EP3573758A1

EP3573758A1 - Pipetting device for pulsed pipetting with a pipetting piston movement regulated on the basis of a determination of the working gas pressure

Info

Publication number: EP3573758A1
Application number: EP18701033.5A
Authority: EP
Inventors: Hanspeter Romer; Reto Ettinger; Fridolin GYSEL; Jürg RAST; Jonas HILTI
Original assignee: Hamilton Bonaduz AG
Current assignee: Hamilton Bonaduz AG
Priority date: 2017-01-24
Filing date: 2018-01-19
Publication date: 2019-12-04
Anticipated expiration: 2038-01-19
Also published as: DE102017201114A1; WO2018138005A1; EP3573758B1

Abstract

The invention relates to pipetting device (10) for the pulsed pipetting of dosing liquids in small dose volumes of less than 2 μΙ, wherein the pipetting device (10) comprises: a pipetting channel (11) at least partially filled with working gas (34), a pipetting tip (26), which is accessible through a pipetting opening (30), such that the volume of dosing liquid drawn into the receiving chamber through the pipetting opening (30) can be varied by means of a change in the pressure of the working gas in the receiving chamber, a pipetting piston (14) for changing the pressure of the working gas (34) and accommodated in the pipetting channel so as to be capable of movement along the pipetting channel (11), a motion drive (20) to drive the pipetting piston (14) for movement along the pipetting channel (11), a control device (24) for controlling the motion drive (20), and a pressure sensor (38) for determining the pressure of the working gas, wherein the control device (24) is designed to control the motion drive (20) in order to generate a pressure pulse in the pipetting channel (11) with a pulse duration of no longer than 40 ms on the basis of the pressure signal output by the pressure sensor (38) in such a manner that the pressure of the working gas (34) during the pulse follows a predefined working gas target pressure pulse characteristic.

Description

A pipetting device for pulse-like pipetting with a pipetting piston movement regulated on the basis of a detection of the working gas pressure

description

The present invention relates to a pipetting device for pulsed pipetting of dosing liquids in small dosing volumes of less than 2 μΙ mediating a pressure-variable working gas.

By means of the control device, the movement drive can be controlled in a targeted manner in order to move the pipetting piston in the desired manner by corresponding activation of the movement drive and, in turn, to change the pressure of the working gas in the desired manner.

A pulse-like dispensation in the sense of the present invention is known from US 2001/0016358 A1. There, however, no overpressure pulse is imparted by a working gas, but a physical impact is directly emitted by the piezoactuator onto the meniscus of the dosing liquid provided in the pipetting device and thereby closer to the opposite longitudinal end of the provided dosing liquid column from the pipetting opening Meniscus thrown off a drop.

The disadvantages of this known method are obvious: Due to the contact of the piezo actuator with the dosing liquid, an increased risk of contamination is given. A pulse-pipetting pipetting device is described in German Patent Application 10 2015 214 566.2, unpublished at the time of application of the present invention.

It is an object of the present invention to provide a pipetting device which is able to pipette as hygienic as possible and in small doses as highly accurate as possible. This object is achieved according to the present invention by a pipetting device of the type mentioned, which comprises:

an at least partially filled with working gas pipetting channel,

a pipetting tip which provides a receiving space filled at least partially with working gas, which communicates with the pipetting pressure channel and which is accessible through a Pipettieroffnung so that by changing the pressure of the working gas in the receiving space through the Pipettieroffnung through the amount of in the receiving space absorbed dosing liquid is changeable,

a pipetting piston movably received in the pipetting channel along the pipetting channel for changing the pressure of the working gas,

a movement drive to drive the pipetting piston for movement along the pipetting channel,

- A control device for controlling the movement drive, and

a pressure sensor for detecting the pressure of the working gas and for outputting a pressure signal indicative of the pressure of the working gas to the control device,

wherein the control device is adapted to the movement drive, for generating a - with respect to a reference pressure in the pipetting at which no fluid passes through the Pipettieroffnung - pressure pulse in the working gas pipetting channel with a pulse duration of not more than 40 ms such based on that of the pressure sensor to trigger the output pressure signal that the pressure of the working gas during the pulse follows a predetermined working gas target Druckimpuls- course.

Due to the design for pipetting in the so-called "Air Displacement" method, in which the pressure of a pipetting piston and dosing liquid existing working gas is changed to dispensing dosing liquid through the Pipettieroffnung or aspirate, it comes with the exception of the inevitable Pipette tip to no contact between a component or component portion of the pipetting device and the dosing liquid. Surprisingly, it has also been found that the control of the piston movement for generating a pressure pulse in the working gas in accordance with the working gas pressure is possible even for very short pressure pulses of less than 40 ms. By regulating the piston movement by the control device based on the pressure in the working gas, it is possible to compensate for leakage effects in the pipetting channel which can occur even in the short duration of the pressure pulse. This enables highly accurate pipetting of even the smallest dosing liquid quantities in the sub-microliter range. A significant contribution to the working gas pressure-based control of the piston movement goes back to the development of high-speed, high-speed pressure sensors. A pressure sensor available at the time of registration, which is sufficiently powerful, is the pressure sensor "TBPDANS001 PGUCV" from Honeywell.

Here, in the present application with a "pressure pulse" or a "pulse-like" pipetting a Dosierflüssigkeitsaufnahme (aspiration) in the pipette tip or a Dosierflüssigkeitsabgabe (Dispensation) from this effecting pressure pulse in the working gas with a total pulse duration of not more than 40 ms , The duration of the pressure pulse is the time interval between leaving a reference pressure, wherein in the pipetting tip any existing dosing liquid is kept substantially motionless and in which no fluid, so neither gas nor liquid, flows through the pipetting, and the renewed permanent return to this.

In order to pipette a small dosing volume of dosing liquid in a single contiguous droplet in a defined manner, in particular to detach it during dispensing, the pressure pulse may preferably comprise an overpressure portion and a negative pressure portion. In the pulse-like dispensing system, which is significantly more important in practice, the overpressure component precedes the negative pressure component in the pressure pulse in time. Since in reality due to the pressure-based control of the piston moving pressure oscillations are to be expected in the working gas, the working gas target pressure pulse course is to use in case of doubt for determining the pressure pulse duration. The latter is exempt from over- and under-swinging of the working gas pressure due to regulation and the quality of the regulation.

Both the overpressure component and the negative pressure component are within the specified time window of a maximum of 40 ms. "Pulse-like" dispensing refers to a dispensing deviating from the conventional pipetting operation. In the pulse-like dispensation, a pressure shock from the working gas is exerted on the side facing away from a metering orifice of the dosing liquid quantity received in a pipetting device by the overpressure pulse of the working gas. This pressure blow propagates through the incompressible Dosierflüssigkeitsmenge to one of the pipetting more near meniscus of the recorded Dosierflüssigkeitsmenge and there leads to the centrifuging of a Dosierflüssigkeitstropfens. The centrifuging of the Dosierflüssig keitstropfens can by a whip-like piston movement, in which a strongly accelerated piston movement in the dispensing a once again strongly accelerated piston movement in the direction of aspiration following immediately in time, defined triggered. The resulting negative pressure component in the pressure pulse course of the working gas is smaller in magnitude and no longer in time, preferably even shorter than the previous excess pressure component. In this way, very small amounts of liquid of less than 2 μΙ or preferably less than 600 nl can be metered with high repeat accuracy and even aliquoted, so dispensed repeatedly from a larger volume in the receiving space of the pipetting tip Dosierflüssigkeitsmenge, without dispensing between individual dispensing liquid to aspirate.

In contrast to pulse-like dispensing, in conventional dispensing, the dosing liquid taken in by the pipetting device is increased by increasing the flow rate Pushed out in the working gas until either a drop from the pipetting orifice dissolves gravity driven or it is metered through the pipetting dosing liquid to a wetted by the dosing liquid substrate or in an existing liquid from which or from which the pipetting opening is lifted after delivery of the predetermined dosage. In conventional quasi-synchronous dispensing usually dosing liquid moves only so long in the dispensing direction through the pipetting, as long as the pipetting in a dispensation sense (increasing the pressure in the working gas) is moved.

