EP2280778A2

EP2280778A2 - Titer plate, reading device therefor and method for detecting an analyte, and use thereof

Info

Publication number: EP2280778A2
Application number: EP09753828A
Authority: EP
Inventors: Walter Gumbrecht; Peter Paulicka
Original assignee: Siemens AG
Current assignee: Boehringer Ingelheim Vetmedica GmbH
Priority date: 2008-05-30
Filing date: 2009-05-22
Publication date: 2011-02-09
Anticipated expiration: 2029-05-22
Also published as: JP5675592B2; US20110076690A1; EP2280778B1; DE102008025992B4; JP2011524004A; CN102046290A; WO2009144173A3; WO2009144173A2; US8383393B2; CN102046290B; DE102008025992A1

Abstract

The present invention relates to a titer plate (10) and a method for detecting an analyte, and the use thereof. According to the invention, it is proposed that a plurality of depressions (12) and a biochip (14) of the titer plate (10) disposed adjacent thereto be surrounded by a wall (16) in order to effectively prevent sample contamination when there is a high degree of spatial integration.

Description

description

Titer plate, reading device therefor and method for the detection of an analyte, and their use

The present invention relates to a titer plate for detecting an analyte and a reading device therefor. Furthermore, the invention relates to a method for detecting an analyte. Furthermore, the present invention involves the use of such methods, titer plates and a combination titer plate reader.

By means of molecular diagnostic analyzes, viral loads of HI viruses, hepatitis C and hepatitis B viruses are determined, for example. 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. Probenmateri ^¬ al and reagents are pipetted by the pipetting robot freely programmable by means of plastic pipette tips or washable, reusable tips in predetermined recesses or wells of the well 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 plant.

An almost indispensable method of molecular diagnostics is an amplification of a so-called target - the analyte - by a thermocycling reaction, such as, e.g. a so-called polymerase chain reaction - short PCR. The 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 contaminations, for example play on an aerosol, with possibly even only 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 consists of hermetically sealing the bottom plate with laminating film before carrying out the PCR, as described in DE 10 2005 059 535 A1. 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 known from WO 99/07879 Al. For this purpose, the resulting PCR product from the reaction ^¬ tion 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. This solution is for routine, diverse At ^¬ application often too expensive 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 disclosed, for example, in DE 102 33 212 A1. Another arrangement for a biochip is known from DE 100 58 397 A1. DE 101 26 341 A1 teaches a biochip which 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 well plate according to the invention preferably has recesses which are arranged at intervals corresponding to the Abstän ^¬ at a standard microtiter plate, so that the titer plate of the invention can be operated by a pipetting robot. This standard distance is eg 9mm. In addition, the titer plate preferably has the length and width of a standard titer plate, eg, it preferably conforms to the standard 12x8 well titer plate format. The thickness of the titre plate according to the invention is preferably larger in order to accommodate the wall, each of which surrounds a biochip and a plurality of recesses, as wei ^¬ ter be 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 this, move it to any of the eg 12x8 = 96 positions of the wells on a standard titer plate, lower them there and aspirate or inject liquid from the well. Moreover, it can take a used pipette tip into ei ^¬ nem waste containers, 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 the detection of an analyte, for example a specific DNA, and is particularly suitable for application of an analyte. senity of the analyte generates an electrical signal. Typically, the biochip to one or more sensitive FLAE ^¬ chen with capture molecules, each specifically binding to an analyte. Several sensitive surfaces with different capture molecules can be arranged on a biochip, eg 8 to 400, particularly 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 the analyte molecules bind to the capture molecules, for example, hybridize with them, for example, is effected a change in the capacitance of the sensitive surface, the electrically who can read ^¬. 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 streptavidin or AG phosphatase is 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.

Preferably / the sensitive surface (s) of the biochip on one side, in particular the upper surface of the biochip are ordered at ^¬ while the contacts, where the electrical Sig- can be read nal, on a duly opposite ^¬ the side of the biochip disposed are. Preferably, 5 to 100, advertising tested in particular 8 to 12 sensitive areas and entspre ^¬ accordingly arranged many contacts to a biochip so that simultaneously a plurality of different analytes the can. The biochip is preferably embedded in the titer plate, for example, the biochip is embedded in a plastic ring, which in turn is inserted into a suitable recess of the titer plate, so that sample or reaction material can be brought into contact with the preferably upwardly facing sensitive surfaces of the biochip , Preferably, the biochip on the side of the sensitive surfaces - for example, upwards - provided with a seal which prevents impurities can penetrate the catcher molecules. The seal is for example a cover of an elastomeric and / or thermoplastic material.

