Abstract:
The microreactor is formed by a sandwich including a first body, an intermediate sealing layer and a second body. A buried channel extends in the first body and communicates with the surface of the first body through a first and a second apertures. A first and a second reservoirs are formed in the second body and are at least partially aligned with the first and second apertures. The sealing layer separates the first aperture from the first reservoir and the second aperture from the second reservoir, thereby avoiding contamination of liquids contained in the buried channel from the outside and from any adjacent buried channels. The sealing layer is perforated during use of the device, but a resilient plug can be used to reseal the device.

Description:
PRIOR RELATED APPLICATIONS 
     This application claims priority to application EP03425771.7 filed on Nov. 28, 2003. 
     FIELD OF THE INVENTION 
     The present invention refers to an integrated chemical microreactor with separated channels for confining liquids inside the channels and to the manufacturing process for making same. The chