WO2001023092A1

WO2001023092A1 - Device and method for absorbing and releasing minute amounts of liquid

Info

Publication number: WO2001023092A1
Application number: PCT/DE2000/003447
Authority: WO
Inventors: Hans Lindner; Hannes Dobler; Claus Kuhn; Hansjörg GRILL
Original assignee: Giesing, Michael; Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 1999-09-28
Filing date: 2000-09-28
Publication date: 2001-04-05
Also published as: DE19946525A1

Abstract

The invention relates to a device and to a method for applying minute amounts of liquid on a very confined area. The invention provides a method and a device that allows application of up to 2500 drops per mm<2> on a planar substrate by dosing minute amounts of liquid by means of a drawn glass capillary in combination with a high-precision positioning device. The inventive method and device are specifically useful for producing biochips.

Description

Device and method for taking up and dispensing the smallest amounts of liquid

The invention relates to a device and method for receiving and dispensing the smallest amounts of liquid, the device comprising a dosing head, a handling device and a device for receiving a substrate. The dosing head has at least one capillary for receiving and dispensing the smallest amounts of liquid and the relative position of the dosing head and the device for receiving a substrate can be changed by the handling device.

For miniaturized analysis, devices, so-called "chips" or "biochips" are required, which consist of a mostly planar substrate or carrier on which various chemical and / or biological substances (e.g. haptens, proteins, oligo) are present in the highest possible density - And / or polymeric nucleic acids, sugar, lipids, cells, other chemical and biological substances) are immobilized. These individual chemical and / or biological substances can be arranged on the substrate in the form of an array or a matrix. The different substances are either synthesized in situ on the substrate or must be applied to the substrate in a correspondingly high density in a dissolved form, the dissolved molecules then being immobilized either covalently or adsorptively on the surface of the substrate. The higher the number of different substances that are applied per unit area of the substrate, the more analysis parameters can be examined within one measurement process.

In the following, substrate is used to denote the surface of a material with or without chemical activation. The material can be, for example, glass, plastic, metal, a semiconductor material or the like. In the following, substance is understood to mean liquids in which the chemical and / or biological substances to be dispensed are dissolved or suspended. In connection with the invention, capillaries are thin bores, regardless of the type of manufacture. It can be drawn glass tubes or drilled or otherwise drilled holes or holes in a solid block.

In order to reduce the required substances and the sample quantities required for the analysis, one of the efforts is to arrange the smallest possible drops in the smallest possible space. A disadvantage of the known devices for receiving and dispensing the smallest amounts of liquid according to the prior art is that the smallest possible meterable drop volumes have a volume of approximately 1 nl (10 ^'9 1). The drop diameter is therefore approx. 100 μm. This means that a maximum of 20 drops per mm ² can be applied.

From DE 40 24 545 A1 a device for the metered supply of a biochemical analysis liquid to a target is known, which works according to the so-called bubble jet method. Such non-contacting metering devices according to the prior art have the disadvantage that, in addition to the actual metering drop, additional smaller droplets, so-called satellite drops, occur in an unforeseeable and unreproducible manner. However, these satellite drops lead to cross-contamination in applications in miniaturized analysis and thus to possible false results. In addition, the tear behavior of the drops cannot be predicted exactly in the non-contacting processes, so that inaccuracies in the trajectory and the positioning of the drops on the substrate arise. Finally, the non-contacting metering devices, which operate according to the "Inkjef process known from inkjet printers, have a large dead volume. To meter drops of liquid in the nanoliter range, at least 1 μl must be taken up.

DE-197 09 348 A1 describes an automatic multi-epitope ligand mapping system in which various reagent solutions are applied to a tissue sample. The automatic pipetting system described there is not touching, as can be seen, for example, from column 3, lines 6-9 of this document. In addition, the pipetting system only has an insufficient positioning accuracy of 100 μm for the production of biochips.

