DE10104957A1 - Method of manufacturing a 3-D micro flow cell and 3-D micro flow cell - Google Patents

Abstract

The invention relates to a method for producing a 3-D micro flow cell and to a micro flow cell produced according to said method. The aim of the invention is to provide a method that is cost-effective and that achieves particularly constant geometric parameters. According to the invention, the flow channel (3), a spacer I defining both sides of said channel (5), and additional spacers (14) consisting of a substantially non-compressible or curable material of a predetermined depth are applied at least to the lower substrate (1). Once applied, said spacers and flow channel are irreversibly fixed to the lower substrate (1) or upper substrate. A pasty adhesive, acting as a spacer-II (6), is applied with a uniform thickness around the outer periphery of spacer-I (5) and the upper substrate (2) is subsequently positioned on the lower substrate (1) and joined thereto by force, heat or light, thus simultaneously sealing the flow channel (3).

Description

The invention relates to a method for producing a 3-D micro flow cell, consisting of a lower and a upper substrate, between which a flow channel is arranged is an electrode structure connected to external contacts penetrates, with at least one of the substrates initially with a trace and electrode structure and at the ends of the Flow channel with vias to connect a Liquid inlet and outlet is provided. The invention also relates to a 3-D micro manufactured using the method flow cell.

Such 3-D micro flow cells are used, for example, as Cell manipulators for handling and optical analysis dielectric biological particles, especially cells and / or bacteria or viruses. For this purpose the micro flow cells equipped with a flow channel tet, at the ends of one or more liquid supply and -flows are provided. These liquid inflows and outflows are, for example, perpendicular to the flow channel extending vias. The amount of Liquid channels are usually in the range of a few Micrometers, with the flow channel passing through the top and bottom Glass substrates and / or silicon substrates and laterally through corresponding channel walls is limited. To single cells  at a given location within the liquid channel "Floating" can be fixed in the liquid speed channel electrodes that are applied when an electrical Generate an electric field. The electrostatic fixed cell can then by suitable lighting illuminated and observed using a microscope.

In order to be able to implement such three-dimensional structures various technologies have become well known the. So z. B. a glass substrate wet-sided on one side be etched to form a flow channel therein and subsequently by means of diffusion welding with a second one Glass substrate can be connected as a cover element. The one for that Handling of cells or biological particles required Electrodes are placed on the first and / or second glass beforehand substrate using known methods of photolithography brought and the second glass substrate then face-down the lower glass substrate mounted.

However, the technology of diffusion welding is rela tiv expensive and the possibilities of usually isotropic Glass structuring is limited. Another disadvantage is to see that only relatively rough electrode structures can be applied to the structured glass surfaces can. For precise handling of individual cells or biologically However, being able to realize shear particles is extremely precise geometric design of the electrodes required, to touch these particles electrostatically at the desired location able to manipulate and hold on without hesitation.

Another technology is from Müller / Gradl / Howitz / Shirley / Schnell / Fuhr in the magazine "BIOSENSORS & ELECTRONICS", No. 14 (1999), pages 247 to 256. This is about it is the application of the purely manual epoxy resin adhesive technology, initially using a polymer spacer on a glass top surface that was previously processed with platinum electrodes and electrical interconnects has been provided. Then will the glass substrate with a synthetic resin, e.g. B. epoxy, as  Spread glue outside the polymer structure and then then a second glass, which also has electrodes is provided, positioned and the composite ver pressed. This assembly step is usually done with one so-called die bonder (cip bonder) executed.

Difficulties can be seen in the fact that it is problema table is to manufacture micro flow cells that are always accurate have the same geometric dimensions and those with Safety during the assembly process no glue in the Penetrates the flow channel, which would partially narrow it. The efficiency of this step is therefore extremely poor and not suitable for mass production.

A so-called underfiller technique is also known in which a polymer-1 (thick varnish) on the with Elek troden provided glass substrate, the Thickness of the spin-on polymer by the height of the front seen channel is specified. This polymer becomes then structured the positive channel system, d. H. the rest Thick varnish becomes complete during this photo structuring away. Then the second glass substrate is then adjusted and pressed on the first glass substrate. This on this 3-D arrangement obtained in this way is achieved by lateral inflow a creepable adhesive (underfiller), a polymer-2, fixed, after which the channel system in polymer-1 with a Lö is washed out again. The solution is allowed do not attack polymer-2. Is particularly disadvantageous here that in this way there is no internal flow in the channel elements can be produced because they are not made of polymer-2 can be achieved. In addition, this technique is extreme time consuming and limited in terms of structure resolution.

The invention is based on the object of a method to create a 3-D micro flow cell, which can be realized inexpensively and with the ins special constant geometric parameters realized can be. The invention is also based on the object  de to create a 3-D micro flow cell that works with the Method according to the invention can be produced inexpensively can.

The object underlying the invention is in one A method of manufacturing a 3-D micro flow cell, be standing from a lower and an upper substrate, between which a flow channel is arranged, the one with the outside contacts connected electrode structure penetrates, wherein at least one of the substrates first with an interconnect and Electrode structure and at the ends of the flow channel with plated-through holes for the connection of liquid supply and -Procedures is solved by the fact that on the lower Substrate defining the flow channel on both sides of the same Spacer-I as well as additional spacers from one essentially non-compressible material or hardenable material predetermined height to be applied after the application irreversibly firmly bonded to the lower substrate, that outside of the flow channel a pasty adhesive as Spacer-II of uniform thickness is applied and that at closing the top substrate on the bottom substrate posi tioned and connected to it with force, the flow channel being sealed at the same time.

This easy-to-implement procedure is guaranteed by one on the one hand an extreme precision of the geometrical dimensions of the flow channel and on the other hand a complete and easy sealing of the same without the risk of that quantities of adhesive penetrate into the flow channel could narrow it.

In a first continuation of the invention, the spacer-II directly next to the spacer-I, which includes this in parallel, applied, the thickness of the spacer-II before assembly is greater than the height of the spacer-I.

Best for the production of the Spacer-I and the spacers different options. So the Spacer-I and  the spacers using screen printing or dispensing applied the lower substrate and then hardened be, the hardening, for example, by the action of heat or can be carried out by light or UV radiation.

Another option is to use the Spacer-I and the Spacers on the lower substrate using photolitogra fischer process and then by annealing to harden. Spacer-I and Spacers made of a photostructurable photoresist, preferably a dry photoresist. Fotolitho graphic processes enable a compared to screen printing less edge roughness and thus greater precision, so that finer structures can be made.

Another option is to use the Spacer-I and the Spacers from a pre-structured, at least one producing adhesive metal or polymer film and stick on the lower substrate.

For attaching the top substrate to the bottom Substrate, d. H. to manufacture the 3-D structure is preferred as an adhesive as Spacer-II based on epoxy resin or silicone rubber used. The production of the verb Formation of the upper with the lower substrate can under effect of pressure and heat and / or light or UV radiation tion.

The problem underlying the invention is further resolved by a 3-D micro flow cell there is a lower and an upper substrate, between the substrates with fluidic vias provided flow channel is arranged, the one with the outside contacts connected electrode system penetrates and the characterized in that on the lower substrate Spacer-I defining the flow channel and additional Ab stands and / or stabilizers from one essentially not compressible material or hardenable material  bener height are arranged with this lower substrate are irreversibly firmly connected, and that the top substrate close the flow channel tightly with the lower substrate by means of a pasty, hardenable adhesive layer, forming a spacer-II is firmly connected.

In a first embodiment of the invention, the Spacer-II on both sides outside of the flow channel on the Outside of the spacer-I, comprising this in parallel.

The thickness of the Spacer-I and the spacers must be the same should be between 10 µm and 1050 µm depending on the intended height of the flow channel.

In continuation of the invention, at least one of the two Glass substrates with a thickness of 150 µm. , , Have 200 µm and the other 500 µm. , , Be 1000 µm thick. So the association receives sufficient mechanical stability and is also for use high-resolution microscopy.

The upper substrate can also be made of a plastic film for example a polymer film with a thickness of 170 microns to 200 microns exist.

A further embodiment of the invention is thereby characterized records that the area of the flow channel at least in Wavelength range from 250 nm to 450 nm optically transparent is. This can be done simply by choosing suitable materials for the lower and the upper substrate.

The invention is in a further special embodiment characterized in that the upper and lower substrates each have metallic microelectrodes in one predetermined three-dimensional geometric relationship to each other stand and that the top substrate face-down on the bottom Substrate is mounted. The microelectrodes of the upper sub strates are provided with contact pads and with the external contact on the lower substrate using conductive adhesive or solder pads  electrically connected.

The microelectrodes consist of a thin film system Platinum, gold, tantalum or titanium.

In a special embodiment of the invention, the elec Trode and connection system on the upper and lower Whole substrate using an inorganic insulator material area insulated, the insulator material inside the Flow channel, on the contact pads and on the contact supports recessed to provide adequate electrical To enable contacting at these points.

To one through the polymer of Spacers-I - the flow channel forms - self-fluorescence caused by light excitation to hide during optical-microscopic detection, is at least on the outside of the upper substrate opaque panel attached in such a way that the Edge area of the flow channel covered, however central area is kept clear. The special advantage Such an aperture is that a fluorescence-based detection tion on biological cells in the flow channel, without the fluorescence of the channel-limiting materials have a disruptive influence would exercise.

The panel can also be used as a shield from the inside and outside for electromagnetic and bioelectric waves be educated, which will certainly prevent it from being regular existing electrosmog has a negative impact on the De tection of the cells.

In the simplest case, the cover is made of metal, and this also from a photolithographically structurable thin film, z. B. can consist of Cu or Al.

This thin film should expediently be removable, so that if necessary, the entire flow channel  can be examined optically.

In order to form an adhesive film on the inside of the Preventing flow channels as possible is in a be special continuation of the invention of the Spacer-I in its Contact surface with a groove or with another along the same recess for receiving adhesive provided during the assembly process.

In special cases it may be desirable that the upper substrate is detachably connected to the lower substrate. In this case, a special variant of the invention characterized in that the Spacer-II made of silicone rubber is printed on the spacer-I and after the vulcanization the upper and lower substrates are non-positively be connected. This makes this 3-D micro Open the flow cell easily and disinfect if necessary adorn.

This is another special variant of the invention characterized that the photolithographically on the bottom Spacer-I produced substrate has a width that in essentially the parallel arrangement of Spacer-I and Spacer-II corresponds and that the upper substrate by adhesive force is attached to the lower substrate. This variant of the However, the invention is only suitable for such cases in which the upper substrate does not contain an electrode structure.

The invention will be based on an embodiment are explained in more detail. In the accompanying drawings:

Figure 1 is a schematic plan view of a 3-D micro flow cell.

FIG. 2 shows a sequence of manufacture of the lower substrate of the 3-D micro flow cell;

Figure 3 shows the assembly sequence for completing the 3-D micro flow cell.

FIG. 4 shows a sectional view of the 3-D micro flow cell corresponding to FIG. 3 as a glass-glass module;

Figure 5 is a sectional view of a 3-D micro flow cell with flip-chip bonding. and

Fig. 6 is provided with a Cu diaphragm 3-D micro flow cell.

From the drawing figure 1, a 3-D micro flow cell according to the invention can be seen, which consists of a lower substrate 1 made of glass with a thickness of about 750 microns and an upper substrate 2 . In the present case, the upper substrate likewise consists of glass with a thickness of approximately 150 μm, whereby other materials can also be used here, which are in the wavelength range between 250. , , 450 nm have sufficient transparency. Between the two substrates 1 and 2 there is a flow channel 3 , each of which is provided at its ends with a fluidic contact 4 for supplying and discharging a liquid. The flow channel 3 is laterally limited in its entire longitudinal extent by a spacer-I 5 and a further spacer-II 6 , which extends on both sides outside the flow channel 3 next to the spacer-I.

Furthermore, there is an electrode structure 7 on the upper substrate 2 and the lower substrate 1 , which is connected via conductive tracks 8 to external contacts 9 .

In contrast to the interconnects 8 on the lower substrate 1 , the interconnects 8 end on the upper substrate 2 in contact pads 10 , which are electrically connected to the external contacts 9 on the lower substrate 1 by means of conductive adhesive or solder pads or μ-balls 18 .

Furthermore, all external contacts 9 on the lower substrate 1 are combined in a contact support 11 , which has the task of additional mutual insulation.

For the electrostatic fixation of cells 12 or biological particles or the like at a predetermined location within the flow channel 3 (see FIG. 5), the electrode structure 7 contains microelectrodes 13 , each on the lower substrate 1 and the upper substrate 2 protrude into the flow channel and are positioned exactly three-dimensionally.

To achieve a constant spacer spacing between the substrates 1 , 2 over the substrate, spacers 14 are also provided.

In order to better illustrate the formation of the individual structures on the lower substrate 1 , FIG. 2 shows a corresponding sequence. For this purpose, the lower glass substrate 1 is first drilled in order to later be able to implement the required fluidic contacts 4 to the flow channel 3 . Subsequently, the lower substrate 1 is provided with the electrode structure 7 and the interconnects 8 , as well as the external contacts 9 , using the usual thin film technology and photolithography. The entire structure is then insulated over the entire surface by means of an inorganic insulator material 15 . This insulator 15 is then removed in the area of the future flow duct 3 , as well as on the external contacts 9 , in order to be able to produce effective electrical structures.

Subsequently, the flow channel 3 is formed on the lower substrate 1 by a first spacer 5 is applied from a polymer on the lower substrate. 1 For the production of the Spacers-I 5 , a dry photoresist can be structured by means of photolithography, or a suitable material can be applied by means of screen printing. The spacer-I 5 is then hardened by the action of heat or UV radiation. It is very important in this step that the spacer-I 5, after hardening, has exactly the thickness that the flow channel 3 is later to have.

The spacer II 6 , surrounding the spacer I 5 , is then applied to the lower substrate 1 by printing or with the aid of a dispenser. The thickness of spacer-II 6 is greater than that of spacer-I 5 . In any case, an adhesive based on epoxy resin or silicone rubber is used as spacer-II 6 .

When the upper substrate 2 3a only an electrode structure 7 is produced in the same manner as connected on the lower substrate and interconnects with contact pads 10 corresponding to FIG.. This structure, too, is subsequently insulated over the entire area with an organic or inorganic electrical insulator material 15 , the electrode structure 7 subsequently being exposed in the region of the future flow channel and the contact pads 10 by removing the insulator material 15 .

The flip-chip assembly according to FIG. 3 then takes place by positioning the upper substrate 2 face-down exactly above the lower substrate and then placing it on. At the same time, heat is supplied to harden the spacer-II 6 and thus to produce the 3-D structure as shown in FIGS. 1, 4, 5.

In order to be able to produce the necessary electrical contacts between the contact pads 10 on the upper substrate and the external contacts 9 on the lower substrate, conductive adhesive 16 is dispensed onto the connections before the flip-chip assembly.

For preventing the penetration of adhesive into the Strö flow duct 3 during the assembly process, can on the upper surface of the first spacer 5 a along the same running, for example. B. V-shaped groove. This is easily possible using the known methods of photolithography. In addition, a higher strength of the overall structure is achieved.

Since the channel walls of the spacer-I 5 already produce a disruptive fluorescence when illuminating a cell 12 spatially fixed in the flow channel 3 during the optical detection, for the optically high-resolution detection on e.g. B. an immersion lens of a microscope, a suitable expansion of the inherent fluorescence of the spacer material.

In order to rule out such disturbances, an opaque screen 17 can be provided according to FIG. 6, which covers the edge of the flow channel 3 and keeps the central area clear. This aperture 17 can be produced from a metallic structurable and adjusted thin film. In order to make such an aperture reversible if necessary, the use of an easily removable layer system makes sense, so that the entire cross section of the flow channel 3 can be observed if necessary.

The particular advantage of such a diaphragm 17 is that a floureszenzbasierte detection of biological cells 12 in the flow channel can be made 3 without the thereby simultaneously induced fluorescence of the channel 3 limiting materials would interfere with a caused by stray light exert the influence of a. Another advantage can be seen in the fact that it is no longer necessary due to the aperture 17 to provide an additional aperture in the optical system, which leads to a higher light intensity of the optical system.

The aperture 17 can advantageously also be designed as a shield from the inside and outside for electromagnetic and bioelectric waves, which reliably prevents the presence of regularly present electrosmog from having a negative influence on the detection of the cells.

In the simplest case, the aperture 17 can be made of a metal, the aperture 17 also made of a photolithographically structurable thin film, for. B. of Cu, Al or another metal.

Thus, the aperture 17 can be easily removed by etching without impairing the micro flow cell.

In the event that only value is placed on an optical shielding through the aperture 17 , this can of course also be made of other materials, e.g. B. a plastic.

In special cases, it may be desirable that the upper substrate 1 is releasably connected to the lower substrate 2 . In this case, a special variant of the invention is characterized in that the spacer-II 6 made of silicone rubber is printed on the spacer-I 5 and after the vulcanization, the upper and lower substrates 2 , 1 are non-positively connected to one another. The non-positive connection can be realized by a simple clamping device.

In the simplest case, ie if the upper substrate has no electrode structure 7 , a significant simplification of the structure of the 3-D micro flow cell can be achieved if the spacer-I 5 produced photolithographically on the lower substrate 1 has a width which is in the essentially the parallel arrangement of Spacer-I 5 and Spacer-II 6 speaks ent ( Fig. 5), wherein the upper substrate 2 is only attached to the lower substrate 1 by adhesive force. The prerequisite for this is that the contact surface of the first spacer-I ( 5 ) with the upper substrate is completely flat.

LIST OF REFERENCE NUMBERS

1

lower substrate

2

top substrate

3

flow channel

4

fluidic via

5

Spacer I

6

Spacer II

7

electrode structure

8th

interconnect

9

outside Contact

10

contact pad

11

Contact Support

12

cell

13

microelectrode

14

spacer

15

insulator

16

conductive adhesive

17

cover

18

μ-Ball

Claims (28)

1. A method for producing a 3-D micro flow cell consisting of a lower and an upper substrate, between which a flow channel is arranged, through which an electrode structure connected to external contacts penetrates, at least one of the substrates initially having an interconnect and electrode structure and the ends of the flow channel are provided with plated-through holes for the connection of liquid inflows and outflows, characterized in that on the lower substrate ( 1 ) the flow channel ( 3 ) defining the spacer-I ( 5 ) on both sides thereof and additional spacers ( 14 ) an essentially non-compressible material or hardenable material of a predetermined height are applied, which are irreversibly firmly connected to the lower substrate ( 1 ) after application, that a pasty adhesive as spacer II ( 6 ) of uniform thickness is applied outside the flow channel and that the upper S ubstrat ( 2 ) positioned on the lower substrate ( 1 ) and connected to it with force, the flow channel ( 3 ) being sealed at the same time. 2. The method according to claim 1, characterized in that the spacer-II ( 6 ) immediately next to the spacer-I ( 5 ), this parallel, is applied, the thickness of the spacer-II ( 6 ) before assembly larger than the height of the spacer-I ( 5 ). 3. The method according to claim 1 and 2, characterized in that the spacer-I ( 5 ) and the spacers ( 14 ) by means of screen printing on the lower substrate ( 1 ) and then hardened. 4. The method according to claim 3, characterized records that curing by exposure to heat and / or by light radiation, such as UV radiation, is made. 5. The method according to claim 1, characterized in that the spacer-I ( 5 ) and the Spacer ter ( 14 ) on the lower substrate ( 1 ) by means of photolithographic processes, or by dispensing and then hardened by annealing. 6. The method according to claim 5, characterized in that the spacer-I ( 5 ) and the spacer ( 14 ) is made of a photostructurable photoresist, preferably a dry photoresist. 7. The method according to claim 1, characterized in that the spacer-I ( 5 ) and the spacer ( 14 ) made of a pre-structured, at least one side adhesive metal or polymer film and glued to the lower substrate ( 1 ). 8. The method according to any one of claims 1 to 7, characterized in that the production of the connection of the upper substrate ( 2 ) with the lower substrate is carried out under the action of pressure and heat and / or UV radiation. 9. The method according to any one of claims 1 to 8, characterized in that an adhesive is used as spacer-II ( 6 ) based on epoxy resin or silicone rubber. 10. 3-D micro flow cell, produced by the method according to one of claims 1 to 9, consisting of a lower and an upper substrate, wherein between the substrates a flow channel provided with fluidic contacts is arranged, through which an electrode system connected to external contacts penetrates, characterized in that on the lower substrate ( 1 ) the spacer-I ( 5 ) defining the flow channel ( 3 ) as well as additional spacers ( 14 ) made of an essentially non-compressible material, or hardenable material, of a predetermined height, are arranged with this lower one Substrate ( 1 ) are irreversibly firmly connected, that the upper substrate ( 2 ) with the lower substrate ( 1 ), the flow channel ( 3 ) tightly sealing, by means of a pasty, curable adhesive layer as Spacer-II ( 6 ) is connected , 11. 3-D micro flow cell according to claim 10, characterized in that the spacer-II ( 6 ) extends on both sides outside the flow channel ( 3 ) on the outside of the spacer-I ( 5 ) in the form of a strip along the same. 12. 3-D micro flow cell according to claim 10 and 11, characterized in that the thickness of the spacer I ( 5 ) and the spacer ( 14 ) is the same and is between ~ 10 microns and ~ 100 microns. 13. 3-D micro flow cell according to claims 10 to 12, characterized in that at least the lower substrate ( 1 ) consists of glass and a thickness of ~ 140 µm. , , 210 µm. 14. 3-D micro flow cell according to one of claims 10 to 12, characterized in that the upper substrate ( 2 ) consists of a plastic film. 15. 3-D micro flow cell according to claim 14, characterized in that the upper substrate ( 2 ) made of a polymer film with a thickness of 170. , , 200 microns exists. 16. 3-D micro flow cell according to claims 10 to 15, characterized in that the loading area of the flow channel ( 3 ) is optically transparent at least in the wavelength range from 250 to 450 nm. 17. 3-D micro flow cell according to one of claims 10 to 16, characterized in that we at least contains the upper substrate ( 2 ) or the lower substrate ( 1 ) metallic microelectrodes ( 13 ) which are in a predetermined geometric relationship to each other and that the upper substrate ( 2 ) face-down is mounted on the lower substrate ( 1 ). 18. 3-D micro flow cell according to claim 17, characterized in that the microelectrodes ( 13 ) of the upper substrate ( 2 ) are provided with contact pads ( 10 ) and with the external contacts ( 9 ) on the lower substrate ( 1 ) by conductive adhesive or solder pads are electrically connected. 19. 3-D micro flow cell according to one of claims 10 to 18, characterized in that the microelectrodes ( 13 ) consist of platinum, gold, tantalum or titanium. 20. 3-D micro flow cell according to one of claims 10 to 19, characterized in that the electrode and connection system on the upper and the lower substrate ( 2 ; 1 ) is insulated over the entire surface by means of an organic or inorganic electrical insulator material, the insulator material is recessed inside the flow channel, on the contact pads and on the contact supports. 21. 3-D micro flow cell according to one of claims 10 to 20, characterized in that on the outside of the upper substrate ( 2 ) an opaque screen ( 17 ) is applied in such a way that the edge region of the flow channel is covered, however the central area is kept clear. 22. 3-D micro flow cell according to claim 21, characterized in that the diaphragm ( 17 ) is designed as a shield from the inside and outside for electromagnetic and bioelectric waves. 23. 3-D micro flow cell according to claim 22, characterized in that the diaphragm ( 17 ) consists of metal. 24. 3-D micro flow cell according to claim 23, characterized in that the aperture ( 17 ) consists of a photolithographically structurable Cu or Al thin film. 25. 3-D micro flow cell according to one of claims 21 to 24, characterized in that the diaphragm ( 17 ) is removable. 26. 3-D micro flow cell according to one of claims 10 to 25, characterized in that in the spacer-I ( 5 ) a groove or other longitudinally extending ben ben is incorporated for receiving adhesive. 27. 3-D micro flow cell according to claim 10, characterized in that the spacer-I ( 5 ) made of silicone rubber is printed on the spacer-II ( 6 ) and after vulcanizing the upper and the lower substrate ( 2 , 1 ) are non-positively connected. 28. 3-D micro flow cell according to claim 10, characterized in that the spacer-I ( 5 ) produced photolithographically on the lower substrate ( 1 ) has a width which essentially corresponds to the parallel arrangement of spacer-I ( 5 ) and spacer -II ( 6 ) and that the upper substrate ( 2 ) is attached to the lower substrate ( 1 ) by adhesive force.