Patent Application
20120314528
This application claims priority from German Patent Application No. 102011077199.9, which was filed on Jun. 8, 2011, and is incorporated herein in its entirety by reference.
The present invention relates to devices, fluidic modules and methods for producing a dilution series and, in particular, to devices and methods for producing a dilution series on a centrifugal-microfluidic platform.
Producing dilution series is a routine task in any biological, chemical or medical laboratory all over the world. Manually diluting reagents or samples by means of a pipette may be done for a multitude of applications and is therefore an integral part of daily laboratory work. Depending on the task set, typically 3 to 10 dilutions having dilution factors of between 2 and 20 are produced. Should a specific dilution series be used very often, automation by means of a pipetting robot may be effected. Possible examples for using dilution series include:
For example, one specific application is typical enzyme kinetics measurement. To this end, a dilution series of a substrate of an enzyme in used in most cases, and the amount of a product produced within a predefined time period and/or the conversion rate of the enzyme within the product per time unit is measured. The dilution series in most cases includes 2 to 4 orders of magnitude of concentrations of the substrate. The amount of enzyme remains constant in this context. Dilution series of similar types are produced in inhibition studies, the amount of enzyme and the amount of substrate being kept constant. It is only the inhibitor that is diluted.
A further specific application comprises using a dilution series for determining a number of bacteria. To this end, the bacteria are diluted, in most cases in a decade logarithmic dilution series, and a small volume of each dilution is plated. The dilution found to be “countable” (several to less than one thousand bacterial colonies) is detected, and the total number of bacteria from the sample is calculated from the values obtained and while taking into account the dilution factor.
A further specific utilization of a dilution series comprises determination of a DNA concentration and/or utilization for calibrating a PCR thermocycler. A dilution series is prepared from a solution containing DNA. Said dilution series is subsequently mixed with a PCR mix, and the corresponding enzymatic reaction is performed. The dilution series of the DNA are measured, and subsequently, the initial concentration of the DNA may be determined from the characteristic slope of the curves obtained. Since the initial concentration is unknown in most cases, a dilution series is produced to determine the point at which no more signal can be produced. Said dilution will then correspond to the concentration of DNA which in purely statistical terms contains no more DNA strand. Thus, the concentration of the DNA may be determined from this “non-occurrence” of the signal. By contrast, a sample having a known DNA may be used for validating the PCR system. For this purpose, too, one produces a dilution series so as to show that the points in time of the characteristic signal rise of the PCR linearly correlate with the concentration of the DNA.
Automated production of dilution series may be effected by means of a pipetting robot, which is, however, not economic due to the cost involved specifically for applications with low and medium throughputs. Moreover, when producing dilution series, one is to take a lot of care to avoid contamination and cross-contamination. This is sometimes very difficult to achieve with automated solutions, when the parts of the pipetting robot that are contaminated may be cleaned, or this signifies a high conversion rate of materials used, such as disposable pipetting tips, while involving a large amount of technical expenditure for the mechanical systems for receiving said tips, checking their correct seats and ejecting them after the dispensing process.
Consequently, dilution series, in particular indirect dilution series, are typically produced by means of manual pipetting. This includes several repetitions of the following steps:
What is particularly problematic here is the exponential propagation of pipetting errors. For example, a pipette that has been set wrongly once or insufficient mixing at the beginning of the dilution series will have repercussions on all of the concentrations derived therefrom. This error occurs once and linearly propagates throughout the dilution series. Should the pipetting step for precharging the solution B or for removing and transferring the dilution AB be faulty, this error will propagate exponentially.
In addition, manual handling of minute volumes of liquid with corresponding precision represents a corresponding challenge. For example, it is useful—in order to prepare a dilution having a dilution factor of 10 and a total volume of 10 μl—to mix 1 μl of a solution A with 9 μl of a solution B. For automation in the volume regime described, specific dispensing systems may be used to ensure the level of precision needed.
In conventional technology, various microfluidic systems for automatic production of discrete dilutions or concentration gradients have been described. Fundamentally, a distinction is made here between centrifugal and pressure-operated microfluidic systems by means of the type of actuation of liquid. While centrifugal systems can switch and move liquids passively by means of targeted rotation and of the centrifugal forces resulting therefrom, liquids in pressure-operated systems are moved by means of an external pressure source, such as a syringe pump or an air-pressure source such as described by D. Mark et al., Chem. Soc. Rev., 2010, 39:1153-1182. The advantage of centrifugally actuated systems is basically the possibility of being able to operate with minute volumes and in a manner that is almost free from any dead volume. On the other hand, maximum volumes are limited to several ml. Pressure-operated systems may basically process larger volumes (up to volumes of m3 in the production of foodstuffs such as multivitamin juices, for example). For large-volume dilutions (>50 μl), manual pipetting errors tend to be negligible.
C.-Y. Chen et al., Proc. MicroTas, 2010, pp. 752-754, describe a PDMS (polydimethylsiloxane) chip comprising five liquid inlets, one outlet, and magnetically controlled valves. Depending on the valve position, dilution stages having ratios of 1:10 may thus be produced over 5 orders of magnitude. As an example of use, tetraethylammonium (TEA) is diluted in a buffer, and the effect on the ion channels of cells is observed. The system is very complex in terms of structure and exhibits very large dead volumes (for filling the tubes).
J. Koehler et al., Assay Drug Develop., 2002, pp. 91-96, describe pressure-operated fluidics for automatically producing dilutions. As an example of use, various substrate liquids (chemical compounds that are converted in a reaction catalyzed by an enzyme) are diluted for enzyme kinetics and dose-response measurements. The system is designed such that the resulting fluorescent signals may be read out in a standard microwell plate reader. The dead volumes are very large, the dilution stages may be programmed, and the dilutions cannot simply be removed. Introduction of air bubbles or the changes of fluidic resistances lead(s) to uncontrollable misadjusting of the flow rates and, in this manner, create(s) errors in the dilutions.
