EP2280778B1 - Titer plate and method for detecting an analyte - Google Patents

Description

The present invention relates to a titer plate for detecting an analyte according to claim 1. Furthermore, the invention relates to a method for detecting an analyte according to claim 12.

The WO 03/087410 A1 discloses an apparatus and method for directly performing biochemical processes on a substrate. The substrate comprises a card which is detachably connected to the substrate, wherein a reaction element is also detachably arranged on the upper side of the substrate.

The WO 2007/076023 A1 relates to a multiwell analysis plate having a plate body and a plurality of wells defined therein, the wells having a binding surface with a capture reagent immobilized thereon and a dry reagent.

The WO 2007/111347 A1 discloses a device for treating a multi-well plate, the device having at least one nozzle head for dispensing and receiving within the respective wells of received fluids.

By means of molecular diagnostic analyzes, e.g. viral loads of HI viruses, hepatitis C and hepatitis B viruses. In a central laboratory today such analyzes are often performed on pipetting robot systems. The reaction vessels used are microtiter plates, in particular so-called 96-well plates with, for example, 8 rows of 12 wells each. These recesses are arranged at standardized intervals of about 0.9 cm from each other. Sample material and reagents are pipetted by the pipetting robot in a programmable manner by means of pipette tips made of plastic or washable, reusable tips into predetermined wells or wells of the titer plates. In addition, some operations such as incubation at a certain temperature, mixing processes or, for example, magnetic separation processes are carried out in the pipetting robot system.

An almost indispensable method of molecular diagnostics is an amplification of a so-called target - the analyte - by a thermocyclization reaction such as a so-called polymerase chain reaction - short PCR. there smallest, not directly detectable amounts of analyte molecules are multiplied exponentially to detectable levels.

Manipulating samples to be amplified or amplified is extremely critical. Smallest contamination, for example Aerosol formation, with possibly even single molecules, would lead to sample material with false positive or increased quantitative results. Therefore, it is common in molecular diagnostics to perform sample preparation and amplification in separate rooms. However, this is very expensive and requires the processing of the samples by laboratory staff.

One approach is to hermetically seal the titer plate with laminating film prior to performing the PCR as in DE 10 2005 059 535 A1 described. The titer plate may then no longer be opened in the same room. This measure of the separated rooms contradicts the trend to integrate and automate analysis processes.

To analyze the resulting PCR product together with the amplified analyte, for example, optical methods based on so-called real-time PCR come into question. However, there are measurement methods in molecular diagnostics for electrical detection in which hybridization reactions are performed. A generic method is from the WO 99/07879 A1 known. For this purpose, the resulting PCR product from the reaction vessel is transferred to another vessel for hybridization. A solution for a contamination-free transfer lies in a hermetic integration of the vessels for PCR and hybridization in a closed cassette, such as in a so-called quicklab® point of care cassette. However, this solution is often too expensive for a routine, diverse application and allows only comparatively low throughputs.

For efficient detection, a product of the hybridization reaction with the amplified analyte can be detected electrically. The exemplary named quicklab® is for example in the DE 102 33 212 A1 disclosed. Another arrangement for a biochip is from the DE 100 58 397 A1 known. The DE 101 26 341 A1 teaches a biochip that changes an electrically detectable property by hybridization with an analyte. It is an object of the present invention to provide a titer plate which allows for improved automated handling of the amplified samples.

This object is achieved by technically simple means according to claim 1.

The invention particularly comprises a first embodiment of a titer plate according to claim 1.

The titer plate according to the invention preferably has depressions which are arranged at intervals which correspond to the distances in a standard microtiter plate, so that the titer plate according to the invention can be operated by a pipetting robot. This standard distance is e.g. 9mm. In addition, the titer plate preferably has the length and width of a standard titer plate, e.g. it preferably conforms to the standard titer plate format with 12x8 wells. The thickness of the titer plate of the invention is preferably greater to accommodate the wall surrounding each one biochip and a plurality of wells, as described in more detail below.

The titer plate according to the invention can preferably be operated by a pipetting robot which has a supply of fresh pipette tips. He can grab these, to any of the e.g. 12x8 = 96 positions of the wells on a standard titer plate, lower them there and aspirate or inject liquid from the well. In addition, he can put a used pipette tip in a waste container, grab a new, clean pipette tip and continue working with it.

