DE10338837A1 - Production of DNA chip arrays, to duplicate and characterize nucleic acids, has a structure of ring-shaped sample chambers mounted on an optically transparent wafer carrier which is divided into separate chip arrays - Google Patents

Abstract

To produce a DNA chip array to duplicate and characterize nucleic acids, as a biochip with a rear surface and a sample array on the upper surface, an optically transparent carrier wafer is prepared with biochip carriers, and the structured biochips are prepared. The biochips are fitted, and the sample chamber body is mounted to the upper side of the biochip carrier in a sealed fit, and the wafer is divided at separation edges (16) into separate DNA chip arrays (15). The ring-shaped sample chamber body is bonded within a holding ring (18), and the upper side of the sample chamber is fitted with a heating unit (14).

Description

The The invention relates to a method for producing a device for duplication and characterization of nucleic acids (DNA chip array) with a Sample chamber in which a biochip with a back and a sample array having top is arranged.

DNA chip arrays have bioactive structures that react to different environmental influences. For this purpose, the chips used for gases and liquids or aerosol sensitive surface structures (sample array) on that especially for biological substances such as lymphatic fluids, blood, Urine and other biological fluids are sensitive. The currently available DNA chip arrays will be Manufactured costly and are therefore very expensive.

On such a DNA chip array is, for example, in WO 01/02094 A1 described. A wide variety of biochemicals also originate from this publication Procedures emerging in the use of these DNA chip arrays Role-play.

to introduction the genetic engineering or biochemical background is referred to in this WO 01/02094 directed.

task the present invention is to provide a manufacturing method with what devices for duplication and characterization of nucleic acids (DNA chip arrays) to provide with which on an industrial scale cost-effective Manufacture of such DNA chip arrays is ensured, so in particular the DNA chip arrays made with it as cheap disposable products can be used.

• – Bereitstellen eines optisch transparenten Trägerwafers mit in Zeilen und Spalten angeordneten Biochipträgern mit Oberseiten und Rückseiten;
• – Bereitstellen von Biochips mit einer Rückseite und einer ein Probenarray aufweisenden Oberseite;
• – Bereitstellen von Probenkammerkörpern;
• – Befestigen der Biochips;
• – dichtendes Befestigen von Probenkammerkörpern auf die Oberseiten der Biochipträger;
• – und Trennen des optisch transparenten Trägerwafers in einzelne DNA-Chip-Arrays.

According to the invention, this object is achieved by a method for producing a device for the duplication and characterization of nucleic acids (DNA chip array) with a sample chamber in which a biochip with a rear side and an upper side having a sample array is arranged, which comprises the following steps:

• - Providing an optically transparent carrier wafer with biochip carriers arranged in rows and columns with top and back sides;
• - Providing biochips with a rear side and an upper side having a sample array;
• - Provision of sample chamber bodies;
• - attaching the biochips;
• - sealingly attaching sample chamber bodies to the tops of the biochip carriers;
• - And separating the optically transparent carrier wafer into individual DNA chip arrays.

the method according to the invention The idea is based on the manufacturing steps, which are essentially in the manufacture of semiconductor components or in the housing of Semiconductor devices are used in the manufacture of DNA chip arrays make usable.

The size Advantage of the method according to the invention lies in the fact that, in contrast to the prior art, a multitude from biochip carriers to DNA chip arrays can be processed.

In a variant of the present invention are made after attaching the sample chamber body the tops of the biochip carriers heating elements still applied. These heating elements are usually necessary to the biological fluid to be examined to those for the amplification (duplication) of those to be examined of deoxyribonucleic acids (DNA) necessary temperature plateaus to bring.

The Sample chamber body preferably consist of a rubber, in particular a silicone rubber, which can be pierced by an injection needle to close the investigating biological fluid to inject or around the injected biological fluid remove again. When puncturing and then removing the Injection needle closes the rubber seals.

It however, sample chamber bodies are also conceivable, that are not made of a rubber or rubber-like material, but from a thermoplastic, which has filling and outlet channels, which are in the form of holes.

The Sample chamber body can doing an annular or a pot-shaped one Have design. If the sample chamber body is ring-shaped the biochips are attached directly to the tops of the biochip carriers, especially glued.

Provided the sample chamber body a pot-shaped Have design, the biochips are on the bottoms of the cup-shaped Sample chamber body attached, especially glued.

When gluing the biochips to the bottoms of the sample chamber bodies or to the tops of the biochip carriers, particular care must be taken to ensure that the adhesives or adhesive films used are biochemically inert. Adhesives that interact chemically with the biochemi Materials that chemically react on the tops of the biochips that form the sample array must be avoided.

at the execution, at the ring-shaped Sample chamber body is used in a further development of the present invention a recess is made in the tops of the individual biochip carriers, which is used to hold the biochips. This has the advantage that when using silicone glue or other biochemically inert glue the depression serves as a reservoir delimiting the adhesive. Furthermore, dead spaces are avoided by the depressions. It’s washing up The sample chamber is advantageous because there is no residual liquid after rinsing should remain in the sample chamber. The recess can be in the Biochip carrier via hot stamping or different punching steps are introduced. On the one hand, the Deepening during of processing. However, it is conceivable and also cheaper, to provide the recesses in a prefabricated carrier wafer.

In a further embodiment of the present invention can be found in the backs the biochip carrier Wells are introduced so that the individual biochip carrier in cross section has the shape of a plane-convex lens. The flat side of this The plane-convex lens forms the top of the biochip carrier to which the biochip is applied. The convex side of the plan-convex Lens is created through the wells. This configuration is advantageous to the optical evaluation in the DNA analysis that with the DNA chip arrays in question, to support. Of It is also possible through the targeted choice of material for the biochip carrier particularly cheap Refractive index for preselect the optical evaluation and the lowest possible natural fluorescence.

How already stated above, can the sample chamber body connected to the biochip carriers in various ways become. Besides connecting via Cohesion, d. H. so about Gluing the sample chamber body with the biochip carrier, it is conceivable the sample chamber body with the biochip carrier form-fitting to connect, for example by a special shape, for example due to a claw between the underside of the sample chamber body and the top of the biochip carrier. It is also conceivable the sample chamber body friction, d. H. especially by clamping or pressing on the top attach the biochip carrier.

In Another step will then be on the bottom or top the sample chamber body Heating / cooling elements applied.

The Application of the heating elements can be either form-fitting or friction-locking or also cohesive, d. H. in particular by gluing. The heating elements consist, for example, of eight individual ones connected in parallel microstructured resistance heating cables through which the under the sample chamber body located sample carrier and the biological fluid to be analyzed contained in it can be heated homogeneously. The individual resistance lines of the heating element are preferably dimensioned such that the optical accessibility the biochemically active top side of the biochips is not impaired. As a substrate for the Heizelemenzte is preferably silicon, because this Material has high thermal conductivity having. This also allows rapid cooling during the temperature cycles is important in the polymerase chain reaction.

The inventive method is preferably with a carrier wafer for biochips performed with biochip carriers arranged in rows and columns, the consists of an optically transparent material. This material can, for example, glass, a polycarbonate, a polypropylene Polymethylacrylat or another transparent plastic. Suitable plastics are sold, for example, under the "Plexiglas", "Makralon", "Topas", etc. brands.

The last process step consists of separating the finished processed optically transparent carrier wafer into individual DNA chip arrays. The type of separation depends on the type of carrier wafer material used. Sawing, punching, laser cutting are particularly suitable as separation processes and water jet separation.

The inventive method is characterized by easy automation with machines from that in semiconductor manufacturing or in semiconductor assembly manufacturing present or very similar are. Because the DNA chip arrays to be manufactured are biochemically sensitive objects the procedure must be carried out in a sterile clean room environment. There according to the present invention, much of that in semiconductor manufacturing or semiconductor assembly facilities used existing machinery the essential clean room conditions result as a benefit inevitably, without an additional Investment made must become.

The produced with the inventive method DNA chip arrays ask for are already biochemically hermetically sealed units, their further processing no longer in a sterile environment, especially in a clean room environment.

The Separate into individual DNA chip arrays, the application of connecting cables to the processed heating elements or another attachment of additional Components can therefore be made in production facilities that do not have clean room conditions or sterile environments.

The Invention will now be discussed in more detail with reference to the accompanying figures. there demonstrate:

1a to d an embodiment of the present invention and

2a to d an alternative embodiment of the present invention.

After 1a the first process step is an optically transparent carrier wafer 1 with biochip carriers arranged in rows and columns 2 provided. The biochip carriers 2 have tops 3 and backs 4 on. In the tops of the biochip carriers 2 are deepening 5 brought in.

As from the 1b can be seen in the next process step into the wells 5 that on the tops 3 the biochip carrier 2 are arranged, biochips 6 brought in. The biochips 6 have a back 7 and a top 8th on. On the top 8th there is a so-called sample array 9 ,

The biochips 6 are typically chips with an area of approximately 3 mm × 3 mm that are on the tops 8th have a DNA sample array.

The individual biochip is used for attachment 6 in the recess 5 with a biochemically inert and transparent adhesive 10 bonded. The deepening 5 serves on the one hand as an adhesive reservoir and on the other hand the dead volumes in the sample chamber are minimized, which makes it easier to rinse the sample chamber. A silicone adhesive is used in the present example.

In a subsequent step ( 1c ) are then on the tops 3 of the individual biochip carriers 2 ring or crescent shaped sample chamber body 11 placed. The ring-shaped sample chamber body 11 again have tops 12 and backs 13 on. In the present case, the backs 13 the sample chamber body 11 with the tops 3 the biochip carrier 2 with a biochemically inert adhesive 10 bonded.

The sample chamber body 11 consists here of a silicone rubber that can be pierced by an injection needle (not shown) without an opening remaining in the silicone rubber material after removal of the injection needle. The attachment of the sample chamber body 11 on the tops 3 the biochip carrier 2 is sealing. It is essential that between the backs 13 the sample chamber body 11 and the tops 3 the biochip carrier 2 a complete seal is made. When using the finished DNA chip arrays later, it is essential that no leaks occur, so that the biological fluid to be analyzed (not shown) can leave the sample chamber body or contamination can occur from outside.

In a subsequent process step, which in the 1d is shown on the tops 12 the annular sample chamber body 11 heating elements 14 applied. In the present embodiment of the invention, the heating elements 14 to the tops 12 the sample chamber body 11 again applied cohesively. The cohesive application is again carried out by gluing with a biochemically inert adhesive 10 ,

After the heating elements have been applied 14 to the tops 12 the sample chamber body 11 are the individual finished DNA chip arrays 15 here by sawing at the designated separating edges 16 separated (singulated).

The 2a to d show an alternative embodiment of the present invention. The shows 2a again an optically transparent carrier wafer 1 with biochip carriers arranged in rows and columns 2 with tops 3 and backs 4 ,

In the present embodiment are in the back 4 of the individual biochip carriers 2 recesses 17 brought in. Through these recesses 17 points out the individual biochip carrier 2 on the back an at least partially convex shape. Since the individual biochip carriers are optically transparent, the biochip carriers shown form 2 in cross section a plane-convex lens. The flat part of the plane-convex lens is the top 3 the biochip carrier 2 represents the convex part of the plane-convex lens with recesses 17 provided backs 4 the biochip carrier.

Furthermore are on the tops 3 the biochip carrier 2 circumferential receiving rings 18 molded on to accommodate the sample chamber body 11 serve. It can be used instead of the mounting rings shown 18 however, other shapes can also be formed, for example crescent-shaped bodies with openings for the injection needles

In a subsequent step, ge according to the 2 B to the tops 3 the biochip carrier 2 in the receiving rings 18 Sample chamber body 11 applied. The sample chamber body 11 again have tops 12 and backs 13 on. The sample chamber body shown 11 consist in turn of a silicone rubber, which can be pierced by an injection needle, without having an opening in the wall of the sample chamber body after removal of the injection needle (not shown) 11 remains. The injection needles are inserted into bores or openings in the wall of the receiving rings (not shown) and pushed through the wall of the sample chamber body.

In the embodiment shown, the backs 13 the annular sample chamber body 11 with the tops 3 the biochip carrier 2 inside the receiving rings 18 using a biochemically inert adhesive 10 cohesively connected.

After the ring-shaped sample chamber body has been applied 11 to the tops 3 the biochip carrier 2 is in the opening of the sample chamber body 11 a biochip 6 brought in. The introduced biochips 6 have backs 7 and tops 8th on. The tops 8th in turn are using a sample array 9 provided that is biochemically active. The backs 7 the biochips 6 turn with the tops 3 the biochip carrier 2 cohesive by gluing with a biochemically inert adhesive 10 cohesively connected.

Then heating elements 14 again on the tops 12 the annular sample chamber body 11 applied. In the present case, the heating elements 14 in a circulating paragraph 19 inside the receiving rings 18 introduced and pressed in frictionally by caulking. This procedure eliminates the time-consuming hardening of the relatively poorly hardening biochemically inert silicone adhesives, which are typically involved in the integral connection of individual components of the DNA chip arrays 15 be used.

The in the 2a shown biochip carrier 2 point to their tops 3 step-like increases on which a receptacle for the individual heating elements to be introduced 14 exhibit.

After the heating elements have been applied 14 the carrier wafer shown is then separated at the separating edges 16 into individual DNA chip arrays 15 ,

The one in the 2a to d carrier wafer shown 1 consists of optically transparent plexiglass that can be easily separated using water jet cutting.

Furthermore, the in the 2a to d shown optically transparent carrier wafer 1 manufactured using an injection molding process so that the recesses 17 on the back 7 and the receiving rings 18 with the paragraphs 19 on the tops 8th can be easily prefabricated.

In the in the 2a to d In the exemplary embodiment shown, the biochip carrier, sample chamber body, biochip, heating elements were integrally connected by gluing. However, it is also possible to frictionally connect the biochip carrier, sample chamber body, biochip, heating elements to one another, so that the long curing times are eliminated.

1

carrier wafer

2

biochip support

3

top biochip support

4

back biochip support

5

deepening

6

biochip

7

back biochip

8th

top biochip

9

sample array

10

adhesive

11

Sample chamber body

12

top Sample chamber body

13

back Sample chamber body

14

heating elements

15

DNA chip array

16

separating edge

17

deepening

18

Afnahmering

19

paragraph

Claims (20)

Method for producing a device for the duplication and characterization of nucleic acids (DNA chip array) with a sample chamber in which a biochip ( 6 ) with a back and a sample array ( 9 ) having the upper side is arranged, with the following steps: provision of an optically transparent carrier wafer ( 1 ) with biochip carriers arranged in rows and columns ( 2 ) with tops ( 3 ) and backsides ( 4 ); - Provision of biochips ( 6 ) with a back ( 7 ) and one a sample array ( 9 ) top side ( 8th ); - Provision of sample chamber bodies ( 11 ) with tops ( 12 ) and backsides ( 13 ); - attaching the biochips ( 6 ); - sealing the sample chamber body ( 11 ) on top ( 12 ) the biochip carrier ( 2 ); - separation of the optically transparent carrier wafer ( 1 ) into individual DNA chip arrays ( 15 ). The method of claim 1, wherein after attaching the sample chamber body ( 11 ) on top ( 3 ) the biochip carrier ( 2 ) on top ( 12 ) the sample chamber body ( 11 ) Heating elements ( 14 ) are applied. The method according to claim 1 or 2, wherein ring-shaped or crescent-shaped sample chamber bodies ( 11 ) to be provided. The method of claim 3, wherein the biochips ( 6 ) on top ( 3 ) the biochip carrier ( 2 ) are attached. The method of claim 1 or 2, wherein cup-shaped sample chamber bodies are provided become. The method of claim 5, wherein the biochips are on the floors the pot-shaped Sample chamber body be attached. Method according to one of claims 1 to 6, wherein for receiving the biochips ( 6 ) or the sample chamber body ( 11 ) in the tops of the biochip carriers ( 2 ) Deepenings ( 5 ) are introduced. Method according to one of claims 1 to 7, wherein the rear sides ( 7 ) the biochip carrier ( 2 ) have an at least partially convex shape. Method according to one of claims 1 to 8, wherein the biochips ( 6 ) with the tops ( 3 ) the biochip carrier ( 2 ) or the floors of the sample chamber body are integrally and / or frictionally connected. The method of claim 9, wherein the biochips ( 6 ) on top ( 3 ) the biochip carrier ( 2 ) or the bottoms of the sample chamber bodies are glued. The method of claim 10, wherein the biochips ( 6 ) with a transparent, biochemically inert adhesive ( 10 ) be glued. The method of claim 9, wherein the biochips ( 6 ) on top ( 3 ) the biochip carrier ( 2 ) are pressed from the sample chamber bodies. The method of claim 12, wherein the biochips ( 6 ) in the wells ( 5 ) are pressed from the sample chamber bodies. Method according to one of claims 1 to 12, wherein the sample chamber body ( 11 ) with the tops ( 3 ) the biochip carrier ( 2 ) be connected frictionally. Method according to one of claims 1 to 12, wherein the sample chamber body ( 11 ) with the tops ( 3 ) the biochip carrier ( 2 ) are firmly bonded. Method according to one of claims 2 to 15, wherein the heating elements ( 14 ) with the tops ( 12 ) the sample chamber body ( 11 ) be positively connected. Method according to one of claims 2 to 15, wherein the heating elements ( 14 ) with the tops ( 12 ) the sample chamber body ( 11 ) be connected frictionally. Method according to one of claims 2 to 15, wherein the heating elements ( 14 ) with the tops ( 12 ) the sample chamber body ( 11 ) are firmly bonded. Carrier wafer ( 1 ) for biochips with biochip carriers arranged in rows and columns ( 2 ) consisting of an optically transparent material. carrier wafer according to claim 19 consisting of an optically transparent, pourable, especially injection moldable plastic.