In addition, various pressure-operated fluidic systems for producing concentration gradients are described by Noo Li Jeon et al., Langmuir, 2000, 16:8311-8316, and Kyle Champbell et al., Lab Chip, 2007, 7:264-272.
Inertia-induced mixing of liquids in a mixing chamber by varying the rotational frequency has been described by M. Grumann et al., Lab Chip, 2005, 5:560-565.
US 2008/0193336 A1 discloses a centrifugal-microfluidic system for producing dilutions. Liquids may be mixed in a central mixing chamber. Here, the dilution factor Z and the volumes created are determined by several channels that transfer, at a defined radial height, liquid from the fill-in chambers to the mixing chamber. Alternatively, several fill-in chambers may be used, the contents of which are serially transferred into the mixing chamber in each case following opening of a valve. The mixture produced may subsequently continue to be passed on into final chambers. In order to open or close the corresponding fluidic paths, the cartridge has wax valves integrated therein which may be actively molten via an external laser. The volumes and the dilutions are predefined by the microfluidic design of the cartridge and cannot be modified later on.
US 2011/0085950 A1 discloses a microfluidic system comprising a spindle motor, via which a carrier may be driven. Cartridges having fluidic structures (46) formed therein may be inserted into the carrier.
U.S. Pat. No. 6,004,515 and U.S. Pat. No. 5,869,004 are directed at methods and devices for producing dilutions, wherein dilutions are produced by means of a main channel, particularly while using an electro-osmotic flow.
U.S. Pat. No. 6,632,655 B1 describes techniques wherein arrays of flowable or solid sets of particles are used in microfluidic systems to perform assays and to modify a hydrodynamic flow.
According to an embodiment, a device for producing a dilution series from a solution to be diluted, which contains a substance to be diluted, and a dilution solution, may have: a body of rotation including fluidic structures, a drive configured to subject the body of rotation to rotations of different rotation protocols, and a controller configured to control the drive so as to pass through the rotation protocols, which fluidic structures may have: a first mixing chamber including at least one fluid outlet, a second mixing chamber including at least one fluid inlet, a fluidic connection between the fluid outlet of the first mixing chamber and the fluid inlet of the second mixing chamber, the fluidic connection between the first mixing chamber and the second mixing chamber being configured such that, when passing through a first rotation protocol, a defined volume of the solution to be diluted and a defined volume of the dilution solution are mixed in the first mixing chamber so as to produce a first mixture having a first dilution ratio, no portion of the first mixture getting into the second mixing chamber, and the fluidic connection between the first mixing chamber and the second mixing chamber being configured such that, when passing through a second rotation protocol, a defined partial volume of the first mixture is transported from the first mixing chamber through the fluidic connection into the second mixing chamber which has a defined volume of the dilution solution located therein, and such that a defined volume of the first mixture remains in the first mixing chamber, the controller being configured to control the drive to pass through the first and second rotation protocols and to pass through a third rotation protocol after having passed through the first rotation protocol and the second rotation protocol, so as to mix, in the second mixing chamber, the defined partial volume of the first mixture with the defined volume of the dilution solution to produce a second mixture having a second dilution ratio.
Another embodiment may have a fluidic module for a device for producing a dilution series from a solution to be diluted, which includes a substance to be diluted, and a dilution solution, which device may have: a body of rotation including fluidic structures, a drive configured to subject the body of rotation to rotations of different rotation protocols, and a controller configured to control the drive so as to pass through the rotation protocols, which fluidic structures may have: a first mixing chamber including at least one fluid outlet, a second mixing chamber including at least one fluid inlet, a fluidic connection between the fluid outlet of the first mixing chamber and the fluid inlet of the second mixing chamber, the fluidic connection between the first mixing chamber and the second mixing chamber being configured such that, when passing through a first rotation protocol, a defined volume of the solution to be diluted and a defined volume of the dilution solution are mixed in the first mixing chamber so as to produce a first mixture having a first dilution ratio, no portion of the first mixture getting into the second mixing chamber, and the fluidic connection between the first mixing chamber and the second mixing chamber being configured such that, when passing through a second rotation protocol, a defined partial volume of the first mixture is transported from the first mixing chamber through the fluidic connection into the second mixing chamber which has a defined volume of the dilution solution located therein, and such that a defined volume of the first mixture remains in the first mixing chamber, the controller being configured to control the drive to pass through the first and second rotation protocols and to pass through a third rotation protocol after having passed through the first rotation protocol and the second rotation protocol, so as to mix, in the second mixing chamber, the defined partial volume of the first mixture with the defined volume of the dilution solution to produce a second mixture having a second dilution ratio, which fluidic module forms the body of rotation or forms the body of rotation when inserted into a carrier, which includes the fluidic structures which include the first mixing chamber including the at least one fluid outlet, the second mixing chamber including the at least one fluid inlet, and the fluidic connection between the fluid outlet of the first mixing chamber and the fluid inlet of the second mixing chamber.
According to another embodiment, a method of producing a dilution series from a solution to be diluted, which includes a substance to be diluted, and a dilution solution, may have the steps of: introducing a defined volume of the dilution solution into a first mixing chamber and introducing a defined volume of the dilution solution into a second mixing chamber, the first and the second mixing chamber being formed in a body of rotation, and a fluid outlet of the first mixing chamber being connected to a fluid inlet of the second mixing chamber via a fluidic connection; introducing a defined volume of the solution to be diluted into the first mixing chamber; subjecting the body of rotation to a first rotation protocol, so that a first mixture having a first dilution ratio is produced in the first mixing chamber without any portion of the first mixture getting into the second mixing chamber; subjecting the body of rotation to a second rotation protocol, so that a defined partial volume of the first mixture is transported from the first mixing chamber into the second fluid chamber which includes the defined volume of the dilution solution located therein, and so that a defined volume of the first mixture remains in the first mixing chamber; and subjecting the body of rotation to a third rotation protocol so as to mix, in the second mixing chamber, the defined partial volume of the first mixture with the defined volume of the dilution solution to produce a second mixture having a second dilution ratio.