In the case of the touching metering devices, the currently achievable density of drops is limited to a few dozen drops per mm ² . With the tip-print process, the previously achievable spot density is at best 10 spots per mm ² .

EP 0 608 423 A1 describes a method for taking up highly viscous liquids, such as red blood cells. In the known method, however, the liquid is not deposited on the surface of an essentially planar substrate. EP 0 508 531 A2 describes a complex method for precise dosing of a liquid, in which an elastic dosing device is first placed on the bottom of a vessel and then lifted off again under constant pressure control, so that there is a connection between the bottom and the tip of the dosing device Forms liquid meniscus. However, the formation of the meniscus requires a liquid volume of a few μl, so that small amounts of liquid which are necessary in the area of the production of biochips cannot be metered.

All previously known devices for receiving and dispensing the smallest amounts of liquid according to the prior art have in common that the handling devices used to move the dosing heads achieve a positioning accuracy of at best 50 μm, so that the drop densities that can be achieved are limited to 400 drops per mm ² .

The object of the invention is to provide a device for taking up and dispensing the smallest amounts of liquid, in which the required amounts of the sample and / or substances to be analyzed are reduced and the number of analysis parameters is increased.

This object is achieved according to the invention by a device for receiving and dispensing the smallest amounts of liquid with a device for receiving a substrate, a dosing head which has at least one capillary for receiving and dispensing the smallest amounts of liquid, at least the dispensing, but preferably also the absorption of liquid by a The tip of the capillary takes place, a handling device by means of which the relative position of the dosing head and the device for receiving a substrate can be changed, the dispensing of liquid taking place by placing the tip of the capillary on the substrate.

This device has the advantages that the smallest droplet volumes of up to 0.1 pl can be deposited on substrates. By placing the drops directly on the substrate, they can be positioned with greater accuracy than with non-contacting methods. As a result, drop densities of up to 2500 drops per mm ^{2 have been} achieved with the device according to the invention. This results in a significant reduction in the required amounts of the sample to be analyzed and the substances. In addition, a larger number of analysis parameters can be examined with a given amount of sample. Furthermore, when the drops are dosed onto substrates, no satellite drops can occur, so that the risk of cross-contamination and the resultant incorrect test results are avoided.

Finally, the dead volume when the liquid to be dosed is taken up by the capillary can be limited to less than 1 nl, which leads to a significant reduction in the amount of substance required. This is made possible by the fact that the substrate is taken up by the tip of the capillary and is also released through this tip. This means that no dead space has to be filled with substrate. Nevertheless, the capillary of the dosing tip can take up a work reserve that is sufficient for stopping at least 100 drops.

It is also possible to fill the capillary via its end facing away from the tip, so that a continuous process control is achieved in the series production of substrates on which substances are immobilized. In series production, the increasing dead volume due to this type of filling is of less importance.

In a variant of the invention, the inside diameter of the capillary is 1 μm to 200 μm at least in the area of the tip, so that sufficiently small drops can be deposited on the substrate.

In addition to the invention, a drawn glass capillary is used, so that capillaries with the required inner diameters can be provided by melting and pulling commercially available glass capillaries, with, if appropriate, a final fire pool.

In a further addition to the invention, it is provided that the outer surface and / or the inner surface of the capillary are coated at least in the area of the tip, so that the behavior of the liquid when it is taken up or released by the capillary can be influenced. The inner and outer surface of the dosing tip can be coated differently.

In another embodiment of the invention, the capillary is coated with polyimide, so that this so-called fused silica capillary has great flexibility. Further additions of the invention provide that the capillary is coated hydrophobically or hydrophilically on the outside and / or inside, so that the behavior of the substance when immobilized on the substrate is suitably coordinated with the properties of the capillary, in particular its surface properties outside and inside, and the Surface of the substrate can be influenced.

Another embodiment of the invention provides that the pressure inside the capillary can be controlled so that the uptake and delivery of the liquid can be actively influenced.

When the invention is supplemented, a plurality of capillaries are combined to form an array or a matrix, so that the capillaries can be charged simultaneously and a plurality of drops can be deposited simultaneously. This embodiment is particularly suitable for series production if substances are to be immobilized on a large number of identically designed substrates.

In a variant of the invention it is provided that the relative position of the dosing head and the device for receiving a substrate can be changed by the handling device in three coordinate directions, so that the dosing head for receiving the liquid from a container and dispensing the liquid on the substrate freely Space is movable.

In addition to the invention, the coordinate directions correspond to the axes of a Cartesian coordinate system, so that straight guides and corresponding drives can be used.

In another embodiment of the invention, the relative angle of rotation of the dosing head and the device for receiving a substrate can be changed by the handling device, so that the substrate and the working direction of the dosing head can be aligned with one another.

In a variant of the invention, the relative position of the dosing head and the device for receiving a substrate can be changed by one or more ball screw drives with a connected hybrid stepping motor, so that large adjustment paths and high adjustment speeds with high positioning accuracy can be achieved. In a further embodiment of the invention, the relative position of the dosing head and the device for receiving a substrate can be changed by one or more piezo-controlled nanomotors, so that space-saving and highly precise positioning is possible.

In another variant, means are available for determining the relative position of the dosing head and the device for receiving a substrate, in particular scales of glass or metal interacting with an optical scanning device, so that the actual position of the capillary or the substrate is very accurate can be recorded in all movement axes.

In a further addition to the invention, a control device for controlling the absorption and dispensing of liquid and the relative position of the dosing head and the device for receiving a substrate are provided, so that the device according to the invention can be operated automatically.

The above-mentioned object is also achieved by a method for taking up and dispensing the smallest amounts of liquid, which can be carried out in particular by means of the inventive device described above. In the method according to the invention, a tip of a capillary is filled with a liquid to be dispensed on a substrate. The tip of the capillary is then positioned precisely above a predetermined point on the substrate at which a spot of the liquid is to be dispensed. Then one contacts the surface of the substrate with the tip of the capillary by lowering the capillary and / or lifting the substrate. The above Advantages, in particular the application of the smallest amounts of liquid in the smallest of spaces, can thus also be realized when carrying out the method.

The capillary can be filled through its tip, for example by immersing the tip in a container filled with the liquid to be dispensed. Capillary forces draw a small amount of liquid into the tip of the capillary. This process can be supported or controlled if, at the same time as the tip is immersed, the pressure inside the capillary is reduced so that liquid is actively taken up from the container. However, it is also possible to fill the tip of the capillary via its end facing away from the tip. For this purpose, liquid from a reservoir is advantageously filled into the capillary under pressure. The pressure is regulated so that the tip of the capillary is just filled without liquid escaping.

In a further addition to the method according to the invention, the pressure inside the capillary is increased when the capillary touches the substrate clamped, for example, in the device for receiving a substrate, so that the amount of liquid which is deposited on the substrate can be controlled.

The invention is explained in more detail below with reference to an embodiment shown in the accompanying drawings.

In the drawings:

Figure 1 is a schematic representation of an embodiment of the device according to the invention for receiving and dispensing the smallest amounts of liquid. and FIG. 2 shows a variant of the holder for the glass capillary of the device of FIG. 1.

1 shows a schematic representation of an embodiment of the device according to the invention for receiving and dispensing the smallest amounts of liquid, which is referred to as a whole as a dosing station 10. The dosing station 10 comprises a dosing head 11, a device 12 for receiving an essentially planar substrate 13, for example a biochip, and a handling device 14, 15, 16, with the aid of which the relative position of the dosing head 11 and the substrate 13 can be changed.

The handling device can be designed in a wide variety of ways. The relative position of the dosing head 11 and the substrate 13 or of the dosing head 11 and the receiving device 12 can preferably be changed at least in all spatial directions. For this purpose, for example, the receiving device 12 can be designed as an xy displacement table, which enables movement in the plane of the planar substrate 13 via a drive device 16. With a stationary dosing head 11, each point on the surface of the planar substrate 13 can be controlled in this way. In addition, a lifting device (not shown in FIG. 1) can be provided, which allows movement of the receiving device 12 and the substrate 13 perpendicular to the plane of the substrate (according to FIG the nomenclature introduced above in the z direction). In the example in FIG. 1, however, the relative movement of dosing head 11 and substrate 13 in the z direction is ensured by a linear drive 14 arranged on dosing head 11, which moves the dosing head perpendicular to the plane of substrate 13. In addition, a rotary adjuster 15 is provided, with the aid of which the angle between dosing head 11 and substrate 13 can be adjusted. According to another variant (not shown), the receiving device 12 and the substrate 13 can be arranged in a stationary manner, while the dosing head can be displaced in the x, y and z directions by suitable positioning devices.

To control the handling device 14, 15, 16, a control device 17 is provided, which usually comprises a computer and suitable measurement and control software. In Fig. 1, the corresponding control lines 18, 19 are shown in dashed lines.

The dosing head 11 has a drawn glass capillary 20 which ends in a thin tip 21 with an inner diameter of a few micrometers. Located in the tip 21 is a fluid 22 to be applied to the surface of the substrate 13, which can be sucked in, for example, by immersing the glass capillary 20 in a fluid reservoir (not shown). For this purpose, the glass capillary 20 is connected via a line 23 to a pressure regulator 24, which makes it possible to generate an overpressure or a vacuum in the glass capillary 20. The pressure regulator 24 is also controlled by the control unit 17 via a control line 25. According to a particularly simple variant, the pressure regulator 24 can be designed as a microdosing syringe, the piston of which is actuated by a coupled stepper motor. The pressure regulator 24 can also have a reservoir (not shown) for the fluid 22, wherein the fluid can be pumped into the glass capillary 20 via the line 23.

The relative movement of dosing head 11 and substrate 13 is specified by control unit 17 (for example when stepper motors are used by specifying the number of steps). For the absolute determination of the position of the tip 21 of the glass capillary 20 above the planar substrate 13, an optical scanning device 26 is provided in the example shown, which includes microscope optics 27 which images the surface of the substrate 13 on a CCD module 28. The signals are transmitted to the control unit 17 and evaluated via a data line 29. For this purpose, high-precision scales made of glass or metal can be used, which are arranged on the substrate 13 or, if a transparent substrate is used, below the substrate in the receiving device 12. It can also be applied to the substrate (for example by embossing, printing or etching), which are recognized and evaluated by a suitable image processing system in the control unit 17. However, the scales can also be arranged on the axes of the positioning devices 14, 16 of the handling device and can be evaluated using separate scanning devices.

In the device according to the invention, fluid is released onto the surface of the planar substrate 13 by bringing the tip 21 of the glass capillary 22 into contact with the surface of the substrate 13. It must be ensured that neither the substrate 13 or, if applicable, coatings applied in a previous dosing process, nor the glass capillary 20 itself are damaged. Due to the usually inevitable tolerances of the substrate surface or the drawn glass capillary, the vertical movement of the glass capillary and substrate is preferably monitored automatically. The tips of drawn glass capillaries are relatively elastic, so that when the glass capillary approaches the surface of the substrate 13 vertically, a desired position lying slightly below the actual substrate surface in the z direction does not necessarily have to result in damage to the capillary. In such a case, however, the elastic tip 21 laterally evades the pressure exerted by the actuating device 14 after contacting the surface, so that the desired positioning accuracy is no longer guaranteed. Therefore, as shown in FIG. 1, the glass capillary 20 is preferably arranged obliquely to the surface of the planar substrate 13. A slightly too low lowering of the glass capillary then leads to a slight bending of the tip 21, but the position of the tip in the plane of the substrate 13 practically does not change and the lateral position of the fluid delivery can be precisely controlled.

1 shows a possibility of how the contact of the tip 21 on the surface of the substrate 13 can be detected. In the example shown, the substrate 13 is provided with metallized tracks 30 which surround the individual spots 31 to be coated. The metallized tracks 30 are grounded via a line 32. In a transition piece 33 made of glass between the glass capillary 20 and the line 23, a fine wire 34 is melted, which leads inside the glass capillary 20 into the area of the fluid 22. Many fluids 22 used in biotechnology have a certain electrical conductivity. A voltage is now applied to line 34 via an ammeter 35. As soon as the tip 21 of the capillary 20 with the surface of the substrate 13 in Is contacted, the fluid spreads 22 by adhesion and by a given _¬ appropriate with the aid of the pressure controller 24 generated in the capillary 20 overpressure in the spot 31 and comes to the edges of the grounded metallization 30 is in contact. A current flow is registered via the measuring device 35 and a signal is sent via the line 36 to the control device 17, which stops the linear drive 14 in the z direction via the line 19.

2 shows a variant for controlling the placement of the glass capillary 20 on the surface of the substrate 13. As in the example in FIG. 1, the glass capillary 20 is fastened in a holder 37. Since the tip 21 of the glass capillary 20 bends slightly when it comes into contact with the substrate 13, a pressure sensor 38 can be arranged in the holder 37, which registers the pressure received by the holder 37 when the capillary is bent and sends a corresponding signal to the line 39 Control unit 17 (not shown in FIG. 2) sends. However, other control concepts are also conceivable. For example, a confocal optic can be provided in the scanning device 26, by means of which the surface of the substrate 13 is imaged with a depth of focus of only a few micrometers. The positioning of the tip 21 of the glass capillary can then be regulated in the control unit 17 with the aid of an image processing system. Accordingly, an optical detection system can also be oriented parallel to the surface of the substrate. The contact of the tip of the capillary with the surface of the substrate is then again determined by an image processing system. An elastic mounting of the glass capillary is also conceivable. In such a case, for example, contact with the surface of the substrate can also be registered by a pressure sensor.

In two further exemplary embodiments, not shown in the figures, the dosing head is arranged to be displaceable in the direction of three Cartesian coordinate axes. In addition, an absolute position measuring system with scales along each axis and optical reading devices cooperating with them is provided. The markings on the measuring rails are optically recorded by the reading device and evaluated by pulse counter cards. Measuring rails with a resolution of 100 nm are used to ensure a positioning accuracy of 1 μm.

In the first embodiment, the dosing head is driven in each axis by a ball screw drive of the precision class with pitch 6 via a hybrid stepper motor with 400 steps per revolution. The 400 full steps are software-technical in each 128 sub-steps divided. This achieves a resolution of 51,200 increments per revolution. This corresponds to a resolution of approx. 120 nm per increment.

The adjustment speeds are up to 120 mm per second, the work area has dimensions of approximately 300 mm x 300 mm x 80 mm. A drawn, hydrophobized capillary made of borosilicate glass with an internal diameter of 5 μm is used as the metering capillary.

The liquid is dosed by adhesion and pressure pulse. Dosing volumes between 0.1 pl and 1000 pl, with a drop diameter between 10 μm and 100 μm, can be sold.

In the second exemplary embodiment, piezo-based nanomotors from Kleindiek are used. In so-called fine mode, you achieve a resolution of 1 nm per 0.1 volt, corresponding to an increment. The control takes place over a range of +/- 15 volts in

Fine operation, which corresponds to a travel distance of 300 nm. For larger travel distances, you have to switch to coarse mode. This enables travel distances in the cm range to be achieved. However, individual steps are lost due to the system. However, this disadvantage is offset by the absolute displacement measurement.

Two piezo-driven nanomotors are connected in parallel for the x and y axes. The positioning accuracy is +/- 100 nm, the adjustment speed is up to 1 mm per second. It is also possible to connect several nanomotors in series to increase the adjustment range.

The work area has dimensions of approximately 10 mm x 10 mm x 10 mm. The liquid is deposited passively through a fused silica capillary with an internal diameter of 5 μm to 150 μm. This results in droplet diameters on the substrate of 8 μm to 300 μm. A metal scale is used to measure the distance.

Claims

claims

1. Device for receiving and dispensing the smallest amounts of liquid with a device (12) for receiving a substrate (13) a dosing head (11) which has at least one capillary (20) for receiving and dispensing the smallest amounts of liquid (22), at least the dispensing of liquid through a tip (21) of the capillary (20), a handling device (14, 15, 16), by means of which the relative position of the dosing head (11) and the device (12) for receiving a substrate can be changed, the Liquid (22) is dispensed by placing the tip (21) of the capillary (20) on the substrate (13).

2. Device according to claim 1, characterized in that the inner diameter of the capillary (20) is at least in the region of the tip (21) 1 μm to 200 μm.

3. Apparatus according to claim 1 or 2, characterized in that the capillary (20) is a drawn glass capillary.

4. Device according to one of the preceding claims, characterized in that the outer surface and / or the inner surface of the capillary (20) is coated at least in the region of the tip (21).

5. Apparatus according to claim 1 or 2, characterized in that the capillary (20) is coated with polyimide.

6. Apparatus according to claim 4 or 5, characterized in that the capillary (20) is coated hydrophobically on the outside and / or inside.

7. Device according to one of claims 4 to 6, characterized in that the capillary (20) is coated hydrophilically on the inside and / or outside.

8. Device according to one of the preceding claims, characterized in that the pressure inside the capillary (20) is controllable.

9. Device according to one of the preceding claims, characterized in that several capillaries (20) are combined to form an array or a matrix.

10. Device according to one of the preceding claims, characterized in that the relative position of the dosing head (11) and the device (12) for receiving a substrate by the handling device (14, 15, 16) can be changed in three coordinate directions.

11. Device according to one of the preceding claims, characterized in that the relative angle of rotation of the dosing head (11) and the device (12) for receiving a substrate can be changed by the handling device (14, 15, 16).

12. The apparatus according to claim 10, characterized in that the coordinate directions correspond to the axes of a Cartesian coordinate system.

13. Device according to one of the preceding claims, characterized in that the relative position of the dosing head (1) and the device (12) for receiving a substrate can be changed by one or more ball screw drives with connected hybrid stepper motor.

14. Device according to one of the preceding claims, characterized in that the relative position of the dosing head (11) and the device (12) for receiving a substrate can be changed by one or more piezo-controlled nanomotors.

15. Device according to one of the preceding claims, characterized in that means for determining the relative position of the dosing head and the device for receiving a substrate are present.

16. The apparatus according to claim 15, characterized in that the means for determining the relative position of the dosing head and the device for receiving a substrate are standards made of glass or metal, which cooperate with an optical scanning device (26).

17. Device according to one of the preceding claims, characterized in that a control device (17) for controlling the absorption and dispensing of liquid and the relative position of the dosing head (11) and the device (12) for receiving a substrate is present.

18. Method for taking up and dispensing the smallest amounts of liquid, in particular using a device according to one of claims 1 to 17, characterized by the following method steps:

Filling a tip of a capillary with a liquid to be dispensed on a substrate,

Positioning the capillary relative to the substrate, and

Contact a surface of the substrate with the tip of the capillary.

19. The method according to claim 18, characterized in that the tip of the capillary is filled by immersing the tip in a container filled with the liquid.

20. The method according to claim 19, characterized in that simultaneously with the immersion of the tip of the capillary in the liquid-filled container, the pressure inside the capillary is reduced.

21. The method according to claim 18, characterized in that the tip of the capillary is filled over its end facing away from the tip.

22. The method according to any one of claims 18 to 21, characterized in that the pressure is increased inside the capillary, if the tip of the capillary the sub ^¬ strat contacted.