For the detection of the analyte, at least one biochip is arranged on the titer plate according to the invention. "Biochip" is understood to mean a chip which is suitable for detecting an analyte, for example a specific DNA, and in particular in the presence thereof the analyte generates an electrical signal. Typically, the biochip has one or more sensitive surfaces with capture molecules that each bind specifically to an analyte. It can be arranged on a biochip several sensitive surfaces with different capture molecules, eg 8 to 400, more preferably 64 or 128 sensitive surfaces or "spots". The biochip is, for example, a CMOS chip with universal zip-code catchers. Preferably, a plurality of biochips, in particular 6 to 24, preferably 12 biochips, are present on a titer plate, so that, for example, 12 random access multiplex assays can be performed.

When analyte molecules bind to the capture molecules, e.g. hybridize with them, e.g. causes a change in the capacitance on the sensitive surface, which can be read out electrically. This is preferably done as follows: The analyte or the target is biotinylated. After analyte molecules bind to the capture molecules, a label enzyme such as e.g. Streptavidin or AG phosphatase added. This enzyme binds to the biotin of the target. When a substrate is subsequently added, a reaction product is produced which generates an electrical signal that can be read by the biochip. Particularly preferably, the biochip is designed for the detection of DNA by means of hybridization to suitable capture molecules.

The sensitive surface (s) of the biochip is / are preferably arranged on one side, in particular the upper side of the biochip, while the contacts on which the electrical signal can be read out are arranged on a side of the biochip opposite thereto. Preferably, 5 to 100, in particular 8 to 12 sensitive surfaces and correspondingly many contacts are arranged on a biochip, so that it is possible to test simultaneously on a plurality of different analytes.

The biochip is preferably embedded in the titer plate, e.g. the biochip is embedded in a plastic ring, which in turn is inserted into a suitable depression of the titer plate, so that sample or reaction material can be brought into contact with the preferably upward-facing sensitive surfaces of the biochip. Preferably, the biochip is on the side of the sensitive areas - e.g. towards the top - provided with a seal, which prevents impurities can penetrate the catcher molecules. The seal is e.g. a cover of an elastomeric and / or thermoplastic material.

The biochip preferably extends over a region on which a standard titer plate has a fixed number, e.g. 2, 4, 6 or 8 wells are arranged. At the standard position of a well, a pipetting robot can stop and suck in or discharge material. This allows the biochip of pipetting robots to be filled with sample material, reaction material or with label enzymes and substrate.

In each case a plurality of depressions or holes are arranged next to each DNA chip, in which e.g. an amplification of the target e.g. can be performed by a PCR reaction or other reagents are provided. Thus, one biochip and several of the wells each form a unit within which a particular analysis is performed. This unit is surrounded by a wall. All necessary steps for detection can be carried out in the unit. The spatial separation of the units allows many units to be accommodated in a very small area without risking contamination of the individual samples. A massively parallel examination of very many samples is possible quickly and inexpensively.

For example, a unit surrounded by a wall includes 2, 4, 6, 8, or 10 wells and one, two, or three biochips. Particularly preferably, 4 wells and 1 biochip form one Unit, where the biochip occupies an area that is also taken up by 4 wells in a standard titer plate. A preferred unit thus corresponds to 8 wells and thus has 8 positions in which a pipetting robot can handle a pipette tip. A titer plate in standard 12x8 format thus has 12 units, each separated by a wall from each other.

The titer plate according to the invention is, for example, a block of about 20-70 mm, preferably of 45 ± 10 mm height, wherein the wall between the individual units is formed by each unit being arranged in a kind of recess in the block. The depressions and the biochip are arranged at the bottom of the recess. The pipetting robot can move a pipette tip from one depression to the next depression or to the biochip within the recess. The wall surrounding the biochip and a plurality of depressions is the wall of the recess and is preferably about as high as the height of the titer plate, ie preferably about 20-70 mm, preferably 45 ± 10 mm high. This effectively prevents individual droplets from being able to pass from one unit to an adjacent unit during pipetting. Thus, the titer plate preferably has as many recesses as units from a plurality of wells and a biochip.

Such a recess is preferably only open to a first side, in particular the upper side of the titer plate. In each unit, a pipette can be introduced via the recess and remains during the transfer of the sample, for example from a recess to the biochip within the enclosed space of the unit. The first side opposite side, preferably the bottom, the titer plate provides a receptacle for the biochip and is provided with bulges, each corresponding to a depression.

Particularly preferably, the recesses are each formed in the form of a slot and have, for example, approximately an "E" shape for each unit. The recesses are adapted to the movement of a pipetting robot. The slot extends over all four recesses and at least one position above the biochip. It virtually forms a single connecting line between the depressions and the biochip. This has the advantage that not only between the individual units, but also partially between the individual wells within a unit walls are arranged. Once a pipette tip comes into contact with sample material, the tip can be left inside this unit. In contrast to known titer plates, the smallest droplets can not get into other analysis units and contaminate them, as a used pipette tip is not led out over other depressions or chips. The recess is designed according to a development so that one or more pipette tips can remain therein.