In accordance with embodiments of the invention, a defined volume of a first mixture having a first dilution ratio may be produced in the first mixing chamber, and a defined volume of a second mixture having a second dilution ratio may be produced in the second mixing chamber. Thus, embodiments enable producing a dilution series which comprises two mixtures having different dilution ratios. In embodiments of the invention, n mixing chambers may be provided, n being a natural number≧3, so that a dilution series may be produced with three or more mixtures of different dilution ratios. The mixing chambers are connected via corresponding fluidic connections, the mixtures being produced serially one after the other in that a defined partial volume, respectively, of a mixture is transferred from a preceding mixing chamber into a subsequent mixing chamber via the fluidic connection by performing a corresponding rotation protocol, in which subsequent mixing chamber said defined partial volume is mixed with a defined volume of the dilution solution.
Embodiments of the invention are based on the finding that dilution series may advantageously be produced in an automated manner in that a plurality of mixing chambers are used on a centrifugal platform. In this manner, it is possible, by passing through corresponding rotation protocols, to provide a centrifugal drive for the substance to be diluted, the dilution solution and the mixtures. Thus, by providing corresponding fluidic structures and by varying the rotational frequency applied to the body of rotation, any desired dilution series, such as logarithmic dilution series, can be implemented.
In embodiments of the invention, the fluidic connection comprises a siphon, said siphon comprising a fluid inlet leading into the first mixing chamber at a first radial position, and a fluid outlet leading into the second mixing chamber at a second radial position, the second radial position being located radially outward of the first radial position. Those parts of the first mixing chamber that are located radially outward of the first radial position may specify a defined fluid volume, so that by means of emptying the first mixing chamber via the siphon, a defined liquid volume may remain in the first mixing chamber. In embodiments of the invention which comprise n mixing chambers, a corresponding siphon may be provided between a preceding mixing chamber and a subsequent mixing chamber in each case, the position where the siphon leads into the preceding mixing chamber being located radially inward of that position where the siphon leads into the subsequent mixing chamber.
In embodiments, the mixing chambers extend radially outward from a position where the fluidic connection leads from the preceding mixing chamber into the mixing chamber, so that a preceding mixing chamber is arranged radially further inward in each case than a subsequent mixing chamber. This enables centrifugal transport of liquid between the mixing chambers in a simple manner.
In embodiments of the invention, each mixing chamber may have a dosing chamber associated with it which is connected to the mixing chamber via a fluidic valve. The dosing chambers may form fingers of an aliquoting structure, via which a defined volume of the dilution solution may be introduced into each of the mixing chambers. The dosing chambers may be configured to introduce identical volumes or different volumes of the dilution solution into the various mixing chambers. In embodiments, the dosing chambers of the aliquoting structure are each filled with a defined volume of the dilution solution in that a fourth rotation protocol is passed through, the defined volumes then being introduced into the mixing chambers via the valves in that a fifth rotation protocol is passed through. In embodiments, the valves may be formed by hydrophobic bottlenecks which extend radially outward and through which the dilution solution may pass only from a predetermined rotational speed.
In embodiments of the invention, the fluidic structures for providing a defined volume of the solution to be diluted may comprise a pre-portioning chamber fluidically connected to the first mixing chamber. The pre-portioning chamber may comprise, in embodiments of the invention, a suitable overflow structure, so that the volume of the solution to be diluted which is passed on to the first mixing chamber is independent of a filled-in volume of the solution to be diluted, provided that the amount of the solution to be diluted that is filled in exceeds the defined volume. In embodiments of the invention, the body of rotation and/or the fluidic module may be provided with several pre-portioning chambers, each of which comprises one inlet and different volumes, so that one of several possible dilution series having different dilution ratios may be selected by choosing a corresponding inlet. In embodiments of the invention, the pre-portioning chamber may be provided in an inset that is replaceable, so that different dilution series may be produced by switching between pre-portioning chambers having different defined volumes.
Embodiments of the invention provide a fluidic module which forms the body of rotation or forms the body of rotation when inserted into a carrier. The fluidic module comprises the fluidic structures which enable the dilution series to be produced, as are described herein.
In embodiments of the inventive method, the volume transferred from the first mixing chamber into the second mixing chamber is dependent on an added volume of the solution to be diluted and/or on the defined volume of the dilution solution.
In embodiments of the inventive method, a body of rotation comprising n mixing chambers, one fluid outlet of a preceding mixing chamber being connected to one fluid inlet of a subsequent mixing chamber via a corresponding fluidic connection, respectively, is used. The method may further comprise subjecting the body of rotation to corresponding rotation protocols so as to transport respective partial volumes of an n−1th mixture into an nth mixing chamber, a defined volume of the n−1th mixture remaining in the n−1th mixing chamber so as to produce a dilution series having n mixtures having n different dilution ratios, n being an integer larger than or equal to three. In embodiments, the defined volumes and partial volumes may be such that the n mixtures represent a logarithmic dilution series.
Embodiments of the invention are directed to automated production of dilution series and aim at small volumes (in the μl to ml ranges), so that the corresponding fluidic structures may be designated as microfluidic structures. In particular, embodiments of the invention aim at applications mostly in the analytical field. Embodiments of the present invention may be employed for producing dilution series for any applications desired, for example the fields of application described at the outset.
Embodiments of the invention use centrifugal forces so as to automatically transport, portion and mix liquids. A body of rotation and/or a fluidic module having incorporated cavities and/or (micro)fluidic structures may serve as a platform. The body of rotation and/or the fluidic module may be referred to as a cartridge, which may consist of plastic. While precise and expensive syringe pumps may typically be used for transporting liquids in non-centrifugal microfluidic systems, a standard laboratory centrifuge may be utilized as a drive and as a controller for producing the dilution series in embodiments of the invention. The body of rotation may then be inserted instead of the standard rotor, and the rotation protocols that may be used may be adjusted manually or by using appropriate software.
