kit according to claim 11, wherein the analysis unit includes two separable subunits, the microchannel system in one subunit and the cavities in the other subunit are arranged.