The titer plate is preferably made of plastic. Since it is preferably equipped with recesses which are open to one side, it is possible to produce the titer plate (without biochips) by injection molding. Preferably, the walls or recesses and the recesses are made as a one-piece plastic injection-molded part, in which later the biochips are embedded. Alternatively, the titer plate can also be formed as a flat plate with recesses and receptacles for the biochips, are placed on the wall elements between the individual units. Since in this case, a seal between wall elements and plate must be taken care of, this embodiment is not preferred.

The biochip is preferably embedded in the titer plate such that each corner of the biochip is located at the corresponding position of a well on the standard format microtiter plate. At least one of these positions is a Einfüllport in an elastomeric cover or seal the biochip present. The elastomeric seal serves to protect the sensitive surfaces of the biochip from contamination. It can be integrated into the titer plate in a two-component injection molding process, for example. Alternatively, the seal or cover is clamped by a plastic ring, in which the biochip is embedded. More preferably, the elastomeric seal is slightly spaced from the sensitive surface of the biochip thereby forming therebetween a biochip chamber in fluid communication with the sensitive surfaces of the biochip.

Via the filling port, liquid can preferably be injected into the biochip chamber, which then comes into contact with the catcher molecules and is thus analyzed by the biochip. Since the filling port of the biochip is, as it were, "aligned" with one of the standard positions of the wells of a titer plate, a pipette can automatically transfer a sample to the biochip by means of the pipetting robot.

Particularly preferably, at least one and preferably two deposition positions for a pipette tip are provided above the elastomeric seal of the biochip, on one or preferably two of the e.g. 4 positions, each corresponding to a well on a standard titer plate and thus accessible by a pipetting robot. A pipette that has come into contact with sample material can be deposited at this point and thus remain inside the unit, even if fresh pipette tips are used in the unit if necessary. In addition, another 4 pipette tips may remain at the 4 well locations.

Further, an overflow reservoir is preferably provided for each unit to receive excess fluid. In a preferred embodiment, the overflow reservoir is connected or connectable directly to the biochip chamber. Consequently If liquid that is filled into the biochip chamber runs directly into the overflow reservoir. Since the biochip is preferably embedded in the bottom of the titer plate, the overflow reservoir is preferably located above the biochip chamber. Therefore, the overflow reservoir is preferably provided with a wick or other absorbent material which draws the liquid from the chip chamber. In this way, all the fluid that is pressed through the chip chamber is received by the overflow reservoir. The overflow reservoir preferably holds 0.5 ml to 5 ml, more preferably 1-2 ml. Furthermore, the overflow reservoir is preferably equipped with an overflow wall, which prevents a backflow of liquid into the biochip chamber. Alternatively, the overflow reservoir could also be filled, for example, through an inlet at one of the standard positions corresponding to the wells on a Stardard titer plate.

According to an alternative embodiment, the invention comprises a titer plate for detecting an analyte, with at least one biochip, wherein the at least one biochip is designed for detecting an analyte and is surrounded by a wall, wherein a seal, preferably an elastomeric seal, on the at least one Biochip (14) is applied, which defines a Biochipkammer together with the biochip and connected to an overflow reservoir directly to the Biochipkammer or connectable. Thus, liquid that is filled into the biochip chamber continues directly into the overflow reservoir. Since the biochip is preferably embedded in the bottom of the titer plate, the overflow reservoir is preferably located above the biochip chamber. Therefore, the overflow reservoir is preferably provided with a wick or other absorbent material which draws the liquid from the chip chamber. In this way, all the fluid that is pressed through the chip chamber is received by the overflow reservoir. According to this alternative embodiment, no further recesses in the titer plate are necessary, so that the entire base area of the titer plate can be completely covered with biochips. All further preferred features of the invention, which are mentioned in connection with the first embodiment of the invention according to claim 1, can also be provided in the alternative embodiment of the titer plate according to the invention.

Optionally, the titer plate according to the invention may be penetrated by at least one bore. This bore extends transversely to the carrier material of the titer plate and preferably opens between the depressions and the biochip. A negative pressure can be applied by means of this, in order to suck off possible droplet impurities from the space above the titer plate. Preferably, each unit or recess is equipped with its own bore.

