DE102015011970B4 - Dispenser and dispensing method - Google Patents

Abstract

The invention relates to a dispensing device for dispensing microdrops (1) with a microdispenser (2) having an outlet nozzle (10) for dispensing the liquid microdroplets (1), wherein the liquid microdroplets (1) each comprise a sample in an aqueous solution (8 ), and with a sample holder (3, 4) for receiving the microdispensers (1) dispensed by the microdispenser (2), in particular as a receiving cup (3) in a microtiter plate (4). The invention provides that the sample receiver (3, 4) contains liquid oil (14), so that the microdispenser (2) injects the microdroplets (1) into the liquid oil (14). Furthermore, the invention comprises a corresponding dispensing method.

Description

The invention relates to a dispensing device or a dispensing method for dispensing microdrops.

For DNA sequencing (DNA: deoxyribonucleic acid), a special form of PCR (PCR: polymerase chain reaction) is often used, namely the so-called emulsion PCR. This emulsion is generated from PCR mix, THEN and oil, to which usually sequencing beads are added. Ideally, you will get exactly one DNA strand per reaction volume and exactly one sequencing bead. In the following PCR, the DNA strand is amplified and immobilized on the sequencing bead. Some next-generation sequencing platforms now use these DNA-loaded beads to place them in small wells of a sequencing chip. A maximum of one bead is deposited per well and thus one DNA sequence per well is determined. A sequencing chip can contain millions of wells and enable the sequencing of complete genomes.

From the prior art (eg WO 2006/128662 A1 Microdispensers are known which can dispense microdrops of a liquid sample onto planar substrates (eg a well of a microtiter plate). The dispensed microdrops then land, for example, on the bottom of the individual wells of the microtiter plate and can then be analyzed. The amplification of DNA sequences from such probes or samples applied to substrates is known from the literature (MITTERER, G., SCHMIDT, WM .: "Microarray-based detection of bacteria by on-chip PCR", Methods Mol Biol. 2006; 345: 37-51). Also known are various types of PCR on solid phases with printed or randomly arranged DNA sequences ( WO 98/44151 A1 . WO 98/44152 A1 ). The use of dispensers according to the invention in the sample preparation of next-generation sequencing in the form of emulsion PCR, however, has not been described hitherto.

Further, NAKANO, Michihiko [et al.]: "Single-molecule PCR using water-in-oil emulsion", Journal of Biotechnology, Vol. 102, 2003, no. 2, pp 117-124, an emulsification method is known in which drops are injected into liquid oil. In this case, however, the emulsification is carried out by subsequent stirring by means of a magnetic stirrer.

Finally, it should be noted on the general technical background of the invention US 2014/0 106 467 A1 ,

The invention is therefore based on the object of correspondingly improving the known dispensing devices or dispensing methods, in particular for providing samples for a polymerase chain reaction, in particular for methods which use a digital PCR or an emulsion PCR.

This object is achieved by an inventive dispensing device or a dispensing method according to the invention in accordance with the subsidiary claims.

The dispensing device according to the invention partially coincides with the known dispensing devices. Thus, the dispensing device according to the invention has a microdispenser with an outlet nozzle in order to dispense liquid microdroplets, where the liquid microdroplets each contain a sample in an aqueous solution.

In addition, the dispensing device according to the invention has a sample receptacle to receive the microdispills dispensed by the microdispenser. In a preferred embodiment of the invention, this sample receptacle is a single receptacle in the form of a commercially available disposable receptacle for laboratory use ("tube") or a receptacle ("well") in a microtiter plate. However, the invention is not limited to individual receiving cups with respect to the sample recording, but in principle can be realized with other constructive designs of sample recordings.

In the case of the known dispensing devices described at the outset, the sample receptacle (eg "tube" or "well") is empty before the dispensing of the microdroplets, so that the dispensed microdrops usually land on the bottom of the sample receptacle. The invention now differs from this prior art in that the sample receptacle is filled with a liquid oil prior to dispensing the individual microdroplets, so that the microdispenser injects the microdroplets into the liquid oil.

In the liquid oil, the dispensed microdrops then form an emulsion, the aqueous solution of the microdrops forming an internal phase of the emulsion while the liquid oil forms an external phase of the emulsion. The emulsified microdroplets can then subsequently be subjected to a polymerase chain reaction, as will be described below.

In a variant of the invention, the microdispenser is submerged in the sample receptacle for dispensing into the liquid oil so that the microdroplets from the outlet nozzle enter directly into the liquid oil. In this variant of the invention, the outlet nozzle of the microdispenser is thus at Dispensing within the liquid oil. In this variant of the invention, therefore, the microdroplets do not pass through an air gap between leaving the exit nozzle of the microdispenser and striking the sample receiver. Immersion of the microdispenser in the liquid oil in the sample holder can be done, for example, by a manipulator by machine, which is known per se from the prior art.

In another variant of the invention, on the other hand, the microdispenser is not immersed into the liquid oil in the sample receptacle for dispensing the microdroplets, but placed at a specific distance above the liquid oil, so that the dispensed microdroplets first emerge from the exit nozzle of the microdispenser Overcome air gap and only then immerse in the liquid oil.

However, the two aforementioned variants of the invention are both suitable for producing an emulsion of the microdroplets consisting of the sample solution in the liquid oil.

In the preferred embodiment of the invention, the outlet nozzle of the microdispenser has a hydrophobic coating. Here, the outlet nozzle is preferably at least partially made of glass, wherein the hydrophobic coating is applied to the glass.

In the context of the invention, the abovementioned coating can be applied to the inner surface of the outlet nozzle (that is to say the walls of the fluid channel), to the end face of the outlet nozzle and / or to the outer surface of the outlet nozzle in order to improve the dispensing behavior.

There is the possibility that the coating on the inner surface of the outlet nozzle on the one hand from a different material than the coating on the end face or the outer surface of the outlet nozzle on the other.

There are various possibilities with regard to the material of the coating. For example, this may be a material having alkyl groups, in particular having a chain length of C3, C8 or C12. In addition, come as a material for the coating and fluorine-substituted groups in question. Other possible materials for coating are (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilanes, trimethoxy (propyl) silanes, trimethoxy (octyl) silanes and trimethoxy (octyl) silanes.

It should also be mentioned with regard to the coating that the layer thickness is preferably in the range from 10 nm to 100 nm. In the context of the invention, however, it is fundamentally also possible for the layer thickness to be in the range from 5 nm to 500 nm or in the range from 1 nm to 1 μm.

It should also be mentioned that the individual microdroplets each have a volume that is in the range from 1 μl to 1 μl.

The ejection of the individual microdroplets from the microdispenser is preferably carried out by an electrically operated actuator, which is known per se from the prior art. For example, this electrically operated actuator may be a piezoactuator, as already described in the patent application mentioned above WO 2006/128662 A1 is described, so that the content of this patent application is fully attributable to the present description.

In this case, the electrically operated actuator (eg piezoactuator) is actuated by a control device with an electrical control signal in order to bring about the ejection of the individual microdroplets from the microdispenser. This electrical control signal preferably comprises pulses with a pulse voltage in the range of 10 V to 500 V, 20 V to 250 V, 50 V to 150 V or 80 V to 110 V. The individual pulses of the control signal are preferably at a pulse frequency in the range of 1 Hz to 10 kHz, 5 Hz to 5 kHz, 50 Hz to 2 kHz, 200 Hz to 1 kHz or 400 Hz to 700 Hz. In addition, to the control signal to mention that the individual pulses preferably have a pulse duration, which is in the range of 1 microseconds to 500 microseconds, 2 microseconds to 250 microseconds, 5 microseconds to 100 microseconds or 10 microseconds to 50 microseconds.

With regard to the filling of the microdispenser with the aqueous sample solution, various possibilities exist within the scope of the invention. One way of filling the microdispenser is that the microdispenser is connected via a refill line with a liquid source, so that the filling takes place via the refill line. Another possibility of filling the microdispenser is that the microdispenser is designed to aspirate the aqueous solution with the sample through the outlet nozzle, in order then to be able to dispense the aspirated sample solution in the form of microdroplets.

In the first variant described above for filling the microdispenser via a refill line, the refill line can be filled with an air volume in order to achieve material separation between the fluid upstream of the air volume on the one hand and the fluid downstream downstream of the air volume. This volume of air may, for example, comprise a volume in the range from 1 μl to 100 μl, 2 μl to 50 μl, 3 μl to 20 μl or 4 μl to 10 μl.

With regard to the filling of the microdispenser via the refill line is also to be mentioned that this filling is preferably carried out with an overpressure, which has proven to be advantageous. This overpressure during filling preferably corresponds to a water column of 1 cm to 30 cm, 2 cm to 20 cm, 3 cm to 10 cm or 5 cm to 7 cm.

It has already been briefly mentioned at the outset that the microdroplets of the sample liquid emulsified in the liquid oil can subsequently be subjected to a polymerase chain reaction. For this purpose, the sample receiver with the emulsified microdroplets is preferably introduced into a thermocycler, which then carries out the polymerase chain reaction. Such thermal cyclers are known per se from the prior art and therefore need not be described in detail.

The introduction of the sample holder (eg "tube" or microtiter plate) into the thermocycler can, for example, be carried out mechanically and automatically by a second manipulator. Alternatively, however, it is also possible that the sample holder (eg microtiter plate) is manually introduced into the thermal cycler.

The device described here offers the possibility for the method to measure the drops delivered during the drop delivery. In this case, parameters such as drop size and drop speed can be determined easily and exactly as known from the prior art, for. B. with optical or acoustic methods. By measuring these quantities, the parameters can be adjusted by changing the applied voltage or pulse shape or pulse width to desired drop volumes in the range of less than 100 picoliters, typically 10 picoliters or even 1 picolitre. This is particularly advantageous when using different formulations for the emulsion PCR, which entail slight viscosity changes (eg denaturing reagents or emulsifiers or separating agents), because this adjustment possibility can be used constantly in an identical volume range.

It should also be mentioned in this connection that the individual microdroplets preferably contain a master mix for the polymerase chain reaction, which is also known per se from the prior art. This master mix preferably contains Taq DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), magnesium chloride (MgCl ₂ ) and reaction buffer in concentrations that allow DNA templates to be amplified by a polymerase chain reaction. In addition, the individual microdroplets preferably contain DNA, in particular a single DNA molecule. In addition, the individual microdroplets may also contain a polymerase chain reaction primer, the above-mentioned materials being known per se in the polymerase chain reaction and therefore need not be described further.

It should also be mentioned that the invention not only claims protection for the dispenser according to the invention, which is generally described above. Rather, the invention also claims protection for a corresponding Dispensierverfahren, as is apparent from the foregoing description.

Other advantageous developments of the invention are characterized in the subclaims or are explained in more detail below together with the description of the preferred embodiments of the invention with reference to FIGS. Show it:

1 a schematic representation of a Dispensiergeräts invention,

2 a schematic longitudinal sectional view through an outlet nozzle of a microdispenser according to the invention,

3 a modification of 2 .

4 another variation of 2 such as

5 a flowchart to illustrate the Dispensierverfahrens invention.

1 shows a schematic representation of an embodiment of a dispensing device according to the invention for dispensing microdrops 1 , The single microdrops 1 be here by a microdispenser 2 in a sample holder 3 dispensed, which is a receiving well ("Well") of a microtiter plate 4 is.

The microdispenser 2 This can be done by a manipulator 5 in several spatial degrees of freedom relative to the microtiter plate 4 be moved, as indicated in the drawing by the double arrows. In this way, the manipulator 5 the microdispenser 2 position one after the other so that the microdrops 1 into the individual wells of the microtiter plate 4 be delivered.

The filling of the microdispenser 2 takes place via a refill line 6 from a liquid source 7 , which is shown here only schematically. The fluid source 7 contains however an aqueous sample solution 8th in which the samples to be analyzed are dissolved. The sample solution 8th will then over the refill line 6 in the microdispenser 2 The filling is carried out with a slight overpressure, which corresponds to a water column with a height Δh ≈ 6 cm.

In the refill line 6 this can be an air volume 9 Separate different sample liquids to avoid contamination.

The microdispenser 2 indicates the ejection of the individual microdroplets 1 an outlet nozzle 10 on, which consists of glass and will be described in detail.

In addition, the microdispenser points 2 a piezoelectric actuator 11 on, over a control line 12 from a controller 13 is driven with suitable electrical pulses to the individual microdroplets 1 eject.

The individual electrical pulses for controlling the piezoelectric actuator 11 For example, a voltage of 105 V, a pulse duration of 50 μs, a pulse frequency of 500 Hz, resulting in a drop volume of the individual microdroplets 1 of 239 pl leads.

Alternatively, there is also the possibility that the individual pulses for controlling the piezoelectric actuator have a voltage of 89 V, a pulse duration of 23 μs, a pulse frequency of 500 Hz, resulting in a drop volume of the individual microdrops of 75 pl.

It should also be mentioned that the sample intake 3 (For example, a "tube" or a receiving cup of the microtiter plate 4 ) with a liquid oil 14 is filled. In a Dispensiervorgang the microdispenser 2 from the manipulator 5 so far into the sample holder 3 introduced that the outlet nozzle 10 of the microdispenser 2 in the oil 14 dips. The single microdrops 1 So then be from the outlet nozzle 10 directly into the oil 14 injected. In such a Dispensiervorgang then forms in the sample holder 3 an emulsion, the microdrops 1 with the sample solution 8th form the inner phase while the oil 14 forms the outer phase of the emulsion.

Now if all the receptacles ("Wells") of the microtiter plate 4 filled in this way and thus contain an emulsion, the microtiter plate 4 in a thermocycler 15 introduced, the thermocycler 15 then performs a polymerase chain reaction, which is known per se from the prior art.

2 shows an enlarged schematic representation of an embodiment of the outlet nozzle 10 of the microdispenser 2 ,

It can be seen that on the inner wall, the outer surface and the end face hydrophobic coatings 16 . 17 . 18 are applied, which may for example consist of trimethoxy (propyl) silanes.

3 shows a modification of 2 ,

A special feature of this modification is that in this case no coating is applied to the inner wall of the outlet nozzle.

4 shows another modification of the embodiment according to 2 ,

A special feature here is that the coating 16 the inner surface of the outlet nozzle 10 made of a different material than the coatings 17 . 18 on the end face or on the outer surface of the outlet nozzle 10 ,

Finally shows 5 a flow chart for illustrating the dispensing method according to the invention.

In a first step S1, first the microdispenser 2 with the aqueous sample solution 8th filled.

In a further step S2 then the individual receiving wells ("wells") of the microtiter plate 4 with the oil 14 attacks.

In a further step S3 then becomes the microdispenser 2 in the oil 14 in the individual wells of the microtiter plate 4 immersed.

In the immersed state of the microdispenser 2 gives the microdispenser 2 then in a step S4 the microdroplets 1 in the oil 14 resulting in an emulsion in the individual wells of the microtiter plate 4 forms.

In a further step S5, the microtiter plate filled in this way becomes 4 then in the thermocycler 15 introduced.

Finally, the thermal cycler leads 15 then in a step S6, a polymerase chain reaction on the emulsified microdrops 1 through to the sample solution 6 to investigate.

The invention is not limited to the preferred embodiments described above. Rather, a variety of variants and modifications is possible, which also make use of the inventive idea and therefore fall within the scope. In particular, the invention also claims protection for the subject matter and the features of the subclaims independently of the claims in each case referred to and in particular without the features of the main claim.

LIST OF REFERENCE NUMBERS

1

microdroplets

2

microdispenser

3

specimen collection

4

microtiter plate

5

manipulator

6

refill

7

liquid source

8th

sample solution

9

air volume

10

exhaust nozzle

11

piezo actuator

12

control line

13

control device

14

oil

15

Thermocycler for PCR

16

Coating the inner surface of the outlet nozzle

17

Coating of the end face of the outlet nozzle

18

Coating the outer surface of the outlet nozzle

.delta.h

Water column according to overpressure

Claims (15)

Dispensing device for dispensing microdrops ( 1 ), with a) a microdispenser ( 2 ) with an outlet nozzle ( 10 ) for dispensing the liquid microdroplets ( 1 ), wherein the liquid microdrops ( 1 ) in each case one sample in an aqueous solution ( 8th ), and b) a sample receiver ( 3 . 4 ) for receiving the microdispenser ( 2 ) dispensed microdrops ( 1 ), in particular as a receiving cup ( 3 ) in a microtiter plate ( 4 ), c) wherein the sample receiver ( 3 . 4 ) liquid oil ( 14 ), so that the microdispenser ( 2 ) the microdrops ( 1 ) into the liquid oil ( 14 ), characterized in that d) that the individual microdrops ( 1 ) each have a volume in the range 1 pl-1 nl. Dispenser according to claim 1, characterized by a first manipulator ( 5 ) for movably positioning the microdispenser ( 2 ), in particular for immersing the microdispenser ( 2 ) into the liquid oil ( 14 ) in the sample receiver ( 3 . 4 ), so the microdrops ( 1 ) from the outlet nozzle ( 10 ) directly into the liquid oil ( 14 ) enter. Dispenser according to one of the preceding claims, characterized in that the dispensed microdrops ( 1 ) in the liquid oil ( 14 ) form an emulsion, the aqueous solution ( 8th ) of the microdrops ( 1 ) forms an inner phase of the emulsion while the liquid oil ( 14 ) forms an outer phase of the emulsion. Dispensiergerät according to any one of the preceding claims, characterized in that a) that the outlet nozzle ( 10 ) of the microdispenser ( 2 ) a hydrophobic coating ( 16 . 17 . 18 ), and / or b) that the outlet nozzle ( 10 ) consists at least partially of glass. Dispensiergerät according to claim 4, characterized in that a) that the coating ( 16 ) on the inner surface of the outlet nozzle ( 10 ), and / or b) that the coating ( 17 ) on the end face of the outlet nozzle ( 10 ), and / or c) that the coating ( 18 ) on the outer surface of the outlet nozzle ( 10 ) is applied. Dispenser according to claim 5, characterized in that a) that the coating ( 16 ) on the inner surface of the outlet nozzle ( 10 ) consists of a different material than the coating ( 17 ) on the face and the coating ( 18 ) on the outer surface of the outlet nozzle ( 10 ), and / or b) that the coating ( 16 . 17 . 18 ) consists at least in part of one of the following materials: b1) a material having alkyl groups, in particular having a chain length C3, C8 or C12, b2) fluorine-substituted groups, b3) (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilanes, b4 ) Trimethoxy (propyl) silanes, b5) trimethoxy (octyl) silanes, b6) trimethoxy (octyl) silanes, and / or c) that the coating ( 16 . 17 . 18 ) has a layer thickness of at least 1 nm, 5 nm or 10 nm and / or at most 1 .mu.m, 500 nm, 200 nm or 100 nm. Dispenser according to one of the preceding claims, characterized by a) an electrically operated actuator ( 11 ), in particular a piezoelectric actuator ( 11 ), to eject the microdrops ( 1 ) from the microdispenser ( 2 ), and b) a control device ( 13 ) for driving the actuator ( 11 ) with an electrical control signal. Dispensiergerät according to claim 7, characterized in that a) that the control signal pulses having a pulse voltage in the range 10 V-500 V, 20 V-250 V, 50 V-150 V or 80 V-110 V, and / or b) that the control signal pulses with a pulse frequency in the range 1 Hz-10 kHz, 5 Hz-5 kHz, 50 Hz 2 kHz, 200 Hz-1 kHz, 400 Hz-700 Hz, and / or c) that the control signal Pulse with a pulse duration in the range 1 μs-500 μs, 2 μs-250 μs, 5 μs-100 μs or 10 μs Has -5 μs. Dispenser according to one of the preceding claims, characterized in that a) that the microdispenser ( 2 ) for filling with the aqueous solution ( 8th ) via a refill line ( 6 ) with a liquid source ( 7 ) or b) that the microdispenser ( 2 ) is adapted to the aqueous solution ( 8th ) with the sample through the outlet nozzle ( 10 ). Dispensiergerät according to claim 9, characterized in that a) that the refill line ( 6 ) between successive filling operations with an air volume ( 9 ) is filled to a mixture of liquids in the microdispenser ( 2 on the one hand and in the sample source on the other hand, b) that the air volume ( 9 ) is preferably in the range of 1 μl-100 μl, 2 μl-50 μl, 3 μl-20 μl and 4 μl-10 μl. Dispensiergerät according to claim 9 or 10, characterized in that a) that the liquid source ( 7 ) the microdispenser ( 2 ) via the refill line ( 6 ) filled with an overpressure, b) that the overpressure during the filling preferably a liquid column (Δh) of 1 cm-30 cm, 2 cm-20 cm, 3 cm-10 cm or 5 cm-7 cm corresponds. Dispenser according to one of the preceding claims, characterized by a) a thermal cycler ( 15 ), the individual microdrops ( 1 ) in the emulsified state in the oil ( 14 ) undergoes a polymerase chain reaction, and / or b) a second manipulator for transferring the filled sample receptacle ( 3 . 4 ) with those in the oil ( 14 ) emulsified microdrops ( 1 ) in the thermal cycler ( 15 ) for carrying out a polymerase chain reaction. Dispensiergerät according to claim 12, characterized in that the individual microdrops ( 1 ) each contain the following: a) a master mix, in particular with Taq DNA polymerase, deoxyribonucleoside triphosphates, magnesium chloride and reaction buffers in concentrations which enable a duplication of DNA templates by a polymerase chain reaction, and / or b) DNA, in particular a single DNA molecule, and / or c) a polymerase chain reaction primer. Dispensing method for dispensing microdrops ( 1 ), in particular with a dispenser according to one of the preceding claims, comprising the following steps: a) filling a microdispenser ( 2 ) with a sample in an aqueous solution ( 8th ), b) providing a sample receptacle ( 3 . 4 ) for receiving microdrops ( 1 ) from the microdispenser ( 2 ), c) dispensing the microdrops ( 1 ) from the microdispenser ( 2 ) in the sample receiver ( 3 . 4 ), d) filling the sample receiver ( 3 . 4 ) with liquid oil ( 14 ) before dispensing the microdrops ( 1 ), so that the dispensed microdrops ( 1 ) into the liquid oil ( 14 ), characterized in that e) that the individual microdrops ( 1 ) each have a volume in the range 1 pl-1 nl. Dispensierverfahren according to claim 14, characterized by the following steps: a) immersion of the microdispenser ( 2 ) into the liquid oil ( 14 ) in the sample receiver ( 3 . 4 ) before starting dispensing so that the microdrops ( 1 ) from the microdispenser ( 2 ) directly into the liquid oil ( 14 ), and / or b) control of an electrically operated actuator ( 11 ) of the microdispenser ( 2 ) with an electrical control signal to the microdrops ( 1 ) through an outlet nozzle ( 10 ), and / or c) inserting the filled sample receptacle ( 3 . 4 ) with those in the oil ( 14 ) emulsified microdrops ( 1 ) in a thermocycler ( 15 ), and / or d) carrying out a polymerase chain reaction on the microdroplets ( 1 ) contained in the liquid oil ( 14 ) in the sample receiver ( 3 . 4 ) are emulsified.

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