DE102006020716B4 - Microfluidic processor - Google Patents

Abstract

Microfluidic processor with integrated controllable elements for handling process media, with elements based on temperature-sensitive hydrogels (4a – 4g) (15a – 15d; 16a – 16f; 19a – 19d), whose sensitivities relevant to media handling change due to deliberately changeable ambient temperature, whereby the ambient temperature can be controlled via electrical or electronics-compatible interfaces (6, 6a, 6b) and the integrated polymer network-based elements (15a-15d; 16a-16f; 19a-19d) are interconnected by hardware in a basic logic configuration and via the interfaces (6 , 6a, 6b) can be controlled by a higher-level system, characterized by at least one fluidic mixing unit which has hydrogel actuator-based pumps (15a-15d; 16a-16f) connected on the output side, which produce mixing ratios for different process media, which are generated by the respective volumes the pump chambers ( 7) the pumps (15a-15d) can be predetermined in terms of design and the resulting mixture in reaction or analysis units (17a-17c; 20a, 20b).

Description

Technical area

The invention relates to an electronically controllable microfluidic processor with integrated controllable elements for handling process media, with temperature sensitive hydrogel based elements whose media handling relevant sensitivities change by selectively variable ambient temperature, wherein the ambient temperature is controlled by electrical or electronics compatible interfaces and wherein the integrated polymer network-based elements are interconnected in hardware in a basic logical configuration and are controllable via the interfaces by a higher-level system.

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. Realizing these devices by integrating functions more or less complex biological, biochemical or chemical processes, so we speak of microfluidic processors or even "labs on a chip" (LOC), "chip labs" or "Micro Total Analysis Systems "(ΜTAS).

The LOC concept offers many advantages. Reducing fluid volumes allows for the analysis of minute sample volumes and economical handling of reagents and samples that are often valuable, rare, harmful or dangerous. This also higher throughputs are achievable, since due to the small quantities shortened provisioning, mixing and reaction times are required with minimal energy consumption. Due to lower system response times, process control can also be easier.

Overall, LOC setups enable significant process rationalization by significantly reducing process time, thereby increasing throughput, and reducing the amount of media required (subjects, analytes, reagents, auxiliary media).

Despite the obvious advantages, there are only rarely implemented LOC applications. The reasons for this are to be found in which partial processes are currently miniaturized (on-chip-integrated).

State of the art

The typical structure of biological, biochemical or chemical processes comprises the tasks of sample preparation, handling and reaction or analysis in specific forms and combinations. At present, on-chip integration of sample preparation as well as reaction or analysis is mainly carried out. However, the economic benefits resulting from the rationalization of these sub-processes have generally proved unsustainable.

Huge rationalization potential offers sample handling because it is particularly time-consuming and labor-intensive. Due to their problematic on-chip integration, sample handling is currently performed manually or with certain special equipment such as diluters, syringe pumps, pipettors, and the like. Ä., performed outside the chips.

However, the very diverse available miniaturized electronically controllable fluidic drives (pumps) and switching elements (valves) have such disadvantages that they either can not be economically integrated into a lab-on-a-chip structure or have inadequate performance characteristics.

Fluidic elements based on solid-state actuators, such as piezoactuators [ US 5,224,843 A . US 2003/0143122 A1 ] and shape memory actuators [ US 5,659,171 A ], 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 B2 ] as well as thermal bubble generators [ US 6,283,718 B1 ] known.

However, aggregate state changers have some inadequate utility characteristics. With the exception of bubble generators, the transducers can not be used in an actuatoric way, so their application is limited to switching elements. Due to the necessary heat fluctuations, the process media are exposed to considerable thermal stress, and in the case of freezing elements also to mechanical stress.

Gas-forming transducers are not suitable for many microfluidic processes because most are gas-sensitive.

A generic microfluidic device is made DE 199 29 734 A1 in which the polymer network-based elements are designed as hydrogel microvalves which control the fluid flow path through the microfluidic device. Hydrogel microvalves block or allow fluid flow, depending on their condition, but they can not pump process media.

From the US 2003/0210997 A1 is a controllable hydrogel-based actuator with valve function known, wherein a plurality of consecutive in a channel and successively controlled valves to realize a peristaltic pump. However, the arrangement is unsuitable for liquid transport since the valve bodies can not displace liquid. They absorb liquid while swelling and release exactly this amount of liquid when shrinking.

In the DE 101 57 317 A1 discloses a microfluidic processor based on a swellable hydrogel, which is easily miniaturized and controllable via electrical or electronically controllable interface sizes. In the fluidic flow channels of the processor, a Aktordot is placed with valve function. Multiple channels can be merged to a common output.

As a result, mixing processes of process media corresponding to the channel cross-sections and channel inlet pressures can be realized. Although a peristaltic pump should also be realized by series connection of several actuator dots, it is not suitable for this purpose, as explained above.

Presentation of the invention

The object of the invention is to provide an LOC device which can be produced with an economically justifiable manufacturing outlay and which contains the sample handling in on-chip-integrated form. Thus, sample preparation and / or sample reaction or sample analysis can be combined with sample handling in a LOC setup. The core task of the microfluidic processor should be to realize controlled mixing ratios by means of hydrogel actuators arranged on the microfluidic processor and interfaces for different process media and to transport the mixtures autonomously either to an output or to feed them to further processing on the microfluidic processor.

According to the invention the object is achieved by the features specified in claims 1 and 2. Advantageous embodiments are specified in claims 3 to 12.

The material basis of the invention are hydrogels. Hydrogels are polymer networks that change their volume, strength and other properties when exposed to aqueous swelling agents. These polymer networks can be subdivided into chemically and physically crosslinked polymer networks or hydrogels according to the type of polymer chain linkage. In chemically crosslinked polymer networks, the individual polymer chains are irreversibly linked by covalent (chemical) compounds. In physically networked polymer networks, the polymer chains are interconnected by physical interactions, which can usually be resolved again.

A special class of hydrogels are those with a "smart" character. Smart hydrogels, as chemically crosslinked polymer networks, perform reversible discontinuous volume phase transitions when exposed to specific environmental parameters. As physically crosslinkable polymer networks, they dewet or crosslink (also gelling) between the maximum crosslinked and fully dissolved states. The environmental quantities can be electric field quantities, radiation, temperature, ion and substance concentrations. An electrical or electron-compatible controllability of smart hydrogels is currently problematic with direct electrical quantities and field sizes, since the corresponding effects with regard to their efficiency are still insufficient and have limited reversibility. With thermal-electronic interfaces, however, temperature-sensitive hydrogels can be controlled very easily, since the necessary heat supply or heat extraction can be implemented in a technically uncomplicated manner.

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 carry out the invention

The invention will be explained in more detail with reference to exemplary embodiments, wherein the reference numbers used have the same meaning. In the accompanying 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 show a storage unit based on a chemically crosslinked 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 There are 0.05 M malonate buffer of pH 5.0. Another hydrogel pump 15c contains a 2 mM 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution as substrate in a 0.05 M pH 5.0 malonate buffer. In a fourth hydrogel pump 15d is a sample of a bioreactor 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 parallelizing several such LOC can a shorter clocking and thus a continuous enzyme activity control corresponding to enzyme production control.

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 15 microns to 100 microns. 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. with conventional Methods and materials of printed circuit board technology can be realized. For this example, a polyester film laminated with 35 micron 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 be glued together, welded or non-positively joined. For example, after a low pressure oxygen plasma treatment PDMS moldings can be bonded very well with PDMS as an adhesive and subsequent temperature hardening.

Hydrogel elements can be produced by various methods. For structuring hydrogel layers, the crosslinking photopolymerization [D. J. 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 [M. E. Harmon et al., Polymer 44 (2003), pp. 4547-4556] and hydrogel particle generation [K.-F. Arndt et al., Polym. Adv. Technol. 11 (2000), 496-505].

Smart hydrogels are currently difficult to control directly with electrical quantities. However, temperature-sensitive hydrogels can be electrically controlled very easily via a thermal interface and can therefore be used as electronically controllable elements. The property profile of electronically compatible hydrogels and their potential applications are therefore presented using 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 (LCST) characteristic, ie, they are swollen at low temperatures and escape when the phase transition temperature is exceeded. The best-known LCST characteristic hydrogel, poly (N-isopropylacrylamide) (PNIPAAm), has a 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 PNIPAAm-based hydrogels are e.g. In A. Richter et al., J. Microelectromech. Syst. 12 (2003) 5, pp. 748-753.

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 are formed by gelling when a certain amount of environmental factors are involved in a polymer solution by linking the individual polymer molecules into a three-dimensional network. This process is usually reversible (sol-gel transition behavior). By acting on the critical environmental size, the linkage points are separated again and the polymer molecules are present dissolved.

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

Chemically cross-linked smart hydrogels change a whole series of properties during the phase transition. For hydrogel elements, it is mainly the changes in their volume, strength, permeability (material permeability) or diffusion properties and storage capacity that can be used. By using the change in volume, for example, valves, fluidic drives (pumps) and actuators can be realized, which are controllable via electronically compatible interface sizes.

Strength and compliance changes can be z. B. for switchable locks and attacks use.

Changes in the permeability or the diffusion properties can also be used for switching or influencing material flows.

Physically crosslinkable hydrogels can also change volume, strength and permeability in the crosslinked state. However, as purely physically crosslinked hydrogels, they do not have a volume phase transition behavior, but change these properties spontaneously upon swelling from drying or drying out. But they can be switched in their networking state, d. h., One can create a hydrogel solid, dissolve or influence its swelling or strength properties.

These properties can be used for hydrogel elements which can be activated once. As in the form of valves, moldings with blocking, locking or actuating function, use the changed over the electronics compatible interface sizes in their strength, dissolved or 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 this hydrogel is 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 swelling 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.

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

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

• - 4 μl of 10 × buffer with 25 mM MgSO ₄ (10 × Pfu polymerase reaction buffer, Fermentas Life Science)
• - 0.2 μl forward primer (FP) final concentration 1 μM
• - 0.2 μl reverse primer (RP) final concentration 1 μM
• - 3.2 μl dNTP (deoxyribonucleoside triphosphate mixture; Fermentas Life Science) final concentration 0.4 mM each
• - 0.8 μl of Pfu DNA polymerase (2.5 U / μl, Fermentas Life Science) and 11.6 μl of 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, the volume information for the master mix is multiplied by the number of applications. The prepared master mix can be stored in a refrigerated storage vessel (4 ° C) outside the LOC. The same applies to the template DNA's.

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

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

By simultaneously pumping the hydrogel pumps 15a . 15b . 15c . 15d Add 10 μL of master mix with 10 μL of template DNA1 (hydrogel pump 15a with hydrogel pump 15b ) and 10 μl of master mix with 10 μl 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 30 s each (template denaturation) and 30 s at 55 ° C (primer annealing)
• - 4 min at 72 ° C (primer elongation).

Finally, 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 10 V / 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 Hydrogelbasierten individual elements can also be determined by appropriate determination of their activation temperatures. When the temperature rises, 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 adapting the mastermix composition (several primers) and the template DNA (sample DNA), this basic structure can be transferred to a variety of DNA analysis methods. There are all applications, eg. B. DNA fingerprint (paternity test), virus analysis u. a, based on the principle of PCR-DNA analysis, realizable 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 Trans kriptasepuffer.

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 fields of application of 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 can be realized on one LOC. 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 field of biotechnology they are, for example, for the enzyme reactor or bioreactor monitoring u. a. suitable for prokaryotes, eukaryotes, yeasts and fungi. As an online, continuous or one-time measurement (also for single use), a rapid screening of the activity of enzymes of different enzyme classes can be realized. By combining several LOCs, multi-rapid screening can be enabled. The online expression level control as well as the online tracking of growth curves and growth factors, also in addition to or combination with flow cytometry, can be carried out with the LOC according to the invention.

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

In the field of medical technology, the LOC technology according to the invention can be used, for example, for on-line cell culture monitoring (eukaryotes, human cell lines and the like) by means of viability tests [eg. B. WST-1 and MTT assay (Conversion of a tetrazolium salt into formazan, e.g., 4- [3- (4-iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolium] -1,3 -Benzendisulfonat) or LDH test (lactate dehydrogenase test)] etc. use.

This is a cell quality, batch and passage control for human cell lines u. a. possible.

Also on-chip blood tests to determine a part of the most important blood parameters, z. As blood sugar, pH, lactate, minerals, creatine, hormones, enzymes, leukocytes, erythrocytes u. a., disease marker, the detection of reactive-oxigen-toxic substances (ROTS-oxidative stress) u. s. w. are feasible.

LIST OF REFERENCE NUMBERS

1

Channel structure support

1a

Channel in the channel structure carrier

2

Aktorstrukturträger

3

flexible membrane

4a

Hydrogel element a for a hydrogel pump

4b

Hydrogel element b for a hydrogel valve

4c

swollen hydrogel element

4d

deswollen hydrogel element

4e

crosslinkable 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

Released active ingredient in the process medium

13

channel structure

14

particle

15a-15d

Hydrogel pumps for fluid intake

16a-16f

Hydrogel pumps for varying mixing ratios

172-17c

Reaction or analysis units

18a-18e

Mischmäander

19a-19d

Hydrogel valves

20a, 20b

PCR chamber

21a, 21b

Gel Electrophoresis

Claims (12)

Microfluidic processor with integrated controllable elements for handling process media, with temperature-sensitive hydrogels ( 4a - 4g ) based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ), whose media handling relevant sensitivities change as a result of a specifically changeable ambient temperature, whereby the ambient temperature is controlled by electrical or electronically compatible interfaces ( 6 . 6a . 6b ) and 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 ) can be controlled by a higher-level system, characterized by at least one fluidic mixing unit, which is connected via outlet-side connected hydrogel actuator-based pumps ( 15a - 15d ; 16a - 16f ), which produce mixing ratios for different process media, which are determined by the respective volumes of the pump chambers ( 7 ) of the pumps ( 15a - 15d ) are constructively specifiable and the resulting mixture in reaction or analysis units ( 17a - 17c ; 20a . 20b ). Microfluidic processor with integrated controllable elements for handling process media, with temperature-sensitive hydrogels ( 4a - 4g ) based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ), whose media handling relevant sensitivities change as a result of a specifically changeable ambient temperature, whereby the ambient temperature is controlled by electrical or electronically compatible interfaces ( 6 . 6a . 6b ) and 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 ) can be controlled by a superordinated system, characterized by at least two first fluidic mixing units, which are connected via output side connected hydrogel actuator-based pumps ( 15a - 15d ), which produce mixing ratios for different process media, which are determined by the respective volumes of the pump chambers ( 7 ) of the pumps ( 15a - 15d ) can be specified constructively and the resulting mixtures of further hydrogel-actuator-based pumps ( 16a - 16f ), wherein at least two of these further hydrogel-actuator-based pumps ( 16a - 16f ) are combined on the output side to form a further mixing unit, and the mixing ratios are likewise determined by the respective volumes of the pump chambers ( 7 ) of the pumps ( 16a - 16f ) can be specified and the resulting mixture via outputs ( 10 . 10a - 10d ) or in analysis or reaction units ( 17a - 17c ; 20a . 20b ). Microfluidic processor according to claim 1 or 2, 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, light or radiation into heat. Microfluidic processor according to claim 1 or 2, characterized in that the elements based on temperature-sensitive hydrogels ( 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 or 2, characterized in that the elements based on temperature-sensitive hydrogels ( 15a - 15d ; 16a - 16f ; 19a - 19d ) consist of chemically cross-linked 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 or 2, characterized in that the elements based on temperature-sensitive hydrogels ( 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 or 2, 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 control ( 6 . 6a . 6b ). Microfluidic processor according to claim 1 or 2, 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 control ( 6 . 6a . 6b ), the elements based on temperature-sensitive hydrogels ( 15a - 15d ; 16a - 16f ; 19a - 19d ) substantially in the actuator structure carrier ( 2 ) are located. Microfluidic processor according to claim 1 or 2, characterized by a sandwich construction with at least one channel structure carrier ( 1 ), a structural support ( 5 ) for an electrical control ( 6 . 6a . 6b ) and a cover ( 9 ) with a flexible area ( 9a ), the elements based on temperature-sensitive hydrogels ( 15a - 15d ; 16a - 16f ; 19a - 19d ) directly in channels ( 1a ) of the channel structure carrier ( 1 ) are placed. Microfluidic processor according to claim 1 or 2, characterized by a sandwich construction, which comprises at least one channel structure carrier ( 1 ), a cover ( 9 ) and a structural support ( 5 ) for an electrical control ( 6 . 6a . 6b ), wherein in the channel structure carrier ( 1 ) or in the cover ( 9 ) the electrical drive structure ( 6 . 6a . 6b ) is integrated. Microfluidic processor according to claim 1 or 2, characterized in that the temperature-sensitive hydrogels ( 4a - 4g ) based elements ( 15a - 15d ; 16a - 16f ; 19a - 19d ) have at least partially different switching or activation temperatures. Microfluidic processor according to claim 1 or 2, characterized in that the microfluidic processor by external devices, such as thermostat, thermal cycler, oven or heat bath, holistic or at certain sections its behavior controlling temperature programs can be suspended.

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