Furthermore, the disclosure is directed to a reading device for a titer plate, which is designed to ensure a further improvement of the automatic handling of the samples. The reading device provides at least one electrical contact or read-out area for the biochip. Preferably, the readout surface is heated, since the readout result of the biochip is usually readable only after appropriate heat treatment.
The reading device also includes trays for the wells, which are preferably designed as heatable recordings (thermoblocks). When placing the titer plate on the reading device in each case a depression is received in a tub, preferably the recess is as closely as possible enclosed by the tub in order to ensure a good heat transfer. Preferably, in each case as many wells as depressions are formed in one unit, form an independent thermo-cycle unit, hereinafter also called thermoblock. By suitable heating of the wells, for example, a PCR can be carried out in the wells.
Preferably, a reading device has mutually independent units each having four trays and a contact surface.

It is a further object of the present invention to provide an improved method for detecting an analyte by means of a biochip. This object is achieved by a method according to claim 12. By using a titer plate according to the invention, a so-called "liquid handling" can take place by means of a pipetting robot.

In addition to introducing the sample into one of the wells of the titer plate, the method preferably comprises an amplification of the analyte and offers the advantage of a spatial integration of a hybridization region of the biochip in a common space. Contamination of other sample materials is effectively avoided as the pipette tip remains in this space.

According to a preferred embodiment of the method, a plurality of pipette tips may be used by the pipetting robot for each unit, which remain within each unit after use. This has the advantage that neighboring units can not be contaminated with amplified material when the pipette tip is transported away over them. Particularly preferably used pipette tips are placed on the storage positions on the cover or seal of the biochip.

For example, not only one, but more than one sample, eg of different tissues of the same patient, are introduced into the different wells of a unit. After a PCR, the reaction mixtures from the different wells with different pipette tips can successively be transferred into the biochip chamber and the biochip read out. The pipette tip is thereby completely emptied over the biochip chamber, wherein excess liquid flows into the overflow reservoir. Thereafter, the pipette tip is preferably deposited on the same well from which the PCR reaction mixture was transferred. For further manipulations, a fresh pipette tip is used, which in turn remains in the unit or recess.

Preferably, after transferring the PCR reaction product, a further liquid, in particular a label liquid, is transferred with a pipette tip onto the biochip or into the biochip chamber. The label is e.g. Streptavidin or AG phosphatase, which binds to the biotinylated analyte. The pipette tip used to transfer the label has not yet been emptied, as it may be reused for further samples, and is therefore parked in one of the storage or parking locations.

Then a substrate is preferably pipetted into the biochip chamber with another pipette tip. This forms a reaction product which triggers an electrical signal on the biochip. The pipette tip with which the substrate has been transferred is not yet emptied, as it may be used again for further samples, and is therefore parked in a second storage or parking position.

Thus, the preferred method is that multiple pipette tips, e.g. remain on different storage positions or in the recesses within the recess.

For the amplification of the analyte, a thermocyclization reaction is preferably used. According to a development of the method according to the invention, a polymerase chain reaction - in short PCR -, an allele-specific primer expression - abbreviated to ASPE - and / or a so-called Amplification Refractory Mutation System - ARMS for short.

In addition to a temperature-controlled amplification of the analyte, a temperature-controlled hybridization by means of the biochip is also provided here.

An improved detection of the analyte is also achieved by a temperature-controlled electrical, in particular electrochemical, detection.

Furthermore, to improve the automatic handling of the samples, an analyzer consisting of a combination of reader and titer plate is provided. This combination is adapted to standard pipetting robots.

Fig. 1

eine perspektivische Ansicht eines Ausführungsbeispiels einer erfindungsgemäßen Titerplatte;

Fig.2

eine perspektivische Ansicht eines Beispiels einer Leseeinrichtung für eine Titerplatte;

Fig. 3

eine perspektivische Ansicht einer Kombination einer Titerplatte gemäß Fig. 1 mit einer Leseeinrichtung gemäß Fig. 2;

Fig.4

eine Draufsicht auf einen Ausschnitt der Oberseite der erfindungsgemäßen Titerplatte;

Fig.5

einen Längsschnitt durch einen Ausschnitt einer Titerplatte und eines Lesegeräts gemäß einer zweiten Ausführungsform;

Fig.6

einen Ablaufplan eines erfindungsgemäßen Verfahrens.