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

In addition to each DNA chip, a plurality of depressions or holes are respectively arranged in which, for example, an amplification of the target can be carried out, for example by means of a PCR reaction, or other reagents can be 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. In a ^¬ standardize all necessary steps for the detection can be carried leads. 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 which are each separated by a wall from each ^¬ .

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 may be a pipette tip from one well to the next well or to the biochip various ^¬ ben 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 can get from a one ^¬ ness in an adjacent unit during pipetting individual droplets. 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 through the recess and remains during the transfer of

Sample, for example, from one well to the biochip within the friedeten to ^¬ space of the unit. The first side opposite ^¬ opposite side, preferably the bottom, of the titer plate provides a housing for the biochip prepared and provided with training bulges which in each case one recess entspre ^¬ chen. Particularly preferably, the recesses are formed respectively in the form ei ^¬ nes elongated hole and have for each unit, for example, in about a "E" shape. The Ausneh ^¬ rules are fit reasonable course of movement of a pipetting robot. The oblong hole extends over all four recesses and at least one position above the biochip, which forms a single connecting line between the recesses and the biochip, which has the advantage that walls are arranged not only between the individual units, but also in part between the individual recesses within a unit Once a pipette tip comes into contact with sample material, the tip can be left inside this unit, unlike known titer plates, the smallest droplets can not get into other analysis units and contaminate them, since a used pipette tip does not penetrate other wells, The recess is according to a development designed 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 such in the titer plate eingebet ^¬ tet that each corner of the biochip is located at the corresponding Posi tion ^¬ a recess on the microtiter plate in a standard format. 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. You can, for example, moved into a two-component injection molding used in the preparation of the titer plate in this integ ^¬ riert. Alternatively, the seal or cover is clamped by a plastic ring, in which the biochip is embedded. Particularly preferably, the elastomeric ^{seal is} slightly spaced from the sensitive surface of the biochip, whereby a biochip chamber is formed therebetween, which is 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.

At least one and preferably two storage positions for a pipette tip are provided above the elastomeric seal of the biochip, namely at one or preferably two of the eg 4 positions, each corresponding to a depression on a standard titer plate and so ^¬ with by a Pipetting robot can be reached. A pipette that has come into contact with the sample material can be deposited at this point and thus remain within the unit, even if fresh pipette tips are possibly present in the unit

Unit to be used. In addition, further 4 Pi ^¬ pettenspitzen remain at the positions of the 4 wells.

Further, an overflow reservoir is preferably provided for each unit to receive excess fluid. In ei ^¬ ner preferred embodiment, the overflow reservoir is connected directly to the biochip chamber or can be connected. 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. Further, the overflow reservoir is preferably provided with egg ^¬ ner overflow wall that prevents back flow of liquid in the biochip chamber. Could ^¬ te the overflow reservoir also be filled, for example through an inlet at one of the standard positions Alternatively, corresponding to the wells of a titer plate stardard.

According to an alternative embodiment, the invention comprises a titer plate for the detection of an analyte, with at least one biochip, wherein the at least one biochip for

Detection of an analyte is designed and is from a wall ^¬ give, with a seal, preferably an elastomeric seal on which at least one biochip (14) is applied, which defines a biochip chamber together with the biochip and with an overflow reservoir directly with the biochip chamber is connected 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 invention is indicated on a reading device for a titer plate which is adapted to ensure a white ^¬ tere improve the automatic handling of samples. The reading device provides at least one electrical contact or read-out area for the biochip. Preferably, the read-out ^{surface can be} heated, since the readout result of the ^{biochip can} usually only be read out after suitable heat treatment.

The reading device also includes trays for the recesses, preferably as heated seats (thermoblocks) konzi ^¬ are piert. When placing the well plate on the Leseeinrich ^¬ tung a recess in a pan is taken in each case, preferably the recess is concluded as closely as possible environmentally from the pan 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. 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 because the pipette ^tip remains in the ^¬ sem 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 adjacent units can not be contaminated with amplified material as the pipette tip is transported away across them. Used pipette tips are particularly preferably stored on the storage positions on an ex ^¬ 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, another liquid, in particular a label liquid, is transferred with a pipette tip onto the biochip or into the biochip chamber. The label is, for example, 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 was transferred has not yet been emptied, as it is used for any additional samples again, and since ^¬ her parked on a second filing or park positions.