Thus, while in conventional dispensing, the pressure change in the working gas, and thus the movement of a pipetting, synchronously or quasi-synchronously with the dispensing of dosing liquid through a pipetting of the pipetting, the pulse-like dispensing on which the present invention is based is asynchronous, ie to a pulse-like, sudden generation of an overpressure pulse in the working gas, the drop of the dosing liquid is usually thrown off only from the recorded Dosierflüssigkeitsmenge, while the pressure pulse is at least subsiding or even subsided. The dispensing of Dosierflüssigkeitstropfens thus does not occur synchronously with a movement of the pipetting. The dosing liquid drops moves in the dispensing direction during pulse-like dispensing, while the pipetting piston moves in the sense of aspiration (reducing the pressure in the working gas) or already stops again. In the pulse-like dispensation, the quantities of liquid to be dispensed are usually delivered as drops, with an acceleration from the quantity of metering liquid received in the pipetting device, which is added to the gravitational force in the direction of gravity when dispensed. This means that the metering liquid droplets detached from the added metering liquid quantity during pulsed dispensing by the pipetting device move faster from the pipetting device when dispensed in the direction of gravity action than merely in free fall. Large amounts of dosing liquid, ie amounts of more than 2 μΙ, are usually pipetted in a synchronous operating mode of the pipetting device, in which the dosing liquid in the pipette tip, more precisely its meniscus pointing to the piston, follows the movement of the dosing-side end surface of the piston synchronously. That is, when the piston is moved in a dispensing direction as a pipetting direction, the meniscus moves in the dispensing direction together with the meter-side end surface of the piston, and when the piston is moved in an aspiration direction as a pipetting direction, moved together with the dosing-side end face of the piston in Aspirationsrichtung. There may be a slight time lag between the movement of the dosing-side end face of the piston and the meniscus of the dosing liquid, since the working gas present between the piston and the dosing liquid initially to overcome friction, capillary, adhesion, cohesion or / and surface effects only by the piston movement must be brought to a pressure level on which the desired pipetting process can proceed. When aspirating, this is a negative pressure relative to the ambient pressure, so that dosing liquid, driven by the differential pressure between the pressure of the working gas and the ambient pressure, flows into the pipette tip from a dosing liquid reservoir in which the pipette opening of the pipetting tip is immersed. When dispensing, this is an overpressure relative to the ambient pressure, so that dosing liquid taken up in the pipetting tip, driven by the differential pressure between the pressure of the working gas and the ambient pressure, exits through the pipetting opening of the pipetting tip. The compressible working gas thus acts like a gas spring. Due to the small but present time offset between the piston movement and the movement of the meniscus of the dosing liquid in the pipette tip, the conventional pipetting of dosing liquid is referred to below as a quasi-synchronous mode of operation.

In conventional dispensing with quasi-synchronous movement of piston and dosing liquid, the demolition of the dosing liquid to be dispensed is effected by the pipetting tip by utilizing inertial forces. The piston is moved for a predetermined time in the dispensing direction and, if a demolition of out of the pipette tip out addition dosing is desired as possible stopped abruptly. The mass inertia of the already displaced metering liquid, which is still in the dispensing movement due to the past piston movement, then leads to constriction of the metering liquid at the pipeline opening and finally to its demolition. The relationship between piston movement and dosing liquid displaced by means of the working gas is usually determined empirically for different liquid classes and is stored in a data memory of the pipetting device. In this quasi-synchronous operating mode, the volume swept by the metering-side piston surface during the movement of the piston in the pipetting direction (general pipetting volume or, depending on the direction of movement of the piston, aspiration volume or dispensing volume) generally exceeds the actual pipetted volume of the dosing liquid by no more than 5 %. The ratio of pipetting volume to actually pipetted metering liquid volume is therefore usually not greater than 1.05.

Due to the inertia-induced liquid separation at the pipetting opening, dosing liquid sometimes adversely adheres to the outside of the pipetting tip in the region of the pipetting opening. In order to avoid that this adhering liquid quantity drips completely or partially uncontrolled, the piston is moved a small distance in the direction of aspiration after the liquid is torn off in order to suck the externally adhering dosing liquid back into the pipetting tip through the pipetting opening.

This dispensing of dosing using inertial forces no longer works depending on the particular dosing liquid for individual dosing volumes of less than 3 to 5 μΙ, since then due to the low mass of the achievable inertial forces other forces, especially due to surface tension, no longer sufficiently safe can overcome in order to ensure a safe, repeatable detachment of such small Dosierflüssigkeitsmengen can.

To be distinguished from the pipetting devices described here are the so-called "dispensers" or "dispensers", which as a rule are dosing liquids dispensing only, but not aspirating. Dispensers receive the dispensing liquid to be dispensed usually via feed channels from a supply, which is in flow communication with a variable by the piston metering chamber of the dispenser.

Further distinguishable from the aforementioned pipetting devices are pipetting devices in which the dosing-side end surface of the piston is in direct contact with the dosing liquid to be pipetted. There is then no working gas between the piston and the dosing liquid.

Because of the direct coupling of movement of piston and dosing liquid in such working gas-free pipetting called their type of pipetting in the art with the English term "positive displacement". Although the omission of compressible in the working gas increases the theoretically achievable pipetting accuracy, in practice it leads to difficulties elsewhere. On the one hand, gas inclusion in the pipetting volume during aspiration can not be excluded completely reliably, so that gas or air bubbles can also occur in the aspirated dosing liquid during positive displacement pipetting, which adversely affects the achievable pipetting accuracy. On the other hand, the pipetting accuracy achievable in positive displacement pipetting is extremely low if the dosing liquid tends to foam. In addition, because of the wetting of the pipetting by the dosing liquid then when the dosing liquid to be pipetted is to be changed, not only a pipette tip, but with this also the pipetting are changed, which means a considerable borrowing assembly costs and consequently significant costs.

In contrast, the pipetting type of generic pipetting devices with a working gas between the piston and the dosing liquid is referred to in the art as "Air Displacement", although the working gas does not necessarily have to be air, but also an inert gas or a quasi-inert gas, such as nitrogen, can be. In this type of pipetting, the pipetting piston is permanently and completely separated from the dosing liquid by a gas column, in particular by an air column. The present pipetting device according to the invention should also be distinguished from those which use a column of a system liquid as a piston. Such system fluids pose a certain degree of contamination risk, since it can sometimes not be ruled out that system fluid, that is to say a part of a liquid piston, passes into the dosing liquid to be pipetted. The piston of the pipetting device of the present invention is at least partially, preferably completely formed as a solid to avoid a risk of contamination. In the case of only a partial formation as a solid body, at least the metering-side end face of the piston facing the metering liquid is designed as a solid body in order to prevent a transfer of liquid to liquid.

In the pulse-like dispensing of small Dosierflüssigkeitsdosen depending on the selected dosing, for example, depending on their viscosity, density and / or surface tension and further depending on the parameters of the pressure pulse and possibly the subsequent negative pressure undesirable side effects occur. For example, instead of merely a single desired dosing drop at the dispensing-near meniscus dispensing the nebulization of dosing liquid or a dispensing of dosing liquid by a dosing droplets accompanied by unwanted satellite drops, which is associated with an undesirable reduction of the achievable dosing quantity accuracy. Dispensing within the meaning of the present application is therefore understood to mean the dispensing of the dosing liquid in a single drop without the need for spraying and nebulization.

In principle, the pipetting device can have a permanently installed pipetting channel with a pipetting tip with pipetting opening formed on the end of the pipetting channel. However, this is less advantageous for hygienic reasons. Preferably, the pipetting device is designed to detachably connect replaceable pipetting tips to the pipetting channel. Accordingly, it is provided according to an advantageous development of the present invention that the pipetting device has a coupling formation interspersed by the pipetting channel for the temporary coupling of a pipetting tip. If a pipette tip is coupled to the coupling formation, the pipette tip lengthens the device-specific pipetting channel and is temporarily, ie during the duration of its coupling, part of the pipetting channel of the pipetting device. The pipetting tip is preferably a so-called "disposable", ie a disposable or disposable pipetting tip, which is disposed of after a single dispensing or aliquoting.

Preferably, the pipetting device is not only designed for pulse-like dispensing, but also for conventional aspiration, so that the provision of dosing liquid in the pipetting device, in particular in a pipette tip picked up by quasi-synchronous aspiration of dosing liquid through the pipetting of the pipetting into a receiving space the pipetting device can be done.

Preferably, the pipetting device is designed both for pulse-like dispensing in asynchronous operation as well as for conventional dispensing in quasi-synchronous operation, so that with the inventive pipetting small Dosierflüssigkeitsmengen of less than 2 μΙ, approximately down to several tens of nano-, as well as large Liquid quantities of several hundred microliters are dispensed repeatable. The changeover between asynchronous and quasi-synchronous operation is very simple by means of selection or / and adjustment of appropriate working gas desired pressure profiles by the control device. The control device can moreover be designed to regulate the piston position only in pulsed pipetting operation in accordance with the pressure signal output by the pressure sensor by corresponding activation of the movement drive. In conventional pipetting operation, because of the close correlation of volume swept from the piston to dispensed or aspirated volume, the control device can actuate the motion drive position-dependently in response to a signal indicative of the position of the pipetting piston of at least one position sensor, thus regulating the piston position. With sufficiently slow piston acceleration and piston movement, the dispensation, as well as the aspiration, quasi-synchronous. The by the control device on the motion drive Values to be set for a desired piston acceleration and / or piston speed can be determined by experiments with little effort for different fluid classes. For example, the control device for realizing a quasi-synchronous pipetting operation may be designed to move the piston for pipetting a predetermined single metering volume of more than 2 μΙ at a tip speed of not more than 1000 μΙ / s. At the specified maximum speed of the piston of not more than 1000 μΙ / s, the dosing liquid follows the piston in a rectilinear motion, if necessary with a slight time offset. The pipetting volume swept by the piston essentially corresponds to the actually pipetted dosing liquid volume. Again, preferably the piston sizes mentioned below in the present application, indicated by the piston area.