Another aspect of the present disclosure relates to the use of the titer plate of the invention. The wells of one unit can be used with four different reference concentrations for quantitative determinations. This is conceivable for expression experiments in the integrated DNA technique or multiple multiplexing in so-called single nucleotide polymorphism (SNP for short).
With reference to the accompanying drawings, preferred embodiments will now be described.
In the drawings show:

Fig. 1

a perspective view of an embodiment of a titer plate according to the invention;

Fig.2

a perspective view of an example of a reading device for a titer plate;

Fig. 3

a perspective view of a combination of a titer plate according to Fig. 1 with a reading device according to Fig. 2 ;

Figure 4

a plan view of a section of the top of the titer plate according to the invention;

Figure 5

a longitudinal section through a section of a titer plate and a reading device according to a second embodiment;

Figure 6

a flowchart of a method according to the invention.

Hereinafter, the embodiments of the present invention will be described with reference to the drawings.

FIG. 1 1 shows a titer plate 10 for detecting an analyte from the underside 22. The titer plate 10 has a plurality of recesses 12 arranged in rows, which are visible from the underside 22 as bulges 12 '. In this case, the depressions 12 are spaced apart and aligned in such a way that a pipetting robot with a pipette tip can introduce necessary reagents, solvents and / or a sample to be tested for the analyte into the depressions 12.

In addition to two rows of wells 12 a number of biochips 14 is arranged. The biochips 14 are also positioned relative to the wells 12 so that the robot can automatically program the pipette tip thereto. These biochips 14 are designed, e.g. to detect a DNA by hybridization, wherein at least one electrical property of the biochip 14 changes. Such biochips 14 may possibly also include a chip card laboratory.

The titer plate 10 is modular, it comprises a block, for example an injection molded part, made of plastic, in which the depressions 12 or bulges 12 'are formed, and the biochips 14. These are accommodated in receptacles 14' in the block. Among the biochips 14, a seal made of a thermoplastic elastomer is preferably arranged, which supports the receptacle 14 'to the top of the titer plate (in FIG Fig. 1 at the bottom). In the seal preferably funnel-shaped openings are arranged, which form an inlet port, an outlet and possibly storage positions. Furthermore, the biochips 14 may each be held in a plastic ring, which is fixed in the receptacle 14 ', eg glued or welded.

In order to be able to examine a plurality of samples on a titer plate 10, a plurality of-in this case four-wells 12, each with a biochip 14, are combined to form a unit 21, which in FIG Fig. 1 is surrounded by a dash-dotted line. At least along this line 21 extends a wall 16 which encloses four recesses 12 and a biochip 14 and protects against contamination. For this purpose, the injection-molded part has a thickness d - ie a wall height - of about 50mm to about 60mm. The titer plate 10 has 16 such units 21.

FIG. 2 shows an example of a reading device 26 for a titer plate 10. On the reading device 26, on the one hand a series of trays 13 are arranged, which serve as heatable receptacles for the wells 12 of the titer plate. In particular, the fit in FIG. 1 The four troughs 13 of a unit 21 are each combined into a thermoblock 11 and preferably independently of each other and in particular heated by the other thermoblocks 11. The illustrated reading device 26 thus comprises 12 independent 4 thermoblocks 11. With a thermoblock 11, an analyte in the wells 12 can be amplified by means of an amplification reaction to a well detectable amount.
The reading device 26 further provides some electrical readout or contact surfaces 15 for the biochips 14. In each case a biochip 14 rests on a readout surface 15, so that the corresponding contacts on the biochip 14 are contacted and read out. Preferably, the temperature of the readout surfaces 15 can be controlled. On a readout surface 15, for example, 8 electrical contacts are arranged.
The contact surfaces 15 and trays 13 are surrounded by a border 17, which has a in FIG. 1 Align the titer plate 10 shown on the reading device 26 and can hold.

The footprint and height of the reading device 26 is preferably compatible with the known STARlet® system, ie the dimensions are for example 150x110x110 mm ³ and fit into a 7-track carrier. The reading device 26 preferably comprises 12 independent 4 thermoblocks or thermal cycler 11, with which an arbitrarily programmable PCR can be performed, as well as 12 independent temperature-controlled electrical biochip read-out blocks. The integrated electronics preferably have a communication interface, 24 independent temperature control units and 12 independent digital interfaces for biochip reading.

In Fig. 3 is the titer plate 10 of the Fig. 1 shown from the top, as directed to a reading device 26 according to Fig. 2 is attached. In this case, the titer plate 10 is supported by the border 17. In the top 20 of the titer plate 10 16 recesses 18 can be seen, each having the shape of an approximately E-shaped elongated hole. The recesses 18 go through to the bottom of the titer plate 10, so they are "milled" as in a block. Each recess 18 is therefore bounded by relatively thick walls 16 which extend to the recesses 12 in the titer plate 10. The walls 16 provide a fencing of the individual units of recesses 12 and the biochip 14 ready. These are arranged on the base 19 of the recess 18 and can be reached by means of a pipetting robot. The pipetting robot moves a pipette tip 40 for transferring an amplified sample mixture from the depression 12 into the biochip 14 along the slot 18.