Within the context of this disclosure, a rotation protocol is understood to mean a rotation of the body of rotation at a rotational frequency or a sequence of rotational frequencies. For example, a rotation protocol may designate a rotation at a specific rotational frequency. A different rotation protocol may include rotations at several different frequencies. Yet another rotation protocol may include stopping the rotation.
What is also advantageous in embodiments of the invention is the possibility of doing without actively controlled valves. An interface with an external periphery is not required. Due to the volumes introduced and the volumes which are predefined (by the fluidic structures) in the body of rotation (the cartridge), the dilutions produced may be unambiguously specified, and the “human” errors occurring during handling as well as the possibility of contamination may be reduced to a minimum.
As compared to systems operated by syringe pumps, embodiments of the invention offer the advantage of clearly reduced dead volumes of the liquids, since tube connections and corresponding fluidic ports for pumping the liquid onto the chip are not required. Thus, embodiments of the invention enable reduced requirement in terms of reagents without employing—as would be the case in syringe pump-operated systems—an immiscible system liquid, which would entail the risk of contamination of the sample and of the dilutions, however. In centrifugal systems, the liquids are controlled by means of inherent inertial forces (centrifugal force). Thus, it is possible to transport even minute amounts of liquid. Consequently, solutions which have only small or no dead volumes whatsoever may be implemented.
Embodiments of the invention enable increased flexibility since they enable the dilution factor to be adjusted, e.g. by switching between inserts having different pre-portioning chambers, as was described above.
Embodiments of the present invention will be detailed subsequently referring to the appended drawings. In the drawings, elements which are identical or have identical actions are designated by identical reference numerals, wherein:
a to 3h show schematic representations for illustrating production of a dilution series in embodiments of the invention;
Before embodiments of the invention will be explained in more detail, partly with reference to the drawings, reference shall first of all be made to the following glossary.
Numerical Example:
Numerical Example:
Numerical Example:
Numerical Example:
Numerical Example:
Embodiments of the invention enable production of dilution series whose the dilution factor Z may be defined by the user. The cartridge that may be used for this may contain exclusively passive geometric elements. Actively controlled valves are not required. A solution A (having a volume VA) and a solution B (having a volume VB) are added. The solution B may initially be split up into several “portions” specified by the fluidic structures (VB1, VB2, VB3 . . . with the sum ΣVBi≦VB). The solution to be diluted A is now added to the portion VB1 and mixed. A mixture having the dilution factor Z1=(VA+VB1)/(VA) is produced. Subsequently, a volume VAB1 of the dilution AB1 is transferred into the portion VB2 and produces a Z2=(VAB1+VB2)/(VAB1), etc. By further successive transferral of a volume VAB(i-1) of a dilution AB(i-1) into a portion VBi for producing the dilution ABi, an indirect dilution series may thus be produced. The respective dilution factor Zi is derived step by step from the preceding dilution factor Z(i-1) and is defined by Zi=(VAB(i-1)+VBi)/(VAB(i-1))*Z(i-1).
In embodiments of the invention, the respectively transferred volume VABi corresponds to the initially added volume VA of the solution A. Likewise, in embodiments of the invention, all of the volumes VBi are identical to one another and, therefore, equal to the first volume VB1 of the solution B in the first mixing chamber. With this configuration, the respective dilution factor of the respective stage results with Zi=(VA+VB1)/(VA)*Z(i-1)=[(VA+VB1)/VA]i. In embodiments of the invention, the user has the possibility of adjusting the dilution factor by adding a defined amount of the solution A.
This shall be explained below with reference to three numerical examples.
Different volumes of VBi, and different transfer volumes VABi:
Three mixing chambers comprising volumes VB1=30 μl, VB2=60 μl and VB3=120 μl;
Transfer volumes VAB1=20 μl, VAB2=45 μl
Addition of VA=15 μl results in Z1=3; Z1=12 and Z1=48
VB≧ΣVBi≧210 μl of the solution B is inserted into the cartridge, and the chambers 1 to 3 are filled with the volumes VB1=30 μl, VB2=60 μl and VB3=120 μl. Any volume of the solution B that is not required is transferred into a waste chamber. The solution A with VA=15 μl is added into the mixing chamber 1. This results in Z1=(15+30)/15=3. VAB1=201 are now transferred from the mixing chamber 1 to the mixing chamber 2. What results is Z2=(20+60)/20*3=12. VAB2=40 μl are now transferred from the mixing chamber 2 to the mixing chamber 3. What results is Z3=(40+120)/40*12=48.
Addition of VA=30 μl results in Z1=2; Z1=8 and Z1=32
VB≧ΣVBi≧210 μl of the solution B is introduced into the cartridge, and the chambers 1 to 3 are filled with the volumes VB1=30 μl, VB2=60 μl and VB3=120 μl. Any volume of the solution B that is not required is transferred into a waste chamber. The solution A with VA=30 μl is added into the mixing chamber 1. What results is Z1=(30+30)/30=2. VAB1=20 μl are now transferred from the mixing chamber 1 to the mixing chamber 2. What results is Z2=(20+60)/20*2=8. VAB2=401 are now transferred from the mixing chamber 2 to the mixing chamber 3. What results is Z3=(40+120)/40*8=32.
Different volumes of VBi, and transfer volume VABi identical to VA:
Three mixing chambers with volumes VB1=30 μl, VB2=60 μl and VB3=120 μl
Transfer volume VAB1=VAB2=VA
Addition of VA=15 μl results in Z1=3; Z1=15 and Z1=135
VB2≧ΣVBi≧210 μl of the solution B is introduced into the cartridge, and chambers 1 to 3 are filled with the volumes VB1=30 μl, VB2=60 μl and VB3=120 μl. Any volume of the solution B that is not required is transferred into a waste chamber. The solution A with VA=15 μl is added into the mixing chamber 1. What results is Z1=(15+30)/15=3. VAB1=15 μl are now transferred from the mixing chamber 1 to the mixing chamber 2. What results is Z2=(15+60)/15*3=15. VAB2=15 μl are now transferred from the mixing chamber 2 to the mixing chamber 3. What results is Z3=(15+120)/15*15=135.