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

For amplification of the analyte, a thermocyclization reaction is preferably used. According to a further development of the method according to the invention, a polymerase chain reaction - in short PCR -, an allele-specific primer expression - ASPE for short - 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 samples, an analysis device of a combination of Leseein ^¬ direction and titer plate is specified. This combination is adapted to standard pipetting robots.

Another aspect of the present invention relates to the use of the titer plate according to 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 now be ^¬ ferred embodiments of the invention are described.

In the drawings show:

Fig. 1 is a perspective view of an embodiment of a titer plate according to the invention;

FIG. 2 shows a perspective view of an embodiment of a reading device for a titer plate according to the invention; FIG.

. Fig. 3 is a perspective view of a combination of a titer plate according to Figure 1 with a Leseein ^¬ direction in FIG. 3;

4 is a plan view of a section of the Obersei ^¬ te the titer plate according to the invention;

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

6 shows a flowchart of a method according to the invention rens.

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

1 shows a titer plate 10 for detecting an analytical th 22 from the bottom of the titer plate 10 includes a plurality of recesses arranged in rows 12 which are visible as bulges 12 'from the lower side ^¬ 22nd The recesses 12 are spaced from each other such and being rich ^¬ tet that a pipetting robot with a pipette tip can contribute to the analyte sample to be tested into the recesses 12 required reagents, solvents, and / or a.

In addition to two rows of wells 12 a number of biochips 14 is arranged. The biochip 14 are also positioned in Be ^¬ train to the recesses 12 such that the Ro ^¬ boter the pipette tip can get moved automatically program-ert. These biochips 14 are designed, for example, to detect a DNA by means of 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 biochip 14 is a seal made of a thermoplastic elastomer is preferably arranged, the 'to the upper surface of the titer plate (un ^¬ th in Fig. 1 lying) towards seals the receptacle 14. 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 having a biochip 14 are combined to form a unit 21, which is surrounded in FIG. 1 by a dot-dash 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 exemplary embodiment of a reading device 26 for a titer plate 10. On the reading device 26, on the one hand, a number of troughs 13 are arranged, which serve as heatable receptacles for the depressions 12 of the titer plate. In particular, the bulges 12 'shown in FIG. 1 fit into the tubs 13. The four tubs 13 of a unit 21 are each combined to form a thermoblock 11 and can preferably be heated independently of one another and in particular 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 good amount to be detected.

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 Ausleseflä ^¬ che 15, for example, 8 electrical contacts are arranged.

The contact surfaces 15 and trays 13 are surrounded by a border 17 which can align and hold a titer plate 10 shown in FIG. 1 on the reading device 26. The base area and height of the reading device 26 is preferably compatible with the known STARlet® system, ie the dimensions are, for example, 150 × 110 × 110 mm ³ and fit into a 7- track carrier. The reading device 26 preferably comprises 12 independent thermo blocks or thermal cycler 11, with which an arbitrarily programmable PCR can be performed, and 12 independent temperature-controlled electrical Bio ^¬ chip read-out blocks. The integrated electronics have preferably ^¬, a communication interface, turkontrolleinheiten 24 independent temperature and 12 independent digital interfaces for the biochip reading on.

In Fig. 3, the titer plate 10 of FIG. 1 is shown from the top, as it is placed on a reading device 26 of FIG. 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 penetrate as far as the bottom of the titer plate 10, ie they are "milled in" as a block, and 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 fenestration of the individual units of wells 12 and the biochip 14. These are located at the bottom 19 of the recess 18 and can be reached by a pipetting robot The pipetting robot moves a pipette tip 40 to transfer an amplified sample mixture from the well 12 in the biochip 14 along the slot 18.

FIG. 4 schematically shows a plan view of a single unit 21 comprising a biochip and four depressions of a titer plate 10 from the upper side 20. 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 covered, for example, by a seal 30, in which a filling port is inserted at 32, by means of which a sample can be brought into contact with the biochip 14 by means of a pipette tip. 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 receptacle may be, for example, a small depression in the seal 30 in which a pipette tip can be received. 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 can still be filled with eg substrate or label enzyme 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.