In FIG. 4 schematically is a plan view of a single unit 21 - comprising a biochip and four wells - a titer plate 10 from the top 20 shown. In the titer plate 10, a recess 18 is introduced. The recess 18 is formed as a slot and enclosed by the walls 16. To the top 20 towards the recess 18 is open.

Through the recess 18 can be seen on the bottom 19 of the titer plate 10, in which four depressions 12 shown on the right are embedded. On the left side of the biochip 14 is below the dashed line. The biochip 14 is e.g. covered by a seal 30 in which at 32, a filling port is inserted, can be brought by means of a pipette tip, a sample in contact with the biochip 14. At positions 33 and 35, the seal 30 on the biochip 14 is not permeable, but provided with a small receptacle for a pipette tip. This shot may e.g. a small recess in the seal 30, in which a pipette tip can be accommodated. However, such depressions are not absolutely necessary. In any case, a pipette tip can be deposited at positions 33 and 35. Therefore, these positions 33, 35 are connected to the slot 18. In this case, the opening of the pipette tip is sealed by the elastomeric material of the seal 30, so that the pipette tip is still filled with e.g. Substrate or label enzyme can be filled when it is parked at one of these storage positions 33 or 35. At the position 34, an opening of an overflow reservoir is arranged. This position is not connected to the slot 18, since it is not necessary to move to this position a pipette tip.

To handle a sample in the recesses 12, the recess 18 is designed in the form of a multi-membered elongated hole, which makes, inter alia, the four wells "accessible from above". That is, pipette tips 40 can be inserted and displaced via the slot 18 by means of a pipetting robot. The inserted pipette tip reaches all the depressions 12 and the biochip 14 without having to leave the recess 18. An amplified sample containing the target can be taken up by the pipette from one of the depressions 12 and transferred via the filling port 32 into the biochip 14. The fill port 32 penetrates the elastomeric seal or coating 30 that is applied to the biochip 14 for protection.

Via the filling port 32, the liquid sample passes from a pipette tip 40 into a biochip chamber 36, which is arranged adjacent to the sensitive surfaces of the biochip 14 and in particular runs between the biochip 14 and the seal 30. From the biochip chamber 36 goes from an overflow reservoir 24, which is open to the top. In the overflow reservoir 24, a wicking element 31 is arranged, in this case a cylindrical piece of absorbent material, e.g. Foam or cotton wool. From the biochip chamber 34, the liquid sample from a pipette tip 40 thus continues to run into the overflow reservoir 24.

A pipette tip may remain in 6 different positions within the recess 18 after use: if the pipette spit is still filled, e.g. With label or substrate, in particular with a liquid that will be needed again later, it can be parked on one of the two storage positions 33, 35, where its opening is closed by the seal 30. An empty, used pipette tip, e.g. after pipetting PCR product from one of the depressions 12 via the filling port 32 into the biochip chamber 36, it is possible to deposit in the respective depression 12. After the transfer of PCR product into the biochip chamber 36, the pipette tip can be completely emptied, since the biochip chamber 36 is connected directly to the overflow reservoir 24, into which all excess liquid is sucked off.

The FIG. 5 shows a combination of a titer plate 10 with a reading device 26 in longitudinal section, wherein again only one unit 21 is shown. The titer plate 10 comprises a plurality of depressions 12, the bulges 12 'of which are introduced into troughs 13 of a thermoblock 11. The introduced sample material is copied several times by means of a thermocyclization reaction, such as a PCR. In order to avoid contamination of sample material from other wells 12, a barrier medium 28 - here a mineral oil film - is provided in one of the wells 12. To the wells 12 close to walls 16, which are open to the first side 20. Thus, pipette tips 40 introduced by a pipetting robot can reach the recesses 12. In each case between the two recesses 12 and the left recess 12 and the biochip 14, the wall 14 is not cut, but recognizable in plan view.

The biochip 14 is surrounded by a plastic ring 41, which preferably has a sealing lip and seals the biochip 14 against the titer plate 10 or the seal 30. At the top, the biochip 14 has as a contamination protection a seal 30, e.g. made of polypropylene. Between the seal 30 and the surface with capture molecules for the analyte on the biochip 14, a biochip chamber 36 is formed, into which a sample can be taken. The amplified sample can be transferred with the pipette tip 40 via a filling port 32 into the biochip chamber 36, wherein the bounded recess 18 is not left.