Addition of VA=30 μl results in Z1=2; Z1=6 and Z1=30
VB≧ΣVBi≧210 μl of the solution B is introduced into the cartridge, and the chambers 1 to 3 are filled with the volumes VB1=30 μl, VB2=60 μl and VB3=120 μl. Any volume of the solution B that is not required is transferred into a waste chamber. The solution A with VA=30 μl is added into the mixing chamber 1. What results is Z1=(30+30)/30=2. VAB1=30 μl are now transferred from the mixing chamber 1 to the mixing chamber 2. What results is Z2=(30+60)/30*2=6. VAB2=30 μl are now transferred from the mixing chamber 2 to the mixing chamber 3. What results is Z3=(30+120)/30*6=30.
Identical volumes VBi=VB1, and transfer volume VABi identical to VA:
This is the configuration for producing logarithmic dilution series.
3 mixing chambers with volumes VB1=VB2=VB3=30 μl
Transfer volume VAB1=VAB2=VA
Addition of VA=15 μl results in Z1=3; Z1=9 and Z1=27 (3̂1; 3̂2; 3̂3)
VB≧ΣVBi≧90 μl of the solution B is introduced into the cartridge, and the chambers 1 to 3 are filled with the volumes VB1=30 μl, VB2=30 μl and VB3=30 μl. Any volume of the solution B that is not required is transferred into a waste chamber. The solution A with VA=15 μl is added into the mixing chamber 1. What results is Z1=(15+30)/15=3. VAB1=15 μl are now transferred from the mixing chamber 1 to the mixing chamber 2. What results is Z2=(15+30)/15*3=9. VAB2=15 μl are now transferred from the mixing chamber 2 to the mixing chamber 3. What results is Z3=(15+30)/15*9=27.
Addition of VA=30 μl results in Z1=2; Z1=4 and Z1=8 (2̂1; 2̂2; 2̂3)
VB≧ΣVBi≧90 μl of the solution B is introduced into the cartridge, and the chambers 1 to 3 are filled with the volumes VB1=30 μl, VB2=30 μl and VB3=30 μl. Any volume of the solution B that is not required is transferred into a waste chamber. The solution A with VA=30 μl is added into the mixing chamber 1. What results is Z1=(30+30)/30=2. VAB1=30 μl are now transferred from the mixing chamber 1 to the mixing chamber 2. What results is Z2=(30+30)/30*2=4. VAB2=30 μl are now transferred from the mixing chamber 2 to the mixing chamber 3. What results is Z3=(30+30)/30*4=8.
The body of rotation 10 comprises the fluidic structures that may be used for producing the dilution series. For example, the fluidic structures may be formed by cavities and channels within the substrate 12. Alternatively, the fluidic structures may be formed by cavities and channels within the substrate 12 and the lid 14. In embodiments, the fluidic structures are formed within the substrate 12, and fill-in openings and venting openings are formed in the lid 14.
In an alternative embodiment shown in
In embodiments of the invention, the body of rotation and/or the fluidic module, which comprises the fluidic structures, may be formed from any suitable material, for example a plastic such as PMMA (polymethyl methacrylate), polycarbonate, PVC (polyvinyl chloride) or PDMS (polydimethylsiloxane), glass or the like, for example.
The body of rotation may be considered as being a centrifugal microfluidic platform.
The fluidic structures 46 comprise five mixing chambers m1 to m5. The first mixing chamber m1 is fluidically connected to a first inlet chamber 52 via an inlet channel 50. An inlet opening 52a and a venting opening 52b for the first inlet chamber 52 are provided, for example within a lid of the body of rotation. The first mixing chamber m1 is connected to the second mixing chamber m2 via a first fluidic connection s1. The second mixing chamber m2 is connected to the third mixing chamber m3 via a second fluidic connection s2, the third mixing chamber m3 is connected to the fourth mixing chamber m4 via a third fluidic connection s3, and the fourth mixing chamber m4 is connected to the fifth mixing chamber m5 via a fourth fluidic connection s4. The fifth mixing chamber m5 is fluidically connected, via an outlet channel 54, to a waste chamber 56 which is fluidically connected to a venting opening 56b via a venting channel 56a. Each of the mixing chambers m1 to m5 is also connected to a venting opening 60 via a corresponding venting channel 58; for clarity's sake, only the venting channel 58 and the venting opening associated with the fifth mixing chamber are provided with a reference numeral in
The fluidic structures 46 further comprise a second inlet chamber 62, for which, in turn, an inlet opening 62a and a venting opening 62b may be provided. The second inlet chamber 62 is fluidically connected to an aliquoting structure comprising a channel 64 and dosing chambers e1 to e5. A radially outer end 66 of the channel 64 is connected to a further waste chamber 68, which is fluidically connected to a venting opening 68b via a venting channel 68a.
A radially outer area of each of the dosing chambers e1 to e5 is fluidically connected, via a respective valve 70, to a radially inner area of an associated one of the mixing chambers m1 to m5. The dosing chambers e1 to e5 represent aliquoting fingers in the form of radially outwardly arranged protrusions of the channel 64, the channel 64 exhibiting a radially falling curve from the inlet chamber 62 to an area 64a located downstream, in terms of the flow direction, from the fifth dosing chamber es. Thus, the aliquoting structure enables, upon rotation of the body of rotation, that a defined liquid volume is retained within each of the dosing chambers e1 to e5, whereas excess liquid is sheared off and gets into the waste chamber 68.
The valves 70 may be formed by a hydrophobic bottleneck, for example, which enables passing of a liquid, e.g. a dilution solution, only from a specific rotational speed. In the embodiment shown, the valves associated with the mixing chambers m2 to m5 are fluidically connected to the mixing chambers via respective fluid channels, a fluid channel associated with the mixing chamber m4 being designated by the reference numeral 72 by way of example.