For handling a sample in the wells 12 is the

Recess 18 designed in the form of a multi-membered elongated hole, which makes, inter alia, the four wells "accessible from above." That means that pipette tips 40 can be introduced and moved by means of a pipetting robot via the slot 18. The inserted pipette tip reached be made without departing from the recess 18 to Müs ^¬ sen both all the wells 12 and the biochip 14. a target containing the amplified sample can be taken up by the pipette from one of the pits 12 transferred via the injection port 32 in the biochip fourteenth 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 wick element 31 is arranged, in this case a zy ^¬ linderförmiges piece of absorbent material, such as 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.

FIG. 5 shows a combination of a titer plate 10 with a reading device 26 in longitudinal section, again showing only one unit 21. 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. Thus, there is 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 introduced into the biochip chamber 36, it passes through the outlet 34 in the seal 30 into an overflow reservoir 24. In order for liquid once in the overflow reservoir 24 not to run back, an intermediate wall 37 is provided as an overflow reservoir. provided as shown in Figure 5. 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 arranged 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 shown in Fig. 4, which can hold about 1 to 2 ml 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 in a titer plate 10 in ^¬ as described above. The method comprises the following steps: introduction of a sample into one of the depressions 12 of the titer plate 10 and introduction of reagents into the depressions 12

Performing an amplification reaction with the reagent-added sample, wherein a PCR reaction, an ASPE and / or an ARMS reaction come into consideration. 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 shown in FIG. 6 as a flow chart. 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 can then be disposed of in the usual way by a pipetting robot.

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 Pi ^¬ pettenspitze introduced by pipetting robot 40 into the recess 18 and preferably thereafter disposed of in the usual way except ^¬ half the recess. Optionally, the sample may be overcoated with mineral oil prior to the amplification reaction. After merging the reagents and the sample becomes

Temperature program 106 performed to run off a PCR reaction ^¬ run. The reaction mixture then contains the analyte in bulk copied form for better detection.

Then, the pipetting robot picks up a new, clean Pi ^¬ pettenspitze 40, step 108. With this pipette tip is transferred in step 110, the reaction mixture having the amplifizier- th sample from a first of the recesses 12 in the biochip fourteenth The reaction mixture is introduced into the Biochipkam ^¬ mer 36th Possibly too much captured running ^¬ reaction mixture through the outlet 34 into the overflow reservoir 24. The used pipette tip is after the complete unloading emptied at the first recess 12, step 114, ie it remains within the recess 18th

Then, the pipetting robot initially picks up another new, clean pipette tip 40, step 116. With this pipette tip is made in step 118 a label enzyme from a befindlichem outside the recess 18 reservoir on ^¬ and transferred to the biochip fourteenth The pipette tip 40 is then not yet empty, and is therefore stored at the first storage position 33, step 120, that is, it remains ^¬ ver within the recess 18th

Thereafter, the liquid-handling robot picks up a further new clean pipette tip 40, step 122. With this pipette tip is received in step 124, a substrate made of a befindlichem outside the recess 18 from ^¬ reservoir and transferred into the biochip fourteenth The pipette tip 40 is then not empty and is therefore deposited at the second storage position 35, step 126, ie it remains within the recess 18th

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 used again and there ^¬ parked again at the storage positions 33, 35.

The invention thus allows, used pipette tips do not have other recesses 18 in which are ^¬ tersucht other samples un having to dispose of time and thus reduces the risk of contamination.

Claims

claims

Characterized in that the at least one biochip (14) is designed for the detection of an analyte and in that at least one biochip (14). is surrounded by a wall (16) together with several of the recesses (12).

2. titer plate (10) according to claim 1, dadurchge ^¬ indicates that the biochip (14) adjacent to the recesses (12) is arranged and that at least one off ^¬ recess (18) by a wall (16) is limited and a plurality of the Wells (12) and a biochip (14) at the bottom (19) are arranged.

3. titer plate (10) according to claim 2, dadurchge ^¬ indicates that it has a plurality of recesses (18).

4. titer plate (10) according to any one of claims 2 to 4, since - by in that the at least ^¬ a recess (18) to a first side (20) of the titre plate (10) is open towards, the opposing

Side (22) with a receptacle (14 ^λ ) for the biochip (14) and with a bulge (12 ^λ ) of the recess (12) is provided.

5. titer plate (10) according to any one of claims 2 to 5, since - by in that the at ^¬ least one recess (18) as an elongated hole (24) is formed which extends over all the recesses (12) and the biochip (14 ) which are surrounded by the wall (16).

6. titer plate (10) according to any one of the preceding claims, characterized in that an elastomeric seal (30) on the at least one biochip (14) is applied, which has a Einfüllport (32).

7. Titer plate (10) according to one of the preceding claims, characterized in that at least one storage position for a pipette tip (40) is provided on the elastomeric seal (30) of the biochip (14).

8. The titer plate (10) according to one of the preceding claims, characterized in that a biochip chamber (36) is in fluid connection with a sensitive surface of the biochip (14).

9. A titer plate (10) according to claim 8, wherein the biochip chamber (36) is connectable to an overflow reservoir (24) to receive a liquid from the biochip chamber (36).

10. The titer plate (10) according to claim 9, characterized in that in the overflow reservoir (24) a wick element (31) for sucking liquid from the biochip chamber (36) is arranged.

11. A titer plate (10) according to any one of claims 1 to 10, wherein a bore (38) penetrating the titer plate (10) is present within a depression (18).

12. The titer plate (10) according to claim 1, wherein the biochip (14) is designed for the detection of DNA by means of hybridization.

13. reading device (26) for a titer plate (10), in particular for a titer plate (10) according to any one of the preceding claims, characterized in that it comprises at least one electrical contact surface (15) for a biochip (14) and troughs (13) for receiving of depressions (12) of the titer plate (10).

14. Reading device (26) according to claim 13, characterized in that the troughs (13) for depressions (12) of the titer plate (10) are selectively heatable.

15. reading device (26) according to claim 13 or 14, characterized in that meh ^¬ rere troughs (13) for recesses (12) of the titer plate (10) are combined to form a thermoblock (11), wherein the Le ^¬ seeinrichtung (26) includes a plurality of heating blocks (11) operable un ^¬ interdependent.

16. An analyzer, comprising at least one titer plate (10) according to any one of claims 1 to 12 and a reading device according to any one of claims 13 to 15.

17. A 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 12 with the following steps:

Transferring (110) a substance, in particular a liquid, from one of the depressions (12) onto the biochip (14), characterized in that the substance is transferred by means of a pipetting robot with a pipette tip (40) onto the biochip (14) and the pipette tip (40) remains within the recess (18).

18. The method according to claim 17 for the detection of an analyte by means of a biochip (14), which is in a titer plate (10) according to any one of claims 1 to 12 is integrated, with ^¬ follow the steps of: a) introducing (102) a sample into a the recesses (12); b) introducing reagents into at least one of the wells (12); z

c) performing an amplification reaction with the reagent-added sample; d) transferring (110) the resulting reaction mixture onto the biochip (14), and e) reading out the biochip (14) which, by hybridization with the analyte, alters at least one electrically detectable property; characterized in that the reaction mixture and / or the sample by means of a pipetting robot with a pipette tip (40) is introduced and is transferred onto the biochip (14) and that the pipettes ^¬ tip (40) within the recess (18) remains ,

19. The method of claim 18, wherein after step d), a liquid, in particular a label liquid, is transferred to the biochip (14) with a pipette tip (40).

20. The method of claim 17, wherein a plurality of pipette tips remain within the recess.

21. The method according to any one of claims 17 to 20, characterized in that a Pipett- tenspitze (40) after pipetting, in particular after transferring (110) of the resulting reaction mixture of one of the recesses (12) on the biochip (14), in the respective recess (12) is turned off, was transferred from the liquid.

22. The method according to any one of claims 17 to 21, characterized in that a pipette tip (40) after pipetting, in particular after the transfer of label liquid on the biochip (14), on a storage position (33, 35) within the recess ( 18) is turned off, which seals the pipette tip (40) down so that no liquid from the pipette tip (40) can escape.

23. The method according to any one of claims 17 to 22, since ^¬ by in that the Schrit ^¬ te a) are performed to e) with a further sample.

24. Use of a titer plate (10) according to one of the preceding claims 1 to 12 for a quantitative determination, an IDT, a SNP (single nucleotide polymorphism) or an expression analysis.