Between the recesses 12 and the filling port 32, a bore 38 is arranged. This bore 38 penetrates the entire titer plate 10. By applying a vacuum or vacuum, a steady stream of air can be generated which further reduces the likelihood of contamination with sample material. In this case, the air flow enters the titer plate 10 via the first side 20. An aerosol, droplets or the like is then removed with the air flow through the bore 38. There is thus a so-called extraction system for the titer plate 10 according to the invention. The bore 38 may also be covered with a filter material 39.

The sample enters the chip chamber 36 via the filling port 32 via the biochip 14. If too much liquid is filled into the biochip chamber 36, it passes through the outlet 34 in the seal 30 into an overflow reservoir 24. For once in the overflow reservoir 24 liquid located can not run back, an intermediate wall 37 is as overflow protection provided as in FIG. 5 shown. In the intermediate space between the intermediate wall 37 and the wall 16 is, for example, a Saugdocht, which can absorb excess sample liquid and conducts it by means of capillary forces in the overflow reservoir 24, which can accommodate about 1.3ml liquid.

According to another embodiment, not shown, the overflow reservoir 24 is located directly above the outlet 34, but separated from the biochip chamber 36 by a gap. The overflow reservoir 24 is almost completely filled with a wicking element, as in FIG Fig. 4 shown, which can hold about 1-2ml of liquid. Liquid occurring at the outlet 34 is absorbed by the wick. Due to the gap, the liquid flow breaks off immediately, if no liquid is replenished. In this way, no return flow, not even by capillary forces.

With the titer plate 10, the method according to the invention for the detection of an analyte by means of a biochip 14 can be performed. The biochip 14 is integrated into a titer plate 10 as described above. The method comprises the following steps: introduction of a sample into one of the wells 12 of the titer plate 10 and introduction of reagents into the wells 12. Subsequently, an amplification reaction is carried out with the reagent-containing sample, a PCR reaction, an ASPE reaction being carried out. and / or an ARMS reaction. Thereafter, the transfer of the resulting reaction mixture to the biochip 14, and readout of the biochip 14, which changes by hybridization with the analyte at least one electrically detectable property. The inventive method is characterized in that the liquids are transferred by means of a pipetting robot with the pipette tip 40 and the pipette tip 40 remains within the enclosed space 18. For this purpose, it is stored in one of the recesses 12 or on one of the storage positions 33, 35.

The use of pipetting robots considerably speeds up the analysis process, is less error-prone and more cost-effective. In particular, the use for quantitative determinations, an integrated DNA technique - IDT short or a multiple multiplexing in the context of SNP possible.

An inventive method for detecting an analyte by means of biochips 14 is in FIG. 6 shown as a flowchart. It first comprises the introduction 102 of one or more sample (s) to be examined into one or more of the wells 12 of a titer plate 10. This titer plate 10 is preferably a titer plate 10 according to the invention as described above. The pipette tip 40 brought into contact with the sample may then be disposed of by a pipetting robot in the usual way.

In a further method step 104, the reagents required for an amplification reaction of the analyte are introduced into the recesses 12. For this purpose, a further pipette tip 40 is introduced by the pipetting robot into the recess 18 and preferably thereafter disposed of in the usual way outside the recess. Optionally, the sample may be overcoated with mineral oil prior to the amplification reaction. After merging the reagents and the sample, a temperature program 106 is performed to run a PCR reaction. The reaction mixture then contains the analyte in bulk copied form for better detection.

The pipetting robot then picks up a new, clean pipette tip 40, step 108. With this pipette tip, the reaction mixture with the amplified sample is transferred from a first of the depressions 12 into the biochip 14 in step 110. The reaction mixture is introduced into the biochip chamber 36. Eventually too much received reaction mixture passes through the outlet 34 into the overflow reservoir 24. The used pipette tip is after complete emptying placed on the first recess 12, step 114, ie it remains within the recess 18th

Thereafter, the pipetting robot first receives another new, clean pipette tip 40, step 116. This pipette tip is used to pick up a label enzyme from a reservoir located outside of the recess 18 and transfer it into the biochip 14 in step 118. The pipette tip 40 is not yet empty thereafter and is therefore deposited at the first deposition position 33, step 120, i. it remains within the recess 18.

Thereafter, the pipetting robot receives another new, clean pipette tip 40, step 122. This pipette tip is used to pick up a substrate from a reservoir located outside of the recess 18 and transfer it into the biochip 14 in step 124. The pipette tip 40 is not yet empty thereafter and is therefore deposited at the second deposition position 35, step 126, i. it remains within the recess 18.

