DE102006020716A1 - Microfluidic-processor for use in e.g. biomedical industry, has integrated polymer network-based units that are interconnected in logical base configuration and controllable over interfaces by superior system - Google Patents

Abstract

The processor has integrated controllable units for the operation of process media, and integrated polymer network-based units, whose relevant sensibilities modify it through specific modifiable surrounding factors for the media operation. The polymer network-based units are made up of hydraulic gel. The surrounding factors are directly or indirectly controllable over electrical or electronic compatible interfaces (6a, 6b) by electrical variables. The polymer network-based units are interconnected in a logical base configuration and controllable over the interfaces by a superior system.

Description

description the invention

Technical area

The The invention relates to an electronically controllable microfluidic processor with integrated controllable elements.

In the (bio) chemical, pharmaceutical and biomedical industries There is a growing need for miniaturization of fluidic process technology. This request is met by microfluidic devices. Realize these devices through feature integration more or less elaborate Biological, biochemical or chemical processes, that's what we call it from microfluidic processors or also "Labs on a Chip" (LOC), "Chip Labs" or "Micro Total Analysis Systems "(μTAS).

The LOC concept offers many advantages. The reduction of Fluid volumes allows the analysis of the smallest sample quantities and an economical handling with reagents and samples that are often valuable, rare, harmful or are dangerous. This also makes higher throughputs achievable because, due to the small quantities, shortened provision, mixing and and reaction times are needed with minimal energy requirements. Due to lower system response times can also facilitate the process control.

All in all enable LOC builds significant process rationalizations by reducing the process time cut considerably and thus the possible Increase throughput, and the quantities of needed Reduce media (subjects, analytes, reagents, auxiliary media).

In spite of The obvious benefits are only realized in exceptional cases LOC applications. The reasons for this are to be found in which sub-processes currently miniaturized (on-chip integrated).

State of the art

The typical structure of biological, biochemical or chemical processes includes the tasks of sample preparation, handling and reaction or analysis in specific forms and combinations. Currently mainly takes place an on-chip integration of sample preparation and reaction or analysis. The results of the rationalization of these sub-processes However, the resulting economic benefits proved in the Normally not as sustainable.

enormous Rationalization potential offers sample handling because they is particularly time consuming and labor intensive. Because of their problematic on-chip integration is the sample handling currently manual or with certain special equipment, such as diluters, syringe pumps, pipetting devices, etc., performed outside the chips.

The quite diverse available miniaturizable electronically controllable fluidic drives (Pumps) and switching elements (valves) but have such disadvantages, that they either can not be economically integrated into a lab-on-a-chip setup are or over inadequate Have functional properties.

Fluidic elements based on solid-state actuators, such as piezoactuators [ US 5,224,843 , US 2003/0143122] and shape memory actuators [ US 5,659,171 ], although they can be easily miniaturized as individual elements, but have a complicated structure, are specified on certain, usually not plastic-based, materials and must therefore be made separately. A possible hybrid integration (eg sticking of the elements to the LOC) is generally uneconomical.

Transducer elements, which are based on changes in the state of matter, can be integrated into the layout of the channel structure supports with sometimes minor interventions and are therefore usually compatible with the production process of the plastic molded parts of the channel structure support. For example, fuses [R. Pal et al., Anal. Chem. 76 (2004) 13, p. 3740-3748] and freezing elements [ US 6,536,476 ] as well as thermal bubble generators [ US 6,283,718 ] known.

Indeed own aggregate state changeable Converter some inadequate Use properties. Leave except the bubble generators the converters do not use actorically, so their application limited to switching elements is. Due to the necessary heat fluctuations are the process media considerable thermal stress, in freezing elements also exposed to mechanical stress.

converter with gas formation are suitable for Many microfluidic processes are not, because most are sensitive to gas bubbles are.

Presentation of the invention

task The invention is to provide a LOC device, which with an economically viable manufacturing cost produced and which sample handling in on-chip integrated form contains. Leave it the sample preparation and / or Sample reaction or sample analysis with sample handling in one Combine LOC structure.

According to the invention Problem solved by the features specified in claim 1. advantageous Embodiments are specified in claims 2 to 20.

The The basis of the invention are hydrogels. Hydrogels are Polymeric networks, the influence of aqueous swelling agents their volume, their Change strength and other properties. These polymer networks can be chemically linked to each other according to the type of polymer chain linkage and physically crosslinked polymer networks or hydrogels. For chemically crosslinked polymer networks, the individual polymer chains by covalent (chemical) compounds irreversibly with each other connected. In physically crosslinked polymer networks, the polymer chains are through physical interactions are interconnected, mostly solved again can be.

A particular class of hydrogels are those of "smart" character. Perform smart hydrogels as chemically crosslinked polymer networks reversible discontinuous Volume phase transitions at Influence of certain environmental parameters. As physically crosslinkable Remove or crosslink polymer networks (also yellowing or gelling) between the maximum networked and the fully resolved state. The environmental quantities can be electric field quantities, radiation, Temperature, ion and substance concentrations. An electric or electronics-compatible controllability of smart hydrogels is with direct electrical quantities and Field sizes currently problematic, since the corresponding effects regarding their efficiency are still insufficient and have a limited reversibility. With thermo-electronic interfaces, however, can be temperature-sensitive Hydrogels are very easy to control because of the necessary heat input or the withdrawal of heat technically uncomplicated feasible.

A first advantage of hydrogels over other transducers is the enormous variety of active functions that can be realized with them. They can be used as electronically controllable fluidic elements in the form of switching elements [ DE 101 57 317 A1 ], fluidic drives, recording and dispensing systems of active substances and other substances, but also for enclosing / fixing or releasing objects (eg by gelling or dissolving) find use. Another advantage of these effect carriers is the easy manufacturability of hydrogel structures. Since hydrogels are themselves plastics, they can be manufactured using the typical procedures for this class of substances. Since most functional elements also have the same or similar basic structures, the hydrogel elements can be produced directly on the channel structure carriers with one or only a few additional manufacturing steps.

Ways to execute the invention

The Invention is based on embodiments be explained in more detail, wherein the reference numerals used have the same meaning. In the associated Drawings show:

1a the diagram of a channel structure carrier of an electronically controllable microfluidic processor based on temperature-sensitive smart hydrogels, the z. B. can be used for the monitoring of bioreactors on the basis of the expression levels of selected growth marker application,

1b a basic structure of an electronically controllable microfluidic processor according to 1a with a separate actuator structure carrier in cutaway view,

2a a basic structure of an electronically controllable microfluidic processor with a separate Aktorstrukturträger in section,

2 B a basic structure of an electronically controllable microfluidic processor without Aktorstrukturträger in sectional view,

3 the swelling behavior of chemically cross-linked, temperature-sensitive, smart hydrogels as a function of the temperature,

4 the principle of operation via thermoelectronic interfaces driven hydrogel elements, wherein

4a and 4b show a positive displacement micropump based on a chemically cross-linked hydrogel with UCST characteristics,

4c and 4d a storage unit based on a chemically cross-linked hydrogel with LCST characteristics,

4e and 4f show a temperature-activatable gelling element for trapping particles,

4g and 4h show a temperature-activatable valve based on a physically crosslinked hydrogel and

5 a possible circuit diagram of a LOC setup for standard biochemical and medical applications based on polymerase chain reactions.

Based on 1a For example, a procedure which can be implemented with the microfluidic processors according to the invention will be explained by way of example. With the 1b such as 2a and 2 B possible design principles and manufacturing processes are presented. 3 illustrates the useful effects of hydrogels as controllable elements, while 4a to 4h introduce the functionality of some controllable hydrogel elements. The 5 demonstrates further typical applications of the LOC according to the invention.

This in 1a The circuit diagram of an LOC channel structure shown is suitable for a whole range of standard chemical, biotechnological and medical applications. Its functionality is explained by the online determination of the enzyme activity (laccase activity) of a bioreactor. In two hydrogel pumps 15a and 15b are 0.05M malonate buffer of pH 5.0. Another hydrogel pump 15c contains a substrate 2MM 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution in a 0.05M malonate buffer of pH 5.0. In a fourth hydrogel pump 15d is a sample of a Bioreak gate product laccase, which z. B. was removed from the current reactor operation.

By simultaneously pumping the hydrogel pumps 15a and 15c as well as the hydrogel pumps 15b and 15d be buffer and substrate (hydrogel pump 15a with hydrogel pump 15c ) as well as buffer and sample (hydrogel pump 15b with hydrogel pump 15d ) through mixed meander 18a . 18b mixed and on hydrogel pumps 16a to 16f distributed, being in the hydrogel pumps 16a to 16c one buffer sample mixture each and in the hydrogel pumps 16d to 16f a buffer substrate mixture is presented. All hydrogel pumps 15a - 15d and 16a - 16f are based on controllable polymer networks.

• – 16a und 16f (Mischungsverhältnis 2:1)
• – 16b und 16e (Mischungsverhältnis 1:2)
• – 16d und 16c (Mischungsverhältnis 1:1)

werden Puffer-Substrat und Puffer-Probe durch weitere Mischmäander 18c bis 18e gemischt und in Reaktions- bzw. Analyseeinheiten 17a bis 17c transportiert. In diesen lässt sich die Enzymreaktion mit optischen Analysenmethoden verfolgen. Als einfachste optische Analyseeinheit kann ein nicht näher dargestellter lichtempfindlicher Widerstand (LDR – light dependent resistor) dienen, der eine einfache Ja-Nein-Antwort (Enzymaktivität vorhanden bzw. nicht vorhanden) gibt. Enzymkinetische Parameter lassen sich mit lichtspektroskopischen Methoden (z. B. UV-VIS Spektroskopie) ermitteln.By simultaneously pumping the hydrogel pumps:

• - 16a and 16f (Mixing ratio 2: 1)
• - 16b and 16e (Mixing ratio 1: 2)
• - 16d and 16c (Mixing ratio 1: 1)

Buffer substrate and buffer sample are passed through further mixing meanders 18c to 18e mixed and in reaction or analysis units 17a to 17c transported. In these, the enzyme reaction can be followed by optical analysis methods. The simplest optical analysis unit can be a light-dependent resistor (LDR), not shown in detail, which gives a simple yes-no response (enzyme activity present or absent). Enzyme kinetic parameters can be determined by light-spectroscopic methods (eg UV-VIS spectroscopy).

By Parallelization of several such LOCs can be a shorter timing and thus a continuous enzyme activity control, which is the enzyme production control corresponds to be achieved.

The Microfluidic processors according to the invention can have two basic designs.

The in 1b The microfluidic processor shown has a separate swelling agent circuit in an actuator structure carrier 2 , in which, depending on their mode of operation, a large part of controllable hydrogel elements 4a . 4b located. This processor type consists of three functional levels. A channel structure carrier 1 contains fluidic channels for the process medium as well as chambers for the individual functional elements. The actuator structure carrier 2 with the swelling agent circuit is located above the channel structure carrier 1 , The channel systems of both structural beams 1 . 2 are by one or more flexible membranes 3 separated from each other. Serves for the hydrogel elements 4a . 4b the process medium as the swelling agent, the channel systems of channel structure carrier 1 and actuator structural support 2 be connected to each other.

As 1b and 2a show schematically contains the Aktorstrukturträger 2 also the hydrogel elements 4a . 4b , The hydrogel element 4a provides in its secondary function as Quellmittelreservoir the necessary swelling agent, while the hydrogel element 4b uses the process medium as the swelling agent. The plane of the actuator structure carrier 2 is through a structural support 5 covered for electrical control and carries heating structures 6a . 6b as well as their electrical contacts.

The hydrogel element 4a has the function of a fluidic drive. Will it with the heating elements 6a driven from left to right, it starts, with solvent uptake from the memory locations of Aktorstrukturträgers 2 to swell, making it the flexible membrane 3 from left to right in a pump chamber 7 of the channel structure carrier 1 deflects and the local process medium from the pump chamber 7 repressed. Because the pump chamber 7 on the left side through the first segment of the hydrogel element 4a is closed, the process medium is through a breakthrough 8th to the acting as a valve hydrogel element 4b promoted. Is this through the heating structure 6b so driven that it is swollen, the process medium can open the hydrogel element 4b happen. The reference numerals 11 designate the inputs / inlets of the pump chambers 7 for the process medium to be pumped, the reference numerals 10 the exits / outlets. With the help of hydrogel valves 19a - 19c the flow of process medium can be passed through the hydrogel pump unit 16a - 16f , Reaction or analysis units 17a - 17c and meanders 18a - 18e to change.

The hydrogel element 4b , which is actually in the process cycle, is from two aspects by means of the breakthroughs 8th in the plane of the Aktorstrukturträgers 2 laid. On the one hand, all hydrogel elements can be used 4a . 4b in the plane of the Aktorstrukturträgers 2 on the other hand, all hydrogel elements 4a . 4b from a single side through the heating structures 6a . 6b of the structural support 5 be controlled.

The structure of the microfluidic processor after 2 B has the same functionality as the one in 2a shown. However, it is simpler since it does not require a separate source agent circuit. The hydrogel elements 4a . 4b use the process medium for swelling. The hydrogel elements 4a . 4b are directly in channels 1a of the channel structure carrier 1 placed and sit on the structural support 5 for the heating structures 6a . 6b , The channel structures are covered by a cover 9 closed, with this cover 9 functional areas such as an elastic spring energy storage 9a can have.

The production of the microfluidic processors according to the invention can be used for the actuator and channel structure carriers 1 . 2 done with the usual methods of mass production of plastic moldings, such as injection molding, hot stamping or the like. Suitable materials are those customary for microfluidics, z. As polycarbonate (PC), cycloolefins (COC), polyamides (PA), polyester (PES), polystyrene (PS), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA) or polytetrafluoroethylene (PTFE).

For the production of small series or unique pieces are methods of rapid prototyping, z. B. milling the channel structures. A very simple variant for the production of small series is the master molding of PDMS. These are negative structures of the channel structure support 1 and the Aktorstrukturträger 2 Photolithographically produced in silicon wafers and then coated with Teflon by sputtering to obtain a good moldability. Thereafter, the PDMS is placed on the molds and cured for one hour at 100 ° C.

The flexible membranes 3 ; 9a can also be prepared in PDMS by spin coating. The layer thicknesses can be adjusted very well with this method between about 15μm to 100μm. Films of the required thicknesses can also be purchased commercially.

The electrical control of the microfluidic processors can also be manufactured with the known methods for the production of wiring carriers. The structural support 5 with the heating structures 6a . 6b can z. B. can be realized with conventional methods and materials of printed circuit board technology. For this purpose, for example, a polyester film laminated with 35 microns thick copper foil, provided with a mask and then wet-chemically structured.

The structural support 5 can subsequently with heating structures 6a . 6b , z. B. in SMD form, are equipped by wave soldering. Alternatively, the heating structures 6a . 6b also be produced by screen printing corresponding thick-film conductive pastes.

The thick-film conductive paste screen printing is also an independent production possibility of electrical control. The printed conductors and heating resistors can be applied directly to the structural support 5 , or, if it is an integral part of the channel structure carrier 1 or the Aktorstrukturträger 2 is, also directly on the actuator or channel structure support 1 . 2 to be printed.

The individual layers of the LOC can glued together, welded or frictionally joined. PDMS molded parts can be, for example, after a low pressure oxygen plasma treatment Bond very well with PDMS as adhesive and subsequent temperature hardening.

Hydrogel elements can be produced by various methods. For structuring hydrogel layers, crosslinking photopolymerization [DJ Beebe et al., Nature 404 (2000), pp. 588-590] and photocrosslinking [A. Richter et al., J. Microelectromech. Syst. 12 (2003) 5, pp. 748-753]. Furthermore, the molding with subsequent polymerization [ME Harmon et al., Polymer 44 (2003), pp. 4547-4556] as well as the generation of hydrogel particles [K. -F. Arndt et al., Polym. Adv. Technol. 11 (2000), 496-505].

smart Hydrogels are currently difficult to handle directly with electrical Control sizes. temperature Sensitive Hydrogels can but very easy about a thermal interface can be controlled electrically and are therefore used as electronically controllable elements. The Property profile of electronically compatible hydrogels and their applications are therefore represented by temperature-sensitive hydrogels.

Chemically cross-linked, temperature-sensitive smart hydrogels can exhibit two modes of volume-phase transition (see 3 ). Most temperature-sensitive smart hydrogels have a Lower Critical Solution Temperature (LOST) characteristic, ie, they are swollen at low temperatures and escape when the phase transition temperature is exceeded. The best-known LOST-characteristic hydrogel, poly (N-isopropylacrylamide) (PNIPAAm), has one Volume phase transition temperature of 32.8 ° C. The position of the phase transition or switching temperature of NIPAAm-based hydrogels can be adjusted by copolymerization and variation of the synthesis parameters in a range from + 5 ° C to about 60 ° C.

Possible synthesis and structuring methods of PNI-PAAm-based hydrogels are e.g. B. in A. Richter et al., J. Microelectromech. Syst. 12 (2003) 5, p. 748-753, described.

Similar good phase transition properties with LCST characteristics are also available in 3 shown poly (methyl vinyl ether) (PMVE). The location of the phase transition temperature of this hydrogel is 37 ° C. PMVE is cross-linked with high-energy radiation (electron irradiation, gamma irradiation) [K. -F. Arndt et al., Macromol. Symp. 164 (2001), pp. 313-322]. Other representatives with LCST characteristic are based z. On hydroxypropylcellulose [B. Kabra et al., Macromolecules 31 (1998), pp. 2166-2173].

Temperature-sensitive hydrogels with an Upper Critical Solution Temperature (UCST) characteristic swell when the phase transition temperature is exceeded and are swollen at low temperatures. A hydrogel with UCST behavior is the copolymer of hydroxyethyl methacrylate (HEMA) and acetoacetoxyethyl methacrylate (AAEM) [V. Boyko et al., Macromol. Chem. Phys. 204 (2003), pp. 2031-2039]. In a 60wt%: 40wt% ethanol / water mixture, its phase transition temperature is 31.5 ° C (see 3 ).

Physically Crosslinkable polymer networks form when exposed to certain Environment sizes off a polymer solution by gelling, by the individual polymer molecules to a three-dimensional Link network. This process is usually reversible (sol-gel transition behavior). By acting the critical environment size the linking points again separated and the polymer molecules are dissolved before.

typical temperature-switchable physically crosslinkable hydrogels are gelatin, Pectins and agarose. Your sol-gel transition temperatures can be adjusted by various measures between about 15 ° C and 95 ° C. To these and other physically networkable polymer networks offers [K. Nijenhuis, Thermoreversible Networks, Adv. Polym. Sci. 130 Springer-Verlag Berlin, Heidelberg, New York 1997]. There are also polymer networks that are chemically networked and physically have crosslinkable components. By acting on the physically crosslinkable components leave additional Forming or canceling networking points, so that the degree of crosslinking and thus also the degree of swelling can be influenced.

chemical Change networked smart hydrogels at the phase transition a whole lot of properties. For hydrogel elements are mainly the changes their volume, strength, permeability (mass permeability) or diffusion properties and the storage capacity available. By using the volume change let For example, valves, fluidic drives (pumps) and actuators realize that via electronics compatible Interface sizes are controllable.

strength and compliance changes can be z. For example switchable barriers and stops use.

amendments the permeability or the diffusion properties are also for switching or influencing of material flows available.

Physically Crosslinkable hydrogels can be used in the Crosslinked state also change volume, strength and permeability. When However, purely physically cross-linked hydrogels do not have a volume phase transition behavior. but change These properties spontaneously when swelling from the dry or drying out. But they can be in their networking state switch, d. h., you can create a hydrogel solid, dissolve or influence in its swelling or strength properties.

These Properties can be for once activatable hydrogel elements, eg. In the form of valves, moldings with blocking, locking or positioning function, which use the Electron-compatible interface sizes changed in their strength, dissolved or be generated by gelation. The gelation or dissolution process can also be used for the controlled inclusion or release of liquids, but also solids (eg particles, cells, etc.).

The 4a to 4h illustrate the principal modes of operation of some hydrogel elements, as they are needed for the LOC structures according to the invention.

In the 4a and 4b shown displacing acting micropump, as z. B. for the hydrogel pumps 15a - 15d and 16a - 16f to 1a are based, for example, on the chemically crosslinked temperature-sensitive UCST hydrogel hydroxyethyl methacrylate (HEMA) and acetoacetoxyethyl methacrylate (AAEM). That in the actuator structure carrier 2 stocked swelling agent 4d is a 60wt%: 40wt% ethanol / water mixture. at Room temperature is this hydrogel 4d unswollen. The pump chamber 7 is filled with the process medium to be pumped. To make a material flow towards an exit 10 To be able to generate, the pump chamber must 7 first at the entrance 11 be closed. This is the first heating element 6 operated, so that the phase transition temperature of the hydrogel is exceeded, the hydrogel 4c swells and by deflection of the membrane 3 the entrance 11 closes ( 4a ). Then the heating elements 6 sequentially towards the exit 10 pressed, leaving the pump chamber 7 by the progressively swelling hydrogel 4c and the corresponding deflected membrane 3 towards the exit 10 is emptied ( 4b ).

In the 4c and in 4d shown storage unit is based z. On the chemically cross-linked hydrogel PNIPAAm. The storage unit can by swelling of the hydrogel element 4c be loaded in a process medium to be stored. Will the swollen hydrogel element 4c ( 4c ) through the heating structure 6 heated above its phase transition temperature of about 33 ° C, it ( 4d ) and puts an active ingredient 12 in a canal structure 13 of the channel structure carrier 1 free. The construction of 4c and 4d can also be used to realize a valve function. At room temperature, this is the PNIPAAm element 4c to the cover 9 swelled and completely closes the valve seat. By heating with the heating element 6 It swells over the phase transition temperature, so that the valve seat opens.

4e and 4f show a temperature-activatable gelling element for enclosing particles 14 , In the canal structure 13 is located next to the particles 14 a gelable solution 4e , If the particle-containing solution by a Peltier element 6 cooled to a temperature below a gelation temperature, the solution solidifies 4e to a gel 4f so that the particles 14 are locally fixed by inclusion. To release the particles 14 can the gel 4f be resolved again by exceeding the gelation temperature. The gelation temperatures can be adjusted by appropriate choice of gelatin or polysaccharide between about 30 ° C and 95 ° C.

The temperature-activated valve based on a physically cross-linked hydrogel according to 4g and 4h For example, one may be in the channel structure 13 of the channel structure carrier 1 embedded hydrogel valve 19a - 19c to 1a be. To a filling of the reaction units 17a - 17c ( 1a ) to ensure with the process medium, the hydrogel valves need 19a - 19c be open at first. This can be achieved by using the hydrogel valves 19a - 19c initially in a dry or moistened state. Are the reaction chambers 17a - 17c filled, the process medium will aspire to the output and thereby reach the valve seat. The hydrogel 4f ( 4g ) begins to swell and the hydrogel valves 19a - 19c close the channels 13 ( 4g . 4h ). The hydrogel opens to open 4f ( 4g ) is heated above its gelling temperature so that it dissolves (hydrogel state 4g in 4h ) and the respective valve 19a - 19c ( 1a ) is open.

5 shows the circuit diagram of a LOC structure for standard biochemical and medical applications based on polymerase chain reaction (PCR). For the polymerase chain reaction, a master mix, a template DNA for the control reaction (template DNA1) and a template DNA for the actual PCR reaction (template DNA2) are presented.

• – 4μl 10x Puffer mit 25mM MgSO₄(10x Pfu-Polymerasereaktionspuffer, Fermentas Life Science)
• – 0,2μl Forward Primer (FP) Endkonzentration 1μM
• – 0,2μl Reverse Primer (RP) Endkonzentration 1μM
• – 3,2μl dNTP (Desoxyribonucleosid Triphosphat Mischung; Fermentas Life Science) Endkonzentration 0,4mM each
• – 0,8μl Pfu-DNA-Polymerase (2,5 U/μl; Fermentas Life Science) und 11,6μl H₂O (molecular biology grade).

The master mix may, for example, have the following composition:

• - 4μl 10x buffer with 25mM MgSO ₄ (10x Pfu polymerase reaction buffer, Fermentas Life Science)
• - 0.2μl forward primer (FP) final concentration 1μM
• - 0.2μl reverse primer (RP) final concentration 1μM
• - 3.2μl dNTP (deoxyribonucleoside triphosphate mixture; Fermentas Life Science) final concentration 0.4mM each
• - 0.8μl Pfu DNA polymerase (2.5 U / μl, Fermentas Life Science) and 11.6μl H ₂ O (molecular biology grade).

If required, the H ₂ O can also be proportionally substituted by additives such as DMSO, glycerol and the like (eg at high GC content).

For multiple Applications will specify the volume specifications for the master mix with the number of Applications multiplied. The master mix prepared in this way can be used in a chilled Storage vessel (4 ° C) outside provided by the LOC become. The same applies to the template DNA's.

The hydrogel pumps 15b and 15c are loaded with 10μl each of a master mix. In hydrogel pump 15a are 10μl template DNA1 (plasmid, about 100ng in H ₂ O - molecular biology grade) for the PCR control reaction. Hydrogel pump 15d contains 10μl template DNA2 (plasmid, ca. 100ng in H ₂ O - molecular biology grade) for the PCR reaction.

• – 5min bei 94°C (initiale Templat-Denaturierung)
• – 30 Zyklen mit jeweils 30s bei 94°C (Templat-Denaturierung) und 30s bei 55°C (Primer-Annealing)
• – 4min bei 72°C (Primer-Elongation).

By simultaneously pumping the hydrogel pumps 15a . 15b . 15c . 15d Add 10μl of master mix with 10μl of template DNA1 (hydrogel pump 15a with hydrogel pump 15b ) and 10μl master mix with 10μl template DNA2 (hydrogel pump 15c with hydrogel pump 15d ) through the meanders 18a and 18b mixed and after opening the hydrogel valves 19a and 19b to PCR chambers 20a respectively. 20b transported. In the chambers 20a and 20b The polymerase chain reactions take place, whereby the temperature programs can be realized by an external thermocycler. A possible PCR temperature program can proceed as follows:

• - 5 min at 94 ° C (initial template denaturation)
• 30 cycles at 94 ° C for each 30s (template denaturation) and 30s at 55 ° C (primer annealing)
• - 4 min at 72 ° C (primer elongation).

Finally done an incubation of 5 min at 72 ° C to complete the primer elongation.

After the PCR, by simultaneously pumping the hydrogel pumps 15a - 15d and opening the hydrogel valves 19c and 19d the PCR products in gel electrophoresis chambers 21a . 21b (usually agarose gel electrophoresis), taking on the gel from the gel electrophoresis chamber 21a the PCR products of the control reaction and on the gel from the gel electrophoresis chamber 21b the PCR products are applied to the actual PCR.

Optionally, the PCR products can also be at the exit 10a or exit 10b for external processing.

By applying a voltage (field strength 10V / cm), the PCR products in the gel electrophoresis chamber 21a respectively. 21b separated electrophoretically and can be at the output 10c or exit 10d z. B. are provided for the external fluorescence analysis. 11 again denote the inputs to the hydrogel pump chambers 15a - 15d ,

The temperature control of the LOC need not necessarily be done with an integrated electrical-resistive or light-based control. It is also possible to control the LOC in their process flow by external devices into which they are incorporated in whole or in part. Such devices may be, for example, correspondingly modified PCR thermal devices, thermostats, thermal cyclers, thermal cabinets or thermal baths capable of realizing given temperature programs. The microfluidic processor can be holistically subjected to the temperature program. However, the heating can also take place locally at certain sections of the LOC and in a specific chronological order. The local sections conveniently comprise elements that are to be operated simultaneously. For the layout after 5 recommend z. B. three different controllable local sections. The first area includes the hydrogel pumps 15a . 15b . 15c . 15d as well as the hydrogel valves 19a . 19b , the second area the PCR chambers 20a . 20b , the third area the hydrogel valves 19c . 19d , The temporal behavior of the hydrogel-based individual elements can also be determined by appropriate definition of their activation temperatures. As the temperature rises, first the elements with the lowest activation temperatures are activated, finally those elements with the highest activation temperatures. To improve heat transfer, the areas to be heated may be designed to have improved thermal conductivity. This is z. B. by reducing the material thicknesses, but also by using materials with appropriate heat conduction properties.

By the adaptation of the master mix composition (several primers) and the template DNA (sample DNA) leaves this basic structure is transferred to a variety of DNA analysis methods. There are all applications, eg. B. DNA fingerprint (paternity test), Virus analysis and others based on the principle of PCR-DNA analysis, feasible on a LOC.

By adapting the architecture (eg additional pumps, mixing chambers, reaction chambers, etc.) even more complex processes, such as those required for reverse transcriptase PCR (RT-PCR), can be realized on an LOC. For this, only an RT master mix has to be put together and another temperature program has to be integrated before the PCR (two-step RT-PCR method). Alternatively, of course, the in 5 described structure using a one-step RT-PCR method use.

• – Total RNA oder mRNA (prokaryontisch oder eukaryontisch) mit der Targetsequenz
• – Reverse Transkriptase(n)
• – dNTPs (vgl. PCR)
• – oligo(dT)-Primer (alternativ: sequenzspezifische- oder random-hexamer Primer) und
• – RNase-Inhibitor in dem zugehörigen Transkriptasepuffer.
• Die cDNA-Synthese findet bei 37°C bis 50°C statt.

The basic composition of an RT master mix for a two-step RT-PCR method shows the following example:

• Total RNA or mRNA (prokaryotic or eukaryotic) with the target sequence
• Reverse transcriptase (s)
• - dNTPs (see PCR)
• Oligo (dT) primer (alternatively: sequence specific or random hexamer primer) and
• RNase inhibitor in the associated transcriptase buffer.
• The cDNA synthesis takes place at 37 ° C to 50 ° C.

Further applications the invention

The examples described represent a variety of other possible applications of the hydrogel-based microfluidic processors of the present invention. By adapting the processor architecture (eg additional pumps, mixing chambers, reaction chambers, etc.) even more complex processes on an LOC can be realized the. It can be miniaturized and automated manifold pipetting and analysis tasks, which not only causes a significant cost and time reduction, but also the process quality z. B. significantly improved by reducing the pipetting error. The LOCs can be used for one-off (disposable), continuous or online tasks. Through miniaturization and automation, a mobile, location-independent use of LOCs is possible.

in the For biotechnology, for example, they are responsible for enzyme reactor and bioreactor monitoring et al For Prokaryotes, eukaryotes, yeasts and fungi. As online, continuous or one-time measurement (also for single use) a rapid screening of the activity of enzymes of different enzyme classes feasible. By combination Multiple LOCs can be used for multi-rapid screening. Also the online expression level control as well as the online tracking of growth curves and growth factors, too in addition or combination for flow cytometry can with the LOC according to the invention carried out become.

In the environmental or water analysis can be z. B. Microorganism analyzes by identifying and assigning an activity profile, but also quick tests to determine the water quality [COD (chemical oxygen demand), BOD (biological oxygen demand), Heavy metals, nitrate, nitrite, etc.].

in the medical technology area the LOC technology according to the invention for example for online cell culture monitoring (eukaryotes, humane Cell lines, etc.) by viability tests [Z. B. WST-1 and MTT test (conversion of a tetrazolium salt in formazan, e.g. B. 4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolium] -1,3-benzenedisulfonate) or LDH test (lactate dehydrogenase test)], etc.

In order to is a cell grade, Batch and passage control for human cell lines, etc. possible.

Also On-chip blood tests to determine part of the main blood cell parameters, z. Blood sugar, pH, lactate, minerals, creatine, hormones, enzymes, Leukocytes, erythrocytes and others, disease markers, evidence of Reactive Oxigen Toxic Substances (ROTS oxidative stress), etc. are realizable.

1

Channel structure support

1a

channel in the channel structure carrier

2

Aktorstrukturträger

3

flexible membrane

4a

hydrogel element a for one Hydrogel pump

4b

hydrogel element b for one Hydrogel valve

4c

swollen hydrogel element

4d

entquollenes hydrogel element

4e

linkable polymer solution

4f

gelled hydrogel element

4g

dissolved hydrogel element (Polymer solution)

5

Structural support of electrical control (printed circuit board)

6

resistive heating element; Peltier element

6a

heating structure

6b

heating structure

7

pump chamber

8th

breakthrough

9

cover

9a

elastic Cover area of the cover

10 10a-10d

Output / outlet

11

Input / inlet

12

Unlocked Active substance in the process medium

13

channel structure

14

particle

15a-15d

Hydrogel pump for fluid intake

16a-16f

Hydrogel pump for variation of mixing ratios

17a-17c

reaction or analysis units

18a-18e

Mischmäander

19a-19d

Hydrogel valves

20a, 20b

PCR chamber

21a, 21b

Gel Electrophoresis

Claims (20)

Microfluidic processor with integrated controllable elements for handling process media, characterized by on polymer networks ( 4a - 4g ) based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ), whose media handling relevant sensitivities are changed by specifically changeable environmental variables, whereby these environment variables are transmitted via electrical or electronically compatible interfaces ( 6 . 6a . 6b ) are directly or indirectly controllable by electrical quantities, the integrated polymer network-based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) are interconnected in a logical basic configuration in terms of hardware and via the interfaces ( 6 . 6a . 6b ) are controllable by a higher-level system. Microfluidic processor according to claim 1, characterized in that the polymer networks ( 4a - 4g ) of the polymer network-based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) are temperature sensitive. Microfluidic processor according to claim 1, characterized in that that the specifically changeable environment size the temperature is. Microfluidic processor according to claim 1, characterized in that the electrical or electronics-compatible interface ( 6 . 6a . 6b ) is a thermal-electrical interface, which converts the electrical variables by heat resistive resistors, Peltier elements or light or radiation exposure to heat. Microfluidic processor according to claim 1, characterized in that the polymer networks ( 4a - 4g ) of the polymer network-based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) consist of chemically cross-linked, temperature-sensitive hydrogels with a Lower Critical Solution Temperature characteristic, in particular hydrogels based on N-isopropylacrylamide and poly (methyl vinyl ether). Microfluidic processor according to claim 1, characterized in that the polymer networks ( 4a - 4g ) of the polymer network-based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) consist of chemically crosslinked temperature - sensitive hydrogels with upper critical solution temperature characteristics, in particular hydrogels based on hydroxyethyl methacrylate and acetoacetoxyethyl methacrylate. Microfluidic processor according to claim 1, characterized in that the polymer networks ( 4a - 4g ) of the polymer network based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) consist of physically crosslinked hydrogels or polymer solutions which gel or dissolve by changes in temperature, in particular gelatins or polysaccharides such as pectin and agarose. Microfluidic processor according to claim 1, characterized by a sandwich construction with at least one channel structure carrier ( 1 ), an actuator structure carrier ( 2 ), a flexible membrane ( 3 ) and a structural support ( 5 ) for an electrical ( 6 . 6a . 6b ) or optical control. Microfluidic processor according to claim 1, characterized by a sandwich construction with at least one channel structure carrier ( 1 ), an actuator structure carrier ( 2 ), a flexible membrane ( 3 ) and a structural support ( 5 ) for an electrical ( 6 . 6a . 6b ) or optical drive, whereby the polymer networks ( 4a - 4g ) of the polymer network-based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) substantially in the actuator structure carrier ( 2 ) are located. Microfluidic processor according to claim 1, characterized by a sandwich construction with at least one channel structure carrier ( 1 ), a structural support ( 5 ) for an electrical ( 6 . 6a . 6b ) or optical control and a cover ( 9 ) with a flexible area ( 9a ), whereby the polymer networks ( 4a - 4g ) of the polymer network-based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) directly in channels ( 1a ) of the channel structure carrier ( 1 ) are placed. Microfluidic processor according to claim 1, characterized by a sandwich construction, which has at least one channel structure carrier ( 1 ), a cover ( 9 ) and a structural support ( 5 ) for an electrical ( 6 . 6a . 6b ) or optical drive, wherein in the channel structure carrier ( 1 ) or in the cover ( 9 ) the electrical ( 6 . 6a . 6b ) or optical drive structure is integrated. Microfluidic processor according to claim 1, characterized by at least two fluidic sequences, wherein a) each sequence via hydrogel pumps ( 15a - 15d ), which premix the various process media according to their respective predetermined delivery volumes by appropriate output-side interconnection and this for mixing pumps (hydrogel pumps 16a - 16f ), b) at least two mixing pumps (hydrogel pumps 16a - 16f ) are connected together from different sequences at their output, c) the interconnected mixing pumps (hydrogel pumps 1a-16f ) represent a mixing ratio determined by their respective given delivery volumes, and d) the resulting mixture is discharged via exits ( 10a - 10d ) or in analysis or reaction units ( 17 ). Microfluidic processor according to claim 1, characterized by at least one fluidic sequence, which via hydrogel pumps ( 15a - 15d ), which premix the various process media according to their respective predetermined delivery volumes by appropriate output-side interconnection and the resulting mixture in reaction or analysis units ( 17a - 17c ; 20a . 20b ). Microfluidic processor according to claim 12 or 13, characterized in that the mixing ratios of the output side interconnected hydrogel pumps ( 15a - 15d ; 16a - 16f ) by the respective sizes or volumes of the pump chambers ( 7 ) are given constructive. Microfluidic processor according to claim 12 or 13, characterized in that it over meh other mixing stages which are based on an appropriate interconnection of hydrogel pumps ( 15a - 15d ; 16a - 16f ). Microfluidic processor according to claim 1, characterized in that the respective structural supports ( 1 . 5 ) in the area of the structures to be tempered ( 6 . 6a . 6b ) Have sections with reduced thermal resistance, in particular in the form of material dilutions or the use of materials with improved thermal conductivity. Microfluidic processor according to claim 12, characterized characterized in that it has the enzyme activity of a biochemical process controlled. Microfluidic processor according to claim 13, characterized characterized in that it processes based on a polymerase chain reaction controlled. Microfluidic processor according to claim 1 to 3, characterized in that the polymer network elements ( 4a - 4g ; 15a - 15d ; 16a - 16f ; 19a - 19d ) have at least partially different switching or activation temperatures. Microfluidic processor according to claim 1 to 3 and 18, characterized in that the microfluidic processor by external devices, like thermostat, thermocycler, heating cupboard or heat bath, holistic or being exposed to temperature programs at certain sections, who control his behavior.