In the embodiment shown in
The fluid inlet of each siphon structure leads into the preceding mixing chamber at a location that is radially further inward than that where the fluid outlet leads into the subsequent mixing chamber. Thus, the siphon structures enable, when passing through a suitable rotation protocol, emptying of the liquid volume of the preceding mixing chamber, which leads into the subsequent mixing chamber radially inward of that position where the fluid inlet of the siphon structure leads into the mixing chamber. In this manner, following such a partial emptying, a defined liquid volume remains in the preceding mixing chamber. Thus, the mixing chambers m1 to m5 are each configured to retain a defined liquid volume following partial emptying into the respectively subsequent mixing chamber.
Partial emptying of the mixing chambers may be effected by passing through a corresponding rotation protocol. If the centrifugal force caused by a rotation is larger than the capillary force acting within the capillary fluid channel of the siphon structure, capillary filling of the siphon will be prevented and no partial emptying will take place. If the rotational frequency is reduced such that the capillary force is larger than the centrifugal force, capillary filling of the siphon structure will take place. Moreover, if, following the capillary filling of the siphon structure, the centrifugal force is sufficient to overcome a meniscus at the fluid outlet of the siphon structure, partial emptying of the preceding mixing chamber, as was described above, will take place. This may be effected by increasing the rotational frequency following the capillary filling of the siphon structure.
As may be seen in
The mode of operation of the embodiment shown in
In the example described, the volumes Vi of the individual dilutions are nominally identical since the respective dosing chambers and mixing chambers are configured to provide identical liquid volumes. However, in alternative embodiments, any volumes Vi are possible by configuring the chambers accordingly.
Controlling of the liquids, i.e. transport, volume determination, mixing, etc., is performed by corresponding frequency protocols of the rotation of the body of rotation (of the cartridge) and is based on the interplay of the forces resulting therefrom, i.e. centrifugal forces, inertial forces and capillary forces. One important advantage of embodiments of the invention consists in that no active components such as valves to be switched actively, for example, are required in the body of rotation. As has already been explained, a standard laboratory centrifuge may be utilized as a drive and as a controller.
As is shown in
As is shown in the left-hand parts of
Subsequently, the controller effects an increase in the rotational speed to a frequency f2 (f2>f1), as may be seen in the left-hand part of
Following this, the controller controls the drive to stop the rotation, see the arrow 110 in the left-hand part of
Once the rotation has been stopped, a volume VA, defined by the user, of the solution A is filled into the inlet chamber A, which, in turn, may be effected manually or automatically. The defined volume VA determines the dilution factor. Following this, acceleration takes place again, e.g. to the rotational frequency f2, as a result of which the solution A is transferred into the first mixing chamber m1, as is shown by the arrow 112 in
Once the solution A has arrived in the first mixing chamber m1, the controller causes the rotational frequencies to alternately switch 114 between f1 and f2, which results in the solution A being mixed 116 with the precharged solution B in the mixing chamber m1 due to inertia, as is depicted in
Once the solutions, or substances, have been homogenously mixed, a defined volume of the mixture produced will be transferred into the neighboring chamber. To this end, the rotational frequency is reduced, by the controller, to such an extent, e.g. stopped 118, that the capillary siphon s1 is capillarily filled, as is indicated by an arrow 120 in
The state following the transfer of the defined partial volume 130 into the second mixing chamber is shown in
The corresponding rotation protocols (rotation protocols 1 to 3) may then be repeated so as to produce further dilutions in the mixing chambers m3 to m5.
The type of transfer of liquid described enables producing dilutions having different dilution factors by using a geometrically specified body of rotation, or cartridge, by adding a variable amount of the solution A. The dilution factor is determined only by the amount of solution A added. It is possible to serially produce several dilutions, the number of dilution stages being limited essentially by the size of the body of rotation. A specific embodiment of producing a dilution series for an addition of 7.5 μl (I) and 15 μl (II), respectively, of the solution A will now be described by means of a numeric example. In the following, solution A is to contain any bacterium at a concentration of c=10,000 items/μl. 200 μl of a solution B are introduced into the inlet chamber 62, and the frequency protocol is started. Due to centrifugal forces, solution B is split up into individual aliquots in the dosing chambers e1 to e5 having a volume of 30 μl each. The supernatant flows into the waste chamber 68. The frequency is increased, and the aliquots are transferred from the dosing chambers e1-e5 into the mixing chambers m1-m5. The volumes of the dosing chambers e1-e5 are determined such that each mixing chamber is now filled up to the lower edge of the capillary siphons s1-s5. Rotation is stopped, and the respective initial volumes 7.5 and 15 μl of the solution A are introduced into the inlet chamber 52. Repeated rotation transfers the solution A into the mixing chamber m1 where it is mixed with the precharged 30 μl of the solution B. Now the liquid level of the chamber is increased, and the capillary siphon s1 may fill up during standstill. After repeated rotation, the initial volumes of the solution A, i.e. exactly 7.5 and 15 μl, respectively, of the produced dilution AB1 are transferred from the mixing chamber m1 into the mixing chamber m2, where they are also mixed with the precharged 30 μl of the solution B, etc., as is set forth in the following Table 1.
Table 1 shows the dilution factors and bacteria concentrations of the individual dilutions in the mixing chambers m1 to m5 for additions of 6 μl and 15 μl, respectively, of the solution A and upon precharging of 30 μl of the solution B in each mixing chamber.
In alternative embodiments of the invention, the fill-in chambers for the dilution solution and the solution to be diluted may be implemented and/or connected to the remaining fluidics such that both the solution to be diluted and the dilution solution may be precharged simultaneously prior to the start of the frequency protocol. An interruption of the rotation once the dilution solution has been processed, i.e. once the dilution solution has been introduced into the mixing chambers, would then no longer be necessary.
Instead of the aliquoting structure shown in
In alternative embodiments of the invention, integrated pre-portioning of the solution to be diluted may be provided. For example, the inlet or the inlet chamber for the solution to be diluted (solution A) of the cartridge may be combined with a fluidic structure for defined volume determination, so that the solution to be diluted need only be added in excess. The starting volume of the solution to be diluted may thus also be determined automatically and without any influence of manual pipetting. In embodiments, several inlets for the solution to be diluted may be implemented on a cartridge, each of the inlets being designed for a different kind of pre-portioning, so that a desired dilution series may be produced by selecting one of the inlets. Thus, the solution to be diluted may be portioned in accordance with the desired dilution series in each case, so that different dilution series may be produced with one single cartridge, as desired by the user.
In the embodiment described, neighboring mixing chambers are connected to one another by means of capillary siphons, which transfer a defined volume of one mixing chamber into the neighboring mixing chamber in each case. Alternatively, neighboring mixing chambers may also be connected to other suitable valves or transitions which enable initially mixing the liquids and then transferring a portion of the mixture into the next chamber. This may be achieved by a corresponding frequency protocol. The principle of producing dilution series is based on sequentially transporting a defined liquid volume from one mixing chamber into the neighboring mixing chamber.
In the embodiment described, a fluidic valve in the form of a hydrophobic bottleneck is provided between the dosing chambers and the mixing chambers. Alternatively, other suitable valves or transitions may be provided which allow or do not allow the dilution solution to pass, depending on the rotational frequency.
In embodiments of the invention, suitable fluidic structures may be provided in the body of rotation which enable discharging of the mixtures from the mixing chambers by means of further fluidic operations (standard operations). For using or processing the produced dilutions further, the individual mixing chambers may be connected, via suitable valves, to further fluidic elements on the body of rotation. In addition, there is the possibility of centrifuging the liquids in the mixing chamber via suitable valves from the body of rotation into collection vessels. As collection vessels, one might use, in particular, standard laboratory containers such as standard tubes (so-called Eppendorf cups having volumes of, e.g., 0.5 ml, 1 ml, 1.5 ml, 2 ml, Falkon tubes containing 15 ml or 50 ml) or microwell plates or vessels similar to microwell plates as well as relatively small sample containers such as PCR tubes. In embodiments of the invention, the dilutions produced may thus be further processed once the mixture has been produced on the body of rotation, and/or may be transferred into cavities located further outward, for example in enzymatics. In embodiments of the invention, the dilutions produced may be transferred, following mixing, from the body of rotation to outwardly located receptacles and/or containers which may be removed. Said receptacles may be standard laboratory receptacles such as Eppendorf cups, microwell plates and the like. In embodiments of the invention, a fluid output may be provided at a radially outer portion of one or more of the mixing chambers, which fluid output may be provided with a valve, so that the mixture in the mixing chamber may be transported out of the mixing chamber by subjecting the body of rotation to a rotational frequency at which the valve allows the mixture to pass.
Embodiments of the invention are suitable for diluting different starting solutions, depending on the application. The following solutions/mixtures to be diluted may be used for this purpose, among others:
Suspensions containing cells and cellular constituents, for determining the number of germs, for determining constituents and/or for establishing calibration standards.
Applications of embodiments of the invention are in the field of enzyme kinetics. Both the enzyme and the substrate as well as inhibitors or activators may be diluted and be mixed with one another in end cavities by the structures described. This enables determining Michaelis-Menten constants, turnover numbers, IC50 values or other typical characteristics of enzyme kinetics. Thus it is possible to accurately characterize the enzyme used, the substrate and the inhibitor or activator.
Applications of embodiments of the invention are in the field of immunoassay calibration, the antigen of the immunoassay being diluted. In this manner, the corresponding immunoassay may be calibrated, and the detection limit or the quantification limit may be determined.
Applications of embodiments of the invention are in the field of the most probably number for germs. Germs such as bacteria, viruses or fungi, for example, are diluted, and the dilutions are aliquoted in end cavities. If the end cavities have entities located therein which are capable of growing, this is detected by a change (e.g. change in color, clouding, etc.). By means of determining the most probable number, one may estimate, from the dilutions produced and from the individual positive sub-volumes, how many germs were contained in the initial mixture.
Applications of embodiments of the invention are in the field of the most probable number for nucleic acids. Nucleic acids such as DNA or RNA are diluted, and the dilutions are aliquoted in end cavities. There, a PCR is performed. If the corresponding nucleic acid is located in the end cavity, a positive signal will be produced. By means of determining the most probable number, one may estimate, from the dilutions produced and from the individual positive aliquots, how many nucleic-acid molecules were contained in the initial mixture.
Embodiments of the invention provide semi-automatic or fully automatic production of discrete dilutions within a cartridge by means of centrifugation, e.g. within a conventional laboratory centrifuge. Since no external equipment (laser, infrared radiator) is required for actuating valves, standard laboratory equipment (centrifuges) are also suitable for operating the cartridge, which has been confirmed experimentally. No specific processing equipment is required.
The concentrations of the dilutions produced and/or the implemented dilution factors Z may be changed both by the layout and by the structures implemented in the cartridge (on the manufacturing side) as well as by the sample volume of the solution A added by means of pipetting (on the user side). This has already been shown experimentally. Thus, the dilution factors Z of the dilution series may be changed by the user, even after manufacturing of the cartridges. Z=(VA+VB)/VA shall continue to apply in this context. VB is specified by the cartridge; VA can either be freely varied on the part of the user or is also specified by the cartridge. Therefore, one does not need different microfluidic layouts in order to implement different Zs, which imparts a high measure of flexibility to the overall system, which also has already been confirmed experimentally.
In particular, however, one may produce dilution series wherein the dilution stages may be implemented fully automatically and without the influence of manual pipetting errors. To this end, fluidic structures for defining the volumes of the solution to be diluted A and of the dilution solution B are integrated into the cartridge in addition to the inlets for said solutions A and B. The solutions then only need to be added in excess. In this case, a highly accurate dilution series which is almost free from any manual pipetting errors (except for the case where an insufficient amount is pipetted in) may be implemented. In such embodiments, the dilution factor Z may be predefined by the fluidics, so that there is no free choice of the dilution factors Z on the part of the user.
Embodiments of the invention thus provide a centrifugal-microfluidic structure which implements a dilution series semi-automatically or fully automatically. In this context, the dilution solution B is split up into individual volumes VB1 to VBn (with n>1). A solution to be diluted A having the volume VA is added and is diluted with VB1. From this dilution, a volume VAB1 is transferred and diluted with a volume VB2. Step by step, one volume VAB(i-1) at a time is transferred and diluted with a volume VBi, and, thus, a dilution series with Zi=((VAB(i-1)+VBi)/VAB(i-1))*Z(i-1) is produced, with i≦n and Z0=1. Said mixing and transferring may be performed both serially and in parallel until all of the dilutions of the dilution series have been produced.
Embodiments of the invention provide a centrifugal-microfluidic structure implementing a dilution series. The dilution solution B is split up into individual volumes VB1 to VBn (with n>1). A solution to be diluted A having the volume VA is added and diluted with VB1. From this, a partial volume VAB1=VA is again transferred into the next volume VB2. Step by step, one volume VAB(i-1)=VA at a time is transferred and mixed with a volume VBi. Said mixing and transferring is performed step by step until all dilutions have been produced. This results in a dilution series with Zi=((VA+VBi)/VA)*Z(i-1) with i≦n. With this layout, too, the user may change the dilution factors Zi of the dilution series by means of choosing the volume VA.
Embodiments of the invention provide a centrifugal-microfluidic structure implementing logarithmic dilution series. The dilution solution B is split up into individual volumes VB1 to VBn (with n>1), and all volumes be identical to VBi=VB1. A solution to be diluted A having the volume VA is added and diluted with VB1. From this, a partial volume VAB=VA is again transferred into the next volume VB2=VB1. Step by step, one volume VAB(i-1)=VA at a time is transferred and mixed with a volume VBi=VB1. Said mixing and transferring is performed step by step until all of the dilutions have been produced. This results in a dilution series with Zi=((VA+VB1)/VA)*Z(i-1) with i≦n. This results in a Zi=((VA+VB1)/VA)̂ i, which corresponds to a logarithmic dilution series, with a dilution of ((VA+VB1)/VA) between individual concentrations. In this layout, too, the user may change the dilution factors Zi of the dilution series by means of choosing the volume VA.
In embodiments of the invention, the volume VBi is specified by microfluidics, whereas the volume VA is determined by the user.
In embodiments of the invention, the volume VA cannot be influenced by the user. The cartridge exhibits one or more inlets so as to therewith implement different dilution factors Z for advantageously logarithmic dilution series.
Embodiments of the invention comprise a rotating substrate having a plurality of microfluidic structures (fill-in chambers, mixing chambers, possibly siphons, possibly aliquoting structures, possibly passive valves). (a1) A fluidic channel connects one of the fill-in chambers for the dilution solution to a plurality of fluidic “fingers” having defined volumes. (a2) Said fingers may split up the initial amount of the solutions from the fill-in chamber into several sub-volumes. The supernatant of the solution is transferred into a supernatant chamber. Each of the fingers is connected to one mixing chamber in each case. As an alternative to (a1) and (a2), the fill-in chamber for the dilution solution may also be directly connected to the first mixing chamber m1. Portioning may also be effected by serially transferring the supernatant of the solution into the respectively neighboring mixing chamber via the capillary siphons. A second fill-in chamber for the solution to be diluted is connected to one of the mixing chambers. A suitable fluidic connection is provided between neighboring mixing chambers in each case, for example a capillary siphon, so as to initially allow mixing and, subsequently, transferring of a portion of the mixture into the next mixing chamber.
In embodiments of the invention, the volumes of the dilution series are transferred from the cartridge to external containers. In embodiments of the invention, the external containers may be removed from the cartridge. In embodiments of the invention, the containers are standard laboratory containers such as Eppendorf cups, for example. In embodiments of the invention, the containers are microwell plates or parts of microwell plates. In embodiments of the invention, they are containers for nucleic-acid analytics or immunoassays. In embodiments of the invention, the volumes of the dilution series are further processed on the cartridge. In embodiments of the invention, the volumes are transferred into such cavities on the cartridge which are located further outward. In embodiments of the invention, the volumes are aliquoted and transferred, in each case, to one or more cavities on the cartridge which are located further outward. In embodiments of the invention, prior to or following transferral of the volumes of the dilution series, one or more further solutions are fed into the cavities located further outward. In embodiments of the invention, the solutions employed are an enzyme, a substrate, an inhibitor or an activator. In embodiments of the invention, the solutions employed are a nucleic acid or a solution containing nucleic acids. In embodiments of the invention, the solutions employed are a solution of molecules, emulsions or suspensions. In embodiments of the invention, the solutions employed contain germs (bacteria, viruses, fungi). In embodiments of the invention, the solutions employed contain particles. In embodiments of the invention, the structure is used for determining biochemical quantities and characteristics. In embodiments of the invention, the solution to be diluted contains nucleic acids, and the initial concentration of the nucleic acid is determined. In embodiments of the invention, the solution to be diluted contains proteins, and the initial concentration of the proteins is determined. In embodiments of the invention, the solution to be diluted contains germs, and the initial concentration of the germs is determined. In embodiments of the invention, the solution to be diluted contains an antigen or an antibody, and characteristic values/indices of an immunoassay are determined. In embodiments of the invention, the solution to be diluted contains an enzyme, and characteristic values of enzyme kinetics are determined (such as the Michaelis-Menten constant, turnover number, kinetics constants and conversion rates). In embodiments of the invention, the solution to be diluted contains an inhibitor or an activator, and characteristic values of enzyme kinetics are determined, such as the IC50 value. In embodiments of the invention, the solution to be diluted contains particles, and the initial particle concentration is determined.
Embodiments of the invention enable numerous substantial advantages over known approaches of producing dilution series.
While this invention has been described in terms of several embodiments, there are alterations, permutations, and equivalents which fall within the scope of this invention. It should also be noted that there are many alternative ways of implementing the methods and compositions of the present invention. It is therefore intended that the following appended claims be interpreted as including all such alterations, permutations and equivalents as fall within the true spirit and scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102011077199.9 | Jun 2011 | DE | national |