Thereafter, the biochip 14 may be read out, step 128.

Steps 110 to 128 may be repeated several more times, with the other reaction mixtures from the other wells 12. The pipette tip used to pipette the reaction mixture from the well 12 is returned to the respective well 12 and remains there until it is disposed of together with the titer plate 10. The pipette tips with which label enzyme and substrate were transferred are reused and then parked again at storage locations 33, 35.

The invention thus does not allow disposing of used pipette tips over other recesses 18 in which other samples are being examined, thus reducing the risk of contamination.

Claims (18)

1. Titer plate (10) for detecting an analyte, having multiple recesses (12) and having at least one biochip (14),
   characterized in
   that the at least one biochip (14) is designed for detecting an analyte and is arranged next to the recesses (12), wherein, respectively, multiple recesses (12) and one biochip (14) form a unit (21) and are arranged at the base (19) of a clearance (18) of the titer plate (10), which is surrounded by a wall (16).
2. Titer plate (10) according to claim 1, characterized in that it has multiple clearances (18).
3. Titer plate (10) according to claim 1 or 2, characterized in that the at least one clearance (18) is open toward a first side (20) of the titer plate (10), wherein the opposite side (22) is provided with a receptacle (14') for the biochip (14) and with a bulge (12') of the recess (12).
4. Titer plate (10) according to any one of claims 1 to 3, characterized in that the at least one clearance (18) is formed as an elongated hole (24) which extends over all recesses (12) and the biochip (14), which are surrounded by the wall (16).
5. Titer plate (10) according to any one of the preceding claims, characterized in that one elastomeric seal (30) is applied on the at least one biochip (14) and has one filling port (32).
6. Titer plate (10) according to any one of the prceeding claims, characterized in that, on the elastomeric seal (30) of the biochip (14), at least one deposit position is provided for a pipette tip (40).
7. Titer plate (10) according to any one of the preceding claims, characterized in that one biochip chamber (36) is in fluid communication with a sensitive surface of the biochip (14).
8. Titer plate (10) according to claim 7, characterized in that the biochip chamber (36) is connectable with an overflow reservoir (24) in order to collect a liquid from the biochip chamber (36).
9. Titer plate (10) according to claim 8, characterized in that a wick element (31) for soaking up liquid from the biochip chamber (36) is arranged in the overflow reservoir (24).
10. Titer plate (10) according to any one of claims 1 to 9, characterized in that a hole (38) penetrating the titer plate (10) is present within the clearance (18).
11. Titer plate (10) according to any one of claims 1 to 10, characterized in that the biochip (14) is designed for detecting DNA by means of hybridization.
12. Method for detecting an analyte by means of a biochip (14) which is integrated into a titer plate (10) according to any one of claims 1 to 11, having the following steps:

    - transferring (110) a substance, in particular a liquid, from one of the recesses (12) onto the biochip (14),

    characterized in
    that the substance is transferred onto the biochip (14) by means of a pipetting robot with a pipette tip (40) and the pipette tip (40) remains within the clearance (18) after the transfer of the substance.
13. Method according to claim 12, characterized by the following steps:

    a) introducing (102) a sample into one of the recesses (12);

    b) introducing reagents into at least one of the recesses (12);

    c) carrying out an amplification reaction with the sample with reagents added;

    d) transferring (110) the resulting reaction mix onto the biochip (14); and

    e) reading the biochip (14), which changes at least one electrically recordable property owing to hybridization with the analyte;

    wherein the reaction mix and/or the sample is introduced, and/or transferred onto the biochip (14), by means of a pipetting robot with a pipette tip (40) and that the pipette tip (40) remains within the clearance (18).
14. Method according to claim 13, characterized in that, after step d), a liquid, in particular a labeling liquid, is transferred onto the biochip (14) with a pipette tip (40).
15. Method according to claim 13 or 14, characterized in that steps a) to e) are carried out with a further sample.
16. Method according to any one of claims 12 to 15, characterized in that multiple pipette tips (40) remain within the clearance (18).
17. Method according to any one of claims 12 to 16, characterized in that, after pipetting, in particular after transferring (110) the resulting reaction mix from one of the recesses (12) onto the biochip (14), a pipette tip (40) is placed down in the respective recess (12) from which liquid was transferred.
18. Method according to any one of claims 12 to 17, characterized in that after pipetting, in particular after transferring labeling liquid onto the biochip (14), a pipette tip (40) is placed down on a deposit position (33, 35) within the clearance (18), said deposit position sealing the pipette tip (40) to the bottom so that no liquid can escape from the pipette tip (40).

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