With the possibility of operating the pipetting device according to the invention both in synchronous or quasi-synchronous as well as in asynchronous pipetting operation, one and the same pipetting device according to the invention can be adapted to a selectable single-dose in a Dosiervolumenbereich from 100 nl to 100 μΙ, preferably 100 nl to 1000 μΙ with a volume deviation of not more than 2% based on the predetermined single dosing volume as a nominal volume to reproducibly pipette. Thus, the pipetting device according to the invention is able to pipette 10000 times the minimum pipetting volume as the maximum pipetting volume. It should of course not be ruled out that, for example, the mentioned lower limit of 100 nl can still be reached. In any case, the functionality of the pipetting device is guaranteed for the said pipetting volume ranges.

For the reasons mentioned, it is advantageous if the pipetting device has a detachable pipetting tip, with a coupling counter-formation for releasably coupling engagement with the coupling formation and with a pipetting opening as passage for dosing liquid during an aspiration operation and during a dispensing operation. In this case, the dosing liquid is if necessary after an aspiration process, provided in the pipette tip. The aspiration process is not pulsed, but as a quasi-synchronous aspiration, ie the generation of an aspirating negative pressure in the working gas and a consequent inflow of dosing liquid through the pipetting opening into the pipetting or in the pipette tip overlap for the most part in time.

One of the great advantages of the pipetting device according to the invention as well as of the dispensing method according to the invention is that a standard pipetting tip can be used with a nominal pipetting space volume which is substantially larger than the dosing liquid dose dispensed in a single pulse-like dispensing operation. The nominal volume of the pipetting tip or nominal volume of the pipetting tip is preferably more than 80 times, more preferably more than 300 times, most preferably more than 500 times the minimum possible volume of a single liquid-like dispensed or dispensed liquid dose. As a result, aliquoting operations with numerous successive pulse-like dispensations can be realized with simultaneously very high repeatability of the dose volume without intermediate aspiration.

For example, in one experiment, a standard pipetting tip with a nominal capacity of 300 μΙ was temporarily coupled to a pipetting device. In this pipette tip 40 μΙ aspirated a dosing liquid, such as glycerol. A gas volume of 4 to 5 μΙ was provided between the pipetting-opening, discharging meniscus and the pipetting opening, which is advantageous, but not absolutely necessary. In this constellation, glycerol was aliquoted as dosing liquid with a single dosing volume of 448 ml 20 times in succession, the individual dispensed dosing volumes differing by no more than 2.96%.

The multiple release of glycerine as a comparatively high-viscosity liquid with a reproducible dosing volume of less than 450 nl from one in the Pi- The 40 μΙ reservoir provided by the petting device is highly unusual.

Constructively, the achievement of pulsed pressure changes in the working gas is thereby possible in a simple and highly accurate manner, that the pipetting piston is a magnetic piston with at least one permanent magnet and that the movement drive has electrically energizable coils. The control device can then be designed to control the supply of the coils with electrical energy. The magnetic piston is preferably a solid-state piston with preferably a plurality of solid-state permanent magnets, which are preferably sufficiently sealed at its longitudinal end in relation to the pipetting channel movably receiving the piston, for example by means of corresponding caps. The provision of a magnetic piston which can be driven by an electromagnetic field in the manner of a linear motor enables highly dynamic whip-like movement processes of the piston in the pipetting channel and thereby the generation of very short overpressure pulses which can be abruptly stopped by equally short vacuum pulses.

The above-mentioned generation of a negative pressure then comprises a displacement of the magnetic piston in a first direction, typically in a direction away from the pipetting opening.

Likewise, the generation of the excess pressure component in the pressure pulse comprises a displacement of the piston in one of the first opposite second direction.

Preferably, only the working gas and no further system fluid or dosing fluid are located between the pipetting piston and the metering liquid quantity provided in the pipetting channel. To increase the achievable dosing accuracy, the pipetting device may have, in addition to the above-mentioned pressure sensor, at least one position sensor, which detects the position of the pipetting piston and outputs a position signal indicating the detected piston position to the control valve. direction is formed. When the pipetting piston is a magnetic piston, as the at least one position sensor, preferably a plurality of Hall sensors arranged along the pipetting channel may be used. However, other position sensors can also be used.

The control device can for high-precision pulse-like change in the pressure of the working gas during a pipetting process, the supply of the coils with electrical energy in the form of a control depending on a detected current state of supply of the coils with electrical energy, depending on the pos sitionssignal the at least one position sensor and depending on the pressure signal of the pressure sensor control. By changing the supply of the coils with electrical energy for changing the piston position and, consequently, changing the pressure of the working gas taking into account at least the above three parameters, the pressure of the working gas can not only accurately but also rapidly change following a working gas target pressure pulse course become.

The pressure pulse duration of 40 ms specified at the beginning is only an upper limit. The duration can also be significantly shorter than 40 ms, about 15 ms, 10 ms, 5 ms, or even just 1 ms - depending on the desired amount to be dosed. In order to achieve the desired high dosing accuracy in the required short time, which is available for a pulse-like change in the pressure of the working gas, the control device according to a particularly advantageous embodiment of the present invention comprises a cascaded control loop structure with at least three control loops. According to an innermost control circuit of the cascaded loop structure, the control device regulates an electric voltage applied to the coils in accordance with a difference between a target current value and a detected current value of a current flowing in the coils. The control device is further adapted to the target current value of the current flowing in the coils current in a more remote control loop of the cascaded control loop structure in accordance with a difference between a desired position value and an indicated by the position signal actual position value of the To determine pipetting. The control device is further configured to determine the nominal position value of the pipetting piston in an even more external control circuit of the cascaded control loop structure in accordance with a difference between a desired pressure value and an actual pressure value of the pressure of the working gas indicated by the pressure signal.

The cascaded control of the piston movement moreover, several disturbances can be compensated quickly and safely: the utmost in accordance with a difference between the target and actual working gas pressure operating loop can unpredictable and different for different pipetting devices and operations individually different leaks in the pipetting and the pipette tip compensate, for example, leaks on a piston seal and / or on a coupling of a releasably coupled pipette tip. The mean in accordance with a difference between the target and actual position of the pipetting a coil target current value determining control loop can compensate for unpredictable and different for different pipetting devices and operations individually different frictional influences, such as friction between piston seal and Pipettierkanalzylinder.

The innermost in accordance with a difference between the setpoint and actual current value of the current flowing in the coil current determining a setpoint voltage control loop can compensate for unpredictable and different for different pipetting devices and operations individually variations of coil resistances and Spuleninduktivitäten.

For high-precision and high-speed change of the working gas pressure with particularly short pressure pulses, such as in the single-digit millisecond range, but not only there, it is also advantageous if the control device comprises a data memory, in which at least an idealized working gas desired pressure pulse course, for a pilot control the at least one idealized working gas desired pressure pulse course causing idealized Pipettkolben-desired position course and an idealized nominal coil set current profile which effects the idealized pipetting piston nominal position profile is stored.

The control device is preferably designed for precontrol of the control circuits in the cascaded control loop structure in accordance with the parameters working gas pressure, pipetting piston position and coil current. The idealized courses can be determined empirically for different liquids or liquid classes. The course is a chronological sequence of at least three parameter values. Instead of absolute parameter values, the curve may also contain differences (delta values) to those parameter values that apply to the above-mentioned reference state. As a result, the idealized curves can be meteorologically compensated.

Due to the physical effects of the pulse-like pipetting, the pipetting tip is not completely emptied during pulse-like dispensing. It remains dosing liquid even after the pulse-like dispensing process in the receiving space of the pipette tip. Preference is therefore given to a pulse-like dispensing of dosing liquid from a dosing liquid absorbed in the receiving space of the pipetting tip, whose volume is at least five times greater than the volume of dosing liquid to be dispensed in pulses.

The pipetting device is designed for pulse-like dispensing in the jet mode, in which the dispensed liquid volume travels a distance in free flight between the dispensing metering liquid quantity in the pipetting tip and a dispensing target.

In the preferred use of a pressure-changing device with pipetting piston, the whip-like movability of the piston typical for pulse-like pipetting is preferably realized by the control device being designed to operate the motion drive in such a way as to dispense a predetermined single dosing volume of less than 2 μΙ. the piston is moved in the direction of dispensing and its dispensing end surface sweeps over a dispensing volume which is not larger than the single dispensing volume by not less than 1.4 times, and that the piston is then in one of the dispensing positions. Pensationsrichtung opposite Aspirationshchtung is moved while its dosing-side end surface covers an aspiration volume, the working gas target pressure pulse course for not more than 40 ms, preferably not more than 30 ms, from the starting and / or the final pressure level deviating pressure values.

The movement of the piston can be detected from any reference point on the piston, for example on the basis of the dosing-side piston surface. The effect of the motion sequence of the piston proposed by the invention on the dosing liquid is not yet fully understood. However, an explanatory model assumes that with the pulse-like movement of the piston in the direction of pipetting, preferably the dispensing direction, the excitation or breakaway energy is transferred to the dosing liquid to be pipetted, which is necessary, more than the predetermined individual dosing volume to be pipetted to initiate their movement in the desired dispensing direction against inertial forces, surface tension, adhesion and cohesion of the dosing liquid.

With the movement of the piston in the pipetting direction, preferably dispensing direction, opposite Gegenpipettierrichtung, preferably Aspirationsrichtung in which the piston again a volume, usually another, preferably in turn larger volume than the actually pipetted single dosing, passes over, the previously excited pipetting movement, preferably dispensing movement, of the dosing liquid is again "de-energized".

It is thus transmitted a very short, sharp pressure pulse predetermined course of the piston on the working gas to the dosing. How exactly the working gas actual pressure pulse course follows the desired working gas pressure pulse course depends on the quality of the control. Good results were obtained even for short pressure pulses with the cascaded control mentioned above based on several parameters with pre-control of the parameters. Surprisingly, the volumes swept by the piston during its movement: dispensing volume and aspiration volume can be the same. The piston can therefore be in the starting position again at the end of the dispensing process. Nevertheless, a single dosing volume is pipetted.

Therefore, according to the present invention, it does not depend on a "displacement balance" of the piston. Rather, tests have shown that the actually dispensed dosing liquid volume depends on the piston-set movement integrated over time.

Under the reasonable assumption that the shape of the end surface does not change during pipetting, the volume swept by the piston, or its dosing-side end surface, is the area of the projection of the dosing-side end surface on a projection plane orthogonal to the channel path multiplied by the piston stroke. Since preferably at least the meter-side end face of the piston is formed as a solid, this assumption is realistic.

The term "dispensing direction" denotes a direction of movement of the piston which causes a discharge of dosing liquid out of the dosing liquid receiving space of the pipetting tip. By "Aspirationsrichtung" a direction of movement of the piston is referred to, which causes a suction of dosing into the dosing liquid receiving space of the pipette tip.

For the purposes of the present application, a single metering volume is always predetermined when the dispensing process takes place with the aim of dispensing a specific known metering volume. The individual dosing volume can be predetermined by manual input at the pipetting device or by data transmission to the pipetting device or by calculation from manually entered data and / or from data stored in a memory device for the pipetting device.

The dispensing volume initially swept by the dosing-side end surface of the piston can not only depend on the predetermined single dosing volume in addition depend on parameters of each dosing liquid to be pipetted and / or on the volume of the working gas between dosing-side piston surface and dosing. In principle, the greater the viscosity of the dosing liquid (measured at room temperature of 20 ° C. at an atmospheric pressure of 1013.25 hPa by means of a rotational viscometer), the greater the ratio of dispensing volume to single dosing volume. Likewise, the larger the volume of working gas, the greater the ratio of dispensing volume to single dose volume. In the case of the preferred replaceable pipetting tips, it is usually possible for a design-related working gas volume between the piston and the metering volume of 100 μΙ not to be fallen below and not to exceed 3000 μΙ. Preferably, the working gas volume is between 180 μΙ and 1000 μΙ, more preferably between 200 μΙ and 800 μΙ.

For example, the dispensing volume may not be less than 1.4 times the single-dose volume. However, it can also be significantly larger than 1, 4 times the Einzeldosiervolumens. Thus, for example, it can be five times the single metering volume if a low excitation energy is sufficient to accelerate the metering liquid for flowing through the generally narrow pipetting opening. Dosing liquids less readily excitable for movement can be excited to move with a piston movement in the dispensing direction and a dispensing volume swept thereby by the dosing-side end surface of not less than ten times the single dosing volume. Since the piston movement is preferably carried out with a high maximum volume velocity than the volume swept over by the dosing-side end surface per unit time, the repeatability of the pipetting of very small single dosing volumes of less than 2 μΙ increases with increasing dispensing volume. Therefore, the dispensing volume may preferably be not less than twenty-five times the single-dose volume. Experiments have shown that, especially for the class of aqueous liquids to be pipetted frequently, these are liquids having a viscosity in the range from 0.8 to 10 mPas, measured at room temperature of 20 ° C. at an atmospheric pressure of 1013, for the purposes of the present application. 25 hPa by means of a Rota tion viscosimeter - a dispensing volume of between ten and sixty times, preferably between ten and twenty-five times, the single dosing volume results in excellent dosing results. A dispensing volume of between ten and twenty-five times the single dosing volume provides excellent dosing results even for dosing liquids outside the above-mentioned viscosity range.

An upper limit of the dispensing volume represents a dispensing volume in which more than the single dispensing volume is moved through the pipetting opening due to the large time required for the piston to sweep the dispensing volume with its meter-side end face. Tests have shown that dispensing volumes of more than 500 times the single dosing volume do not allow meaningful dispensing of dosing volumes of less than 2 μΙ.

The amount of the maximum pressure difference to the reference pressure at which no fluid flows through the pipetting orifice during the phase of the overpressure portion is preferably less than 50,000 Pa, more preferably less than 25,000 Pa, and most preferably less than 10,000 Pa. These values apply to a variety of different liquids and liquid funds. For the particularly relevant class of aqueous liquids, as defined in this application, the amount of the maximum pressure difference to the reference pressure in the overpressure phase is preferably less than 2200 Pa and more preferably none than 1 .800 Pa. Preferably, the amount of the maximum pressure difference to the reference pressure during the phase of the overpressure portion is greater than 500 Pa, preferably greater than 600 Pa.

The amount of the maximum pressure difference from the reference pressure at which no fluid flows through the pipetting orifice during the negative pressure portion phase is preferably less than 30,000 Pa, more preferably less than 15,000 Pa, and most preferably less than 7,500 Pa. Preferably, the amount of the maximum pressure difference to the reference pressure during the phase of the negative pressure portion is greater than 200 Pa, preferably greater than 400 Pa. The occurring during a dispensing pressure pulse maximum pressure difference values with respect to the reference pressure from which the pressure pulse emanates depends on a not yet final determined plurality of parameters such. B. to be dispensed single-dose volume, and the liquid which is characterized by density, viscosity and surface tension. It is apparent, for example, that for one and the same fluid both the amount of the maximum pressure difference to the reference pressure in the overpressure phase and the amount of the maximum pressure difference to the reference pressure in the negative pressure phase decrease with increasing single metering volume. It should be made clear at this point that, in spite of the above-described large piston movement during dispensing, the pipetting device designed according to the invention moves only the predetermined single metering volume of metering liquid through its pipetting opening. There is no overdosage or overdiscentration with subsequent correction in aspiration direction. Dosing liquid is moved according to the invention during a Dispensationsvorgangs pensationsrichtung only in the desired direction. A dispensing operation in the sense of the present application is completed when the piston movement ends in the direction of aspiration. The aspiration volume swept by the piston during its movement may be equal to the volume dispensed even when aliquoting. However, then, as the number of dispensing operations in the aliquoting operation increases, the meniscus nearer to the pipetting orifice can move ever further into a dispensing liquid receiving space of the pipetting device, which can impair the accuracy of further dispensing processes.

Therefore, the aspiration volume around the single dosing volume may be smaller than the dispensing volume. Thus, it can be ensured that the pipetting nearer meniscus of recorded dosing remains despite several performed dispensing in a constant as possible place. Also, the aspiration volume can thus be much larger than the single dosing according to the above information.

However, at the beginning of the dispensing process, the piston can also initially be returned to its initial position of the piston in the direction of aspiration towards the end of the generation of the overpressure pulse, and then readjusted in the dispensing direction by the single dosing volume. The tracking movement can then take place considerably more slowly than the piston movement during the pulse-like dispensing process and no longer counts as a dispensing process itself.

The correct dispensing and aspiration volume to be swept by the piston for a dispensing process for dispensing small quantities of dosing liquid can be determined simply by tests for a given single dosing volume.

Thus, in contrast to the quasi-synchronous pipetting operation described above in connection with the prior art, an asynchronous pipetting operation is used according to the present invention in which a significant portion of the piston movement does not correlate with the movement of the dosing liquid. While in the previously described quasi-synchronous pipetting operation there is only a slight time offset between movements of piston and dosing liquid in the same direction, in the present case described asynchronous pipetting operation in one and the same time or in one and the same period mutually oppositely directed movements of the piston and Dosing liquid occur or it can only begin a movement of dosing liquid through the pipetting after the piston has completed its movement in Aspira- tion direction and has come to a standstill again. However, the pipetting device according to the invention is due to its preferred design with permanent magnetic piston in addition to the conventional quasi-synonymous trained chronological Dispensationsbetrieb and conventional quasi-synchronous aspiration.

Regardless of when in a dispensing process, the single dosing volume of dosing liquid begins to move through the pipetting opening, however, it is common to most dispensing operations that during dispensation the piston is driven to reverse the direction of movement and, as a rule, the direction of movement of the piston is actually reversed before the predetermined volume of liquid has detached from the pipetting opening.

Thus, the whip-like movability of the plunger-type pipetting piston can be effected by the moving drive comprising a linear motor and the controller and the agitation driver for pipetting a predetermined single metering volume of less than 2 μΙ are designed to tip the plunger at a tip speed of at least 5000 μΙ / s, preferably of at least 10000 μΙ / s, and of not more than 25000 μΙ / s to move.

The volumetric velocity of the piston, that is, the volume swept by the dosing-side end surface of the piston per unit time, is of more importance for pulse-like pipetting than the linear velocity of movement of the piston or a piston rod. Although a smaller stroke is sufficient for pistons with a larger piston area in order to cover the same volume for which a piston with a smaller piston area requires a larger stroke. Thus, to realize increasing volumetric velocities, it would simply be possible to move a piston with a larger piston surface along the channel path than a piston with a smaller piston surface. However, the necessary for a movement initiation of the piston Losbrech force, such as to overcome the static friction, increases significantly with the piston size, so that pistons are increasingly difficult to control with increasingly large piston area for the dispensing of individual dosing volumes of less than 2 μΙ.

The present invention preferably relates to pipetting devices whose pistons have a piston area of between 3 and 80 mm ² , that is to say which in the case of a circular piston area has a diameter of between 2 and approximately 10 mm. sen. In order to be able to arrange a plurality of pipetting channels in a row and column-shaped grid with the smallest possible screen width, the present invention particularly preferably relates to pipetting devices whose pistons have a piston area of between 3 and 20 mm ² , which in the case of a circular piston area has a diameter of between 2 and approximately 5 mm corresponds.

Preferably, the pipetting device has a plurality of pipetting channels, of which in each case a pipetting piston formed as described above is movably received along the pipetting channel axis. Furthermore, each pipetting channel can each have a coil arrangement that can be energized by the control device, which forms a linear motor for driving the pipetting piston with the magnetic pipetting piston.

While dispensing at too high maximum piston speeds of, for example, more than 25,000 μΙ / s, there is still fluid movement out of a dosing liquid receiving space, however, the individual dosing volume is then generally dispersed or atomized into a plurality of partial volumes. which is unacceptable for highly accurate dispensing of the small single-dose volumes discussed here of less than 2 μΙ. In principle, it can be stated that as the piston speed and / or piston acceleration increase, the tendency increases to undesirably pipette the predetermined dosing liquid quantity in several sub-quantities. According to the current state of knowledge, at least for aqueous metering liquids, as defined above, very excellent results are achieved at maximum piston speeds of about 10,000 μΙ / s in terms of accuracy and repeatability of the pipetted liquid quantity.

To give an impression of the piston speed: Preferably, the piston needs for its movement in the dispensing direction and then in Aspira- tion direction of the location of the halfway distance, which is half the distance between the starting point of the pipetting and its first reversal dead center in a whip-like pipetting for generating a pressure pulse in the working gas, until this half-distance is reached again less than 30 ms, preferential less than 20 ms, most preferably less than 16 ms. Even motion times in the single-digit millisecond range are conceivable.

A complete piston movement in the dispensing and Aspirationsrichtung, with which a single-dosing volume of 950 nl of an aqueous dosing at a swept by the dosing end surface dispensing volume of 30 μΙ and a swept aspiration volume of 29.05 μΙ can with a piston with a circular piston surface and a Diameter of 4.3 mm easily expire in about 15 ms.

However, the kinematic aspect of the whip-like piston movement is based not only on the achievable maximum piston speed, but also on the time required for the motion drive to accelerate the piston to the desired piston speed and / or decelerate from the desired piston speed. Preferably, therefore, the control device and the movement drive are adapted to the piston with an acceleration of at least 2 x 10 ⁶ μΙ / s ² , preferably of at least 6 x 10 ⁶ μΙ / s ² particularly preferably even of at least 8 x 10 ⁶ μΙ / s ² and not more than 1 x 10 ⁸ μΙ / s ² to accelerate movement along the channel path and / or delay. The information given above on the preferred piston size, stated as piston area, applies.

Surprisingly, it has further been shown that the pipetting of dosing liquids, in particular of aqueous dosing liquids, with the pipetting devices proposed here according to the invention is independent of the particular pipetting tip used. With the same pipetting parameters, the same pipetting result can always be achieved repeatably for one and the same dosing liquid on one and the same pipetting device with different pipetting tips. In particular, the pipetting result is independent of the nominal receiving volume of the respective pipetting tip coupled to the pipetting device. The pipetting result achievable with a set of pipetting parameters is all the better between pipetting tips having different nominal receiving space volumes. be transferred, if the pipetting tips have the same Pipettieroffnungen and the same dead volumes.

The present invention will be explained in more detail with reference to the accompanying drawings. It shows:

FIG. 1 shows a pipetting device according to the invention in which a pulse-like dispensing method according to the invention takes place immediately after the aspiration of a predetermined amount of dosing liquid,

FIG. 2a shows the pipetting device of FIG. 1 after generation of a first negative pressure in the working gas, based on the holding reference pressure of FIG. 1, for forming a gas volume between pipetting opening and aspirated dosing liquid;

FIG. 2b shows the pipetting device of FIG. 2a after increasing the pressure of the working gas between pipetting piston and aspirated dosing liquid, in order to shift the meniscus closer to the pipetting opening to the pipetting opening;

FIG. 2c shows the pipetting device of FIG. 2b after generation of a second negative pressure in the working gas, based on the holding reference pressure of FIG. 1, for forming a gas volume between pipetting opening and aspirated dosing liquid;

FIG. 3 a shows the pipetting device of FIG. 2 c, which is shown repeatedly for the sake of clarity on the third figure sheet,

FIG. 3b shows the pipetting device of FIG. 3a during the sudden generation of a pressure pulse, FIG. 3c shows the pipetting device of FIG. 3b after completing the whip-like piston movement for dispensing a single dispensing volume of 500 nl, a roughly schematic course of the volume swept by the pipetting piston in the pulse-like exemplary dispensing of approximately 1 .mu.Ι dosing liquid;

5 shows an exemplary control structure, such as the control device of the pipetting device according to the invention for controlling the movement of the

Used pipetting piston depending on a detected pressure of the working gas

6 is an exemplary diagram of a working gas desired pressure pulse course and a working gas-actual pressure pulse course for a pulse-like dispensation of a dosing liquid volume of 500 nl,

7 shows an example diagram of a desired working gas pressure pulse course and a working gas-actual pressure pulse course for a pulse-like dispensing of a dosing liquid volume of 1 μΙ and

8 shows an example diagram of a desired working gas pressure pulse course and a working gas-actual pressure pulse course for a pulse-like dispensing of a dosing liquid volume of 1.5 μΙ.

In FIGS. 1 to 3c, a pipetting device according to the invention is designated generally by 10. This comprises a pipetting channel 1 1, comprising a cylinder 12 which extends along a channel path K designed as a straight-line channel axis. In this pipetting channel 1 1, a piston 14 along the channel path K is borrowed borrowed.

The piston 14 comprises two end caps 16 (for reasons of clarity, only the lower one is provided with reference numerals in FIGS. 1 to 3 c), between which Chen a plurality of permanent magnets 18 (in the present example, three permanent magnets 18) are added. The permanent magnets 18 are polarized along the channel axis K to obtain a separating magnetic field along the channel path K and are arranged in pairs with poles of the same name facing each other. This arrangement results in a magnetic field emanating from the piston 14, which is substantially uniform around the channel axis K, ie substantially rotationally symmetrical with respect to the channel axis K and which has a high gradient of the magnetic field strength along the channel axis K, so that unlike polarization zones are separated along the channel path K alternately alternate. Thus, for example, by Hall sensors of a position sensor arrangement 39, a high position resolution in the position detection of the piston 14 along the channel axis K can be achieved and it can be a very efficient coupling of an external magnetic field to the piston 14 can be achieved. The end caps 16 are preferably formed from low-friction material comprising graphite or mica, as is known, for example, from commercially available caps from Airpot Corporation of Norwalk, Connecticut, (US). To be able to exploit the low friction provided by this material as fully as possible, the pipetting channel 1 1 preferably comprises a cylinder 12 made of glass, so that during a movement of the piston 14 along the channel axis K the material comprising graphite or mica slides on a glass surface with extremely low friction ,

The piston 14 thus forms a rotor of a linear motor 20, the stator of the pipetting channel 1 1 surrounding coils 22 (only four coils are exemplified here) is formed.

It should be expressly understood that FIGS. 1 to 3c merely show a roughly schematic longitudinal section of a pipetting device 10 according to the invention, which by no means is to be understood to scale. Furthermore, multiples of components are represented by any number of components, such as three permanent magnets 18 and four coils 22. In fact, both the number of permanent magnets 18 and the number of coils 22 may be larger or smaller than the number shown. The linear motor 20, more precisely its coils 22, are controlled via a control device 24, which is connected to the coils 22 in signal transmission terms. The signal also includes the transmission of electrical current to energize the coils and thus to generate a magnetic field through them.

At the metering-side end 12a of the cylinder 12, a pipetting tip 26 is releasably attached in a manner known per se. The connection of the pipette tip 26 with the metering-side longitudinal end 12a of the cylinder 12 is likewise only shown roughly schematically.

The pipette tip 26 defines a pipetting space 28 in its interior, which is accessible at the coupling-remote longitudinal end 26a exclusively by a pipetting opening 30. The pipette tip 26 extends the pipetting channel 1 1 during its coupling to the cylinder 12 to the pipetting 30th

In the example of the pipetting device 10 shown in FIG. 1 immediately after completion of a conventional aspiration operation in quasi-synchronous pipetting operation by the same pipetting device 10, a quantity of dosing liquid 32 is received in the pipetting space 28 - and thus in the pipetting device 10.

Between the piston 14 and the metering liquid 32 is permanently working gas 34, which serves as a force transmitter between the piston 14 and the metering liquid 32. Preferably, between the piston 14 and the dosing liquid 32, only the working gas 34, optionally in its chemical composition, is negligibly changed by the absorption of volatile constituents from the dosing liquid 32.

The working gas 34 is arranged even with completely empty pipette tip 26 between the piston 14 and a dosing liquid 32, since the pipetting tip 26 is dipped for aspiration of dosing liquid 32 into a corresponding Dosierflüssigkeitsvorrat, so that in this state, at least at the pipetting 30 a meniscus of the dosing liquid 32 is present is. Thus, working gas 34 is in permanently relevant to a pipetting process state of the pipetting device 10 completely between the piston 14 and a dosing liquid 32 and separates them from each other. More specifically, the working gas 34 is located between a dosing-side end face 14a of the piston 14, which in the present example is formed by an end face of the end cap 16 pointing in the axial direction with respect to the channel path K to the dosing opening 30 and a meniscus 32a remote from the pipetting opening in the pipetting space 28 taken as a liquid column dosing liquid 32nd

A pressure sensor 38 can detect the pressure in the interior of the pipetting channel 11, to which the pressure communicating receiving space 28 belongs, ie the pressure of the working gas 34 between the dosing liquid 32 and the dosing-side end face 14a of the piston 14, and via a signal line transmitted to the controller 24. Thus, when using sufficiently fast pressure sensors 38, such as the Honeywell sensor TBPDANS001 PGUCV, a movement control of the piston dependent on the pressure of the working gas 34 is possible for carrying out a whip-like piston movement as presented above. The position sensor arrangement 39 for detecting the piston position is provided on the pipetting channel 11 and is connected to the control device 24 in terms of signal transmission.

Starting from the state shown in FIG. 1, a preparation for a pulse-like dispensing process of the pipetting device 10 according to the invention and the pulse-like dispensing process itself are described below:

With reference to FIGS. 2a to 2c, a preparation of the pipetting device 10 is described, with which the accuracy of the pulse-like dispensing process illustrated in FIGS. 3b and 3c can be considerably increased. This essentially means that lower minimal dispensing doses can be delivered with high repeat accuracy than without appropriate preparation. Starting from the state of the pipetting device 10 immediately after aspiration of the predetermined metering liquid quantity 32 into the pipetting tip 26 in the conventional quasi-synchronous pipetting operation (see FIG. 1), the control device 24 energizes the coils 22 in such a way that the pipetting piston 14 is generated in the manner of generating a (FIG. first) negative pressure in the working gas 34 is moved, that means away from the pipetting opening 30. This negative pressure is not generated in a pulse-like manner, but with piston accelerations and speeds which ensure a quasi-synchronous displacement of the dosing liquid quantity 32 received in the receiving space 28.

As a result, the metering liquid quantity 32 provided in the pipetting device 10, more precisely in the pipetting receiving space 28 of the pipetting tip 26, is displaced along the channel axis K away from the pipetting opening 30 into the pipetting device 10, more precisely into the pipetting tip 26. The dosing liquid 32 provided is limited to the pipetting piston 14 through a meniscus 32a which lies further away from the pipetting opening 30 and is bounded toward the pipetting opening 30 by a meniscus 32b nearer the opening of the pipetting opening. As a result of the displacement of the dosing liquid 32 away from the pipetting opening 30, a gas volume 35 is formed between the pipetting opening 30 and the meniscus 32b near the pipetting opening.

In an exemplary recorded Dosierflüssigkeitsmenge 32 of 40 μΙ, the gas volume 35 is preferably 4 to 10 μΙ, particularly preferably 4 to 6 μΙ immediately before triggering the pulse-like dispensing overpressure pulse. As a result of the displacement of the meniscus 32b, which is closer to the pipetting opening and therefore discharges the subsequent metering drop 36, away from the pipetting opening 30, the meniscus 32b present after aspiration with undefined shape, in particular undefined curvature, receives a more defined shape. Although the shape of the pipetting-open-close meniscus 32b is not completely defined after the generation of the gas volume 35 according to FIG. 2a, its shape varies only to a slight extent by a conventionally expected shape. The shape of the pipetting-closer meniscus 32b depends, for example, on the surface tension of the dosing liquid 32, on its density, on its viscosity and on the wettability of the wall of the pipetting tip 26. According to FIG. 2b, the control device 24 can move the spools 22 to move the pipetting piston 14 in FIG To drive sense of an increase in pressure in the working gas 34, ie the pipetting piston 14 in the direction of Pipettieroffnung 30 towards shift. As a result, the dosing liquid 32 provided in the pipetting tip 26 is displaced back toward the pipetting opening 30, but not beyond. The gas volume 35 between Pipettieroffnung 30 and pipettieröffnungs- closer meniscus 32b is thereby less or even disappears completely. This change in the working gas pressure is not pulse-like, but according to conventional, quasi-synchronous operation. Further, the controller 24 may again drive the spools 22 to move the pipetting piston 14 to reduce the pressure of the working gas 34, ie, move away from the pipetting orifice 30 in an aspiration sense, again causing a gas volume 35 between the pipetting orifice 30 and the meniscus 32b closer to the pipetting orifice Dosing liquid 32 is formed or / and increased. This also happens in conventional quasi-synchronous pipetting operation. As a result of the reciprocating movement of the dosing liquid 32 in the pipette tip 26, as shown in FIGS. 2 a to 2 c, an identically shaped meniscus 32 b is formed for one and the same dosing liquid 32 at the end of the generation of the second negative pressure according to FIG. which is advantageous for the subsequent pulse-like dispensing operation, as illustrated and described in FIGS. 3a to 3c. The advantage lies in the reduction of the minimum dispensable amount of liquid and the achievable repeatability of the same when aliquoting.

FIG. 3a shows the pipetting device 10 of FIG. 2c on a separate sheet of figures in order to be better able to compare the comparison of the different states of the pipetting device 10 immediately before and during the generation of a pressure pulse. Central point of the inventive idea of the present application is a whip-like movement of the piston 14. This whip-like movement is expressed in several forms. Due to the provided preferred linear motor 20, the piston 14 can be moved with enormous dynamics of movement along the channel axis K. To dispense a small amount of liquid, about 500 nl of the dosing liquid 32, the piston 14 is first accelerated and moved rapidly in the sense of generating a pressure increase in the working gas 34 (here: dispensing direction) to the dosing 30. The control device 24 controls the coils 22 of the linear motor 20 in accordance with a detection result of the pressure sensor 38 such that the piston 14 generates a pressure pulse in the working gas 34, which follows a predetermined in a data memory of the control device 24 working gas target pressure pulse course. The piston 14 carries out a stroke P of such a size that the metering-side end face 14a of the piston 14 sweeps along the stroke P by a multiple, say 40 times, of the predetermined single metering volume 36 (see FIG. 3c). The piston is then in the position shown in FIG. 3b at the bottom dead center of its movement in the dispensing direction, whereupon the piston 14 is driven to an opposite movement in the aspiration direction, ie in the sense of a reduction of the pressure of the working gas 34 (see arrow G) ,

Also in this section of the piston movement in Aspirationsrichtung the movement of the piston 14 is controlled in accordance with a detection result of the pressure sensor 38 such that the pressure caused by the piston movement of the working gas 34 follows a predetermined in a data memory of the control device 24 working gas target pressure pulse course.

The initial impulse or whip-like movement of the piston 14 in the dispensing direction lasts less than 10 ms in the present example. When the piston 14 reaches its bottom dead center, usually no part of the dosing liquid 32 has yet detached from the pipetting tip 26. The pipette-open-close meniscus 32b is shown in a drop-preparatory form. The shape of the meniscus 32b is chosen for purposes of illustration only. clearly indicate that delivery of a dosing liquid drop 36 (see Fig. 3c) is imminent. The pipetting-opening-remote meniscus 32a is shown concavely curved in order to illustrate the effect of the overpressure pulse on the dosing liquid 32. The piston is moved in the dispensing direction approximately at a maximum speed of approximately 10,000 μΙ / s and accelerated and decelerated with an acceleration of up to 8 × 10 ⁶ μΙ / s ² . However, the maximum speed only occurs for a short time. This means that the piston in the said case in which its dosing-side end face 14a in the course of the dispensing movement sweeps over a volume of about 40 times the single dosing volume 36, ie about 20 μΙ, for this dispensing movement about 6 to 8 ms needed.

The metering liquid 32 is too slow here to follow this piston movement. Instead, the piston 14 transmits a pressure pulse across the working gas 34 to the metering liquid 32 in the pipette tip 26. Starting from the representation shown in Figure 3b, the piston 14 is accelerated as soon as possible back into the aspiration, wherein the movement stroke G in aspiration in the present case is so far less than the stroke P of the movement in the dispensing, that the end-side piston surface 14a in the course of movement Aspiration in the direction of aspiration overflows, which is lower by the single metering volume 36 than the swept dispensing volume.

However, this does not have to be the case. The aspiration volume can also be just as large as the dispensing volume. However, an aspiration volume reduced by the single dosing volume has the advantage that the position of the pipetting-closer meniscus does not change after pipetting, which is advantageous, above all, in the aliquoting mode.

In the end position of the pipetting device 10 shown in FIG. 3c after the end of the pulse-like dispensing process, the dosage-side end surface 14a is a resultant stroke H away from the initial position of FIG. 3a, in the illustrated example the piston area of the piston 14 multiplied by the resulting stroke H. the single metering volume 36 corresponds. The movement in the direction of aspiration in the context of the pulse-like dispensation runs at the said maximum speed, so that this movement also takes about 6 to 8 ms. With additional dwell times at the bottom dead center, which can arise by overcoming the static friction limit, as well as possibly occurring movement overshoots of the piston 14 about its desired position, the entire piston movement takes place until reaching the end position, as shown in Figure 3c, in about 14 to 30 ms. Only after the reversal of the piston movement from the dispensing direction in the aspiration direction is a defined single metering volume 36 in the form of a drop ejected from the pipetting opening 30. This droplet moves along the extended imaginary channel path K to a metering target placed below the pipetting opening 30, for example a container or a well. The meniscus 32b nearer to the opening of the pipetting opening 36 can still vibrate for a short time after the dosing liquid drop 36 has been spun off.

The pipetting tip 26 may have a nominal pipetting volume substantially exceeding the single metering volume, approximately 200-400 μΙ, preferably 300 μΙ.

The movement of the piston 14 in the direction of aspiration in turn proceeds so fast that a pressure reduction pulse is transmitted from the dosing-side end face 14a to the dosing liquid 32 in the pipetting chamber 28. The pressure increase pulse of the piston movement in the dispensing direction forms the steep rising edge of a pressure pulse whose steep falling edge of the pressure reduction pulse of the piston movement forms in the direction of aspiration. The faster the individual piston movement takes place, the steeper the flank of the pressure change pulse associated with it. The two pressure change pulses acting in opposite senses can thus define a "hard" pressure pulse with steep flanks. The impact of the thus formed "hard" pressure pulse leads to the extremely precise reproducible dispensing result.

During the entire piston movement to generate the pressure pulse with excess pressure and negative pressure portion, the piston is moved by the control device 24 by applying a corresponding voltage to the coils 22 controlled such that the pressure of the working gas during the pressure pulse a predetermined working gas target pressure pulse follows , Taking into account position detection signals of the position sensor arrangement 39, the pipetting piston 14 can be brought into a defined end position at the end of the dispensing process.

Surprisingly, the dispensing process presented here is independent of the size of the selected pipette tip 26. The same piston movement described above would lead to exactly the same result even with a significantly smaller pipetting tip of about a nominal pipetting volume of 50 μΙ, provided the same working gas and same dosing liquid will continue to be used with unchanged dispensing parameters.

Thus, the present pipetting device according to the invention and the presented pulse-like dispensing method are eminently suitable for aliquoting liquids from even large amounts of dosing liquid 32 received in pipette tips 26. Even over many aliquoting cycles, the dispensing behavior of the pipetting device 10 changes under otherwise identical conditions Not. The dispensing behavior of the pipetting device 10 according to the invention is thus independent of the degree of filling of a coupled to the cylinder 12 pipetting tip 26, as long as it is sufficiently filled for a pulse-like dispensing.

The piston movement may not be perfectly accurate due to the inertia of the motion-based control signal. In places of great dynamic forces - especially in the reversal of the direction of movement of the Dispensationsnchtung in the Aspirationsrichtung, but also when stopping the piston - the piston may tend to overshoot to the desired position around. In case of doubt, therefore, the decisive factors are the control signals that form the basis of the movement, which are the image of a desired movement.

It should be expressly noted that a pulse-like dispensation can also take place starting from the state according to FIG. H. without prior formation of the pipettieröffnungsnahen gas volume 35. In Figure 4 is a roughly schematic and merely exemplified a time course 42 of the movement of the piston 14 (dashed line) shown schematically, as they could be present in a Dispensationsvorgang of Figures 3a to 3c.

The current piston position at the beginning of the dispensing process, ie the piston position shown in FIG. 3a, is selected as the zero point line in FIG.

The abscissa of the representation of Figure 4 shows the time in milliseconds, with a screening of 10 ms is selected. The ordinate shows the volume in microliters, and with respect to the time-of-place curve 42 of the piston 14, the volume of the ordinate axis indicates the volume swept by the metering-side end surface 14a of the piston 14.

With 46 and 48, the locations of the so-called "halfway distance" of the piston 14 between its start position at 0 μΙ and its reversal point of the direction of movement at about -22.5 μΙ are designated. The halfway distance is therefore approximately at -1 1, 25 μΙ.

The time integral of the time-place curve of the piston 14 - represented approximately by the time-place curve of the dosing-side piston surface 14a as a reference point of the piston 14 - between the passage through the location of the half-distance in the dispensing movement and the re-passage through this location during its movement in aspiration direction is a measure of the actually metered with the piston movement impulsively single-dosing volume 36. A surface formed by this integral is hatched as area 50 in FIG. The relationship between the surface area of the surface 50 and the actually pipetted individual dosing volume 36 can be determined empirically for different liquid classes and stored in a data memory of the pipetting device 10.

Thus, very small individual metering volumes 36 of 2 .mu.Ι or less can be dispensed in a highly repetitive manner with the same pipetting apparatus 10, with which even large pipetting volumes of several 100 .mu.s can be aspirated and dispensed in conventional quasi-synchronous pipetting operation ,

FIG. 5 shows a roughly schematic representation of a control structure 52, as may be used in the control device 24.

The control structure 52 is a cascaded control structure having an outermost control circuit 54, a middle control circuit 56, and an innermost control circuit 58.

In a data memory of the control device 24, a working gas desired pressure pulse course 60 is stored which stores desired values of the pressure in the working gas as a function of time for a pulse-like dispensing process for dispensing a predetermined volume of liquid.

Indeed, a plurality of desired working gas pressure pulse waveforms may be stored in the data memory of the controller 24, multi-dimensionally ordered for different classes of fluids and within different fluid classes for different metered fluid volumes.

Depending on the amount of dosing liquid required by manual data entry or data transfer from another device, the control device 24 selects the dosing fluid level indicated also by manual data entry or automated data transfer. Sized liquid amount of true idealized working gas target pressure pulse path 60 and supplies it to an outermost comparator 62.

The extreme comparator 62 is also supplied with a detection signal from the pressure sensor 38, so that the comparator 62 determines the difference between the working gas target pressure value valid for the detection time and the actual working gas pressure detected by the pressure sensor 38 and outputs it to a first controller 64 can. The first controller 64 is advantageously a PID controller, which transmits the determined pressure difference for each detection time point into a desired value for the position of the pipetting piston 14 due to the transfer functions stored in it. This position setpoint of the first controller 64 of the outermost control circuit 54 is supplied to a second comparison 66.

The second comparator 66 is likewise supplied with the detection result of the position sensor arrangement 39 and thus with the actual position of the pipetting piston 14. The second comparator 66 thus outputs a pipetting piston position difference value, which is a measure of the difference between the calculated pipetting piston setpoint position and the detected pipetting piston actual position for each detection instant.

For advantageously fast precontrol of the movement of the pipetting piston 14, an idealized pipetting piston desired position profile 68 is stored in the data memory of the control device 24, which results from the empirically determined working gas desired pressure pulse course 60. The idealized value of the pipetting piston position according to the idealized profile 68, which is valid for the respective detection time, is likewise supplied to the second comparator 66 by means of the pilot control known per se.

A value representing the difference between the pipetting piston setpoint position and the pipetting piston actual position is supplied by the second comparator 66 to the second or middle controller 70, which advantageously again takes the form of a PID controller is. Its transfer function determines from the difference value representing the difference between the setpoint and actual positions of the pipetting piston 14 a desired value for the current flowing in the coils 22 of the movement drive 20 at the time of detection. This current setpoint is fed to a third comparator 72. The third comparator 72 is also supplied with the actual current value at the time of detection, which is readily determinable at the coils 22 in a manner known per se.

The third comparator 72 thus determines a value representing the difference between the current setpoint and the current actual value at the time of detection and supplies this to the third or innermost regulator 74. Advantageously, the third or innermost controller 74 has a PI control behavior.

In the data memory of the control device 24, an idealized coil nominal current value profile 76 is stored, which results from the idealized working gas desired pressure pulse course 60 or / and the idealized pipetting piston desired position profile 68.

The idealized coil set current value valid for the respective detection time is supplied to the third comparator 72 by way of a known pilot control from the idealized desired coil current value profile 76 in order to obtain the fastest possible control of the movement of the pipetting piston 14 in that the pressure profile generated in the working gas 34 by the movement of the pipetting piston 14 coincides as exactly as possible with the desired working gas pressure pulse course 60 selected for the respective pipetting operation.

The transfer function of the third or innermost regulator 74 determines, from the input value representing the difference between the current reference value and the actual current value at the time of acquisition, a coil desired voltage value at the detection time point which is applied to the coils 22 ,

The control loop structure 52 may be separate for each phase of the coils 22. Again, to achieve the fastest possible and highly accurate control of the piston position for precontrol, the coil voltage in the data memory of the control device 24 is also subject to an idealized desired coil voltage curve 78, which is derived from the idealized desired working pressure pulse course 60 or / and the idealized Pipettierkolben target position profile 68 and / or the idealized coil nominal current value curve results.

A fourth comparator 80 is shown to indicate the feedforward control of the coil voltage by means of the idealized coil desired voltage waveform.

With the cascaded control structure shown in FIG. 5, the pipetting piston 14 can be moved so accurately by applying a detected working gas pressure, the detected pipetting piston position and the detected coil current in the range of a few milliseconds that the pressure curve in the working gas substantially follows a predetermined pressure pulse course ,

In FIGS. 6 to 8, working gas setpoint pressure pulse profiles and working gas actual pressure pulse curves are plotted for different dosing liquid quantities 36 to be dispensed in a pulse-like manner. The abscissa of the graphs of FIGS. 6 to 18 shows the time in seconds, wherein in each of FIGS. 6 to 8 a period of 5 to 10th of a second is shown.

The ordinate of the graphs of Figs. 6 to 8 indicates a pressure difference from a reference pressure in Pascal. The reference pressure is the pressure which prevails when the pipetting piston is stationary in the pipetting channel, with no fluid passing through the pipetting opening at the reference pressure.

In FIGS. 6 to 8, the desired working gas pressure pulse course is plotted in dashed lines and designated by the reference symbols 61 (FIG. 6), 63 (FIG. 7) and 65 (FIG. 8). Likewise, in the cited figures, the working gas-actual pressure pulse course is recorded by a solid line and denoted by reference symbols 71 (FIG. 6), 73 (FIG. 7) and 75 (FIG. 8). FIG. 6 shows the pressure pulse courses 61 and 71 for a dosing liquid volume of 500 nl to be dispensed in a pulse-like manner. At about 5 ms, the pressure of the working gas 34 begins to increase until it reaches its maximum value at about 9 ms and drops due to a reversal of the motion of the pipetting piston, which is then moved in the aspiration direction.

The pressure of the working gas 34 reaches its original value approximately between 12 and 13 ms, but continues to fall until it reaches its maximum setpoint negative pressure in the range between 13 and 14 ms. From then on, the pipetting piston is again moved in the dispensing direction in order to return the working gas pressure to its original value until the time of approximately 20 ms.

The target pressure pulse thus lasts from about 5 ms to the time 20 ms and thus extends over 15 ms. In the overpressure portion of the pressure pulse, the pressure of the working gas 34 follows the working gas desired pressure pulse course very well. In the phase of the reduction of the negative pressure, ie in the period of about 13.5 to 20 ms, the actual pressure of the working gas 34 oscillates a little by the desired working gas pressure. The separation of the dosing liquid droplet takes place in the falling pressure flank between the maximum overpressure and the maximum negative pressure. This also applies to the dispensations of FIGS. 7 and 8.

FIG. 7 essentially shows the desired working gas pressure pulse course 63 and the working gas actual pressure pulse course 73 for a dosing liquid volume of 1 μΙ to be dispensed in a pulse-like manner. As can be seen from the desired working gas pressure pulse curve 62, the ideal pressure pulse for this dispensing process lasts about 19 ms, namely from about 4 ms to about 23 ms. Again, the Working gas setpoint pressure pulse course for the dispensation initially a positive pressure portion, such as between the times 4 ms and 15 ms, and has a negative pressure portion, namely in the range of 15 ms to 23 ms. FIG. 8 shows the working gas desired pressure pulse course 65 and the working gas actual pressure pulse course 75 for a pulse-like dispensing of a dosing liquid drop of 1.5 μΙ.

Due to the onset of movement of the pipetting piston, the pressure of the working gas 34 begins to increase at about 4 ms, rises steeply to about 8 ms, and then flattens from about 8 ms to 21.5 ms. Subsequently, the desired working gas pressure pulse drops until approximately 27 ms, where it reaches its maximum negative pressure. From there it rises again to its reference pressure until the time of about 32.5 ms. Ideally, the entire pressure impulse for the pulse-like dispensing of 1, 5 μΙ of the dispensed dosing liquid does not last even 30 ms.

The achievable with this rule rule repeatability of dispensed dosing liquid volumes are in the range of less than 3%.

Claims

claims

Pipetting device (10) for pulsed pipetting of dosing liquids in small dosing volumes of less than 2 μΙ mediated by a pressure-variable working gas (34), wherein the Pipettiervornchtung (10) comprises: at least partially filled with working gas (34) pipetting

(1 1).

a pipetting tip (26) which provides a receiving space (28) at least partially filled with working gas (34) which communicates with the pipetting channel (11) to communicate and which is accessible through a pipetting opening (30) so as to change the pressure of the working gas in the receiving space through the pipetting opening (30) through the amount of dosing liquid received in the receiving space is variable,

a pipetting piston (14) movably accommodated in the pipetting channel along the pipetting channel (11) for changing the pressure of the working gas (34),

a movement drive (20) for driving the pipetting piston (14) for movement along the pipetting channel (1 1),

a control device (24) for controlling the movement drive (20), and

a pressure sensor (38) for detecting the pressure of the working gas and for outputting a pressure signal indicative of the pressure of the working gas (34) to the control device (24),

wherein the control device (24) is adapted to the movement drive (20) for generating - with respect to a reference pressure in the pipetting (1 1) in which no fluid passes through the pipetting - pressure pulse in the pipetting channel (1 1) with a pulse duration of to control no more than 40 ms on the basis of the pressure signal (38) output by the pressure signal, that the pressure of the working gas (34) during the pulse follows a predetermined working gas target pressure pulse course. Pipetting device (10) according to claim 1,

characterized in that the pressure pulse - based on the reference pressure in the pipetting channel (1 1) in which no fluid passes through the pipetting - has a positive pressure and a negative pressure component.

Pipetting device (10) according to claim 1 or 2,

characterized in that the pipetting piston (14) is a magnetic piston (14) with at least one permanent magnet (18) and that the movement drive (20) comprises electrically energizable coils (22), wherein the control device (24) is adapted to supply the coils (22) to control with electrical energy.

Pipetting device (10) according to one of the preceding claims,

characterized in that it comprises at least one position sensor which is designed to detect the position of the pipetting piston (14) and to output a position signal indicating the detected piston position to the control device (24).

Pipetting device (10) according to claims 3 and 4,

characterized in that the control device (24) controls the supply of the coils (22) with electrical energy as a function of a detected current state of supply of the coils (22) with electrical energy, depending on the position signal and dependent on the pressure signal.

Pipetting device (10) according to claim 5,

characterized in that the control device (24) comprises a cascaded control loop structure (52) according to its innerstem control circuit (58), the control device (24) applied to the coils (22) electrical voltage in accordance with a difference between a desired current value and a sensed current value of a current flowing in the coils (22), wherein the control device (24) is adapted to the desired current value of the current flowing in the coils (22) in an outer loop (56) in accordance with a difference between a desired Position value and one Determining the actual position value of the pipetting piston (14) indicated by the position signal, wherein the control device (24) is adapted to set the desired position value of the pipetting piston (14) in an even more external control circuit (54) in accordance with a difference between a Target pressure value and an indicated by the pressure signal actual pressure value of the pressure of the working gas (34) to determine.

Pipetting device (10) according to claim 6,

characterized in that the control device (24) comprises a data memory in which for a feedforward control at least one idealized working gas desired pressure pulse course (60, 61, 63, 65), the at least one idealized working gas desired pressure pulse course (60, 61 , 63, 65) of the idealized pipetting piston nominal position profile (68) and an idealized nominal coil configuration (76) which effects the idealized pipetting piston desired position profile (68) are stored.

Pipetting device (10) according to one of the preceding claims,

characterized in that a pulse-like dispensing of dosing liquid from a in the receiving space (28) of the pipetting tip (26) recorded Dosierflüssigkeitsmenge (32), the volume of which is at least five times greater than the volume of the dosing liquid to be dispensed pulse-like (36).

Pipetting device (10) according to one of the preceding claims,

characterized in that it is designed for the conventional aspiration of dosing liquid.

0. Pipetting device (10) according to one of the preceding claims, including claim 2,

characterized in that the effective piston surface (14a) of the pipetting piston (14) during the pulse-like dispensing of dosing during the generation of the excess pressure portion of the pressure pulse at least the 1, 4 times the volume of the pulse-like dispensed dosing liquid (36) sweeps over.

Pipetting device (10) according to one of the preceding claims,

characterized in that it is designed for pulse-like dispensing in the jet mode, in which the dispensed liquid volume travels a distance in free flight between the dispensing metering liquid quantity (32) in the pipetting tip (26) and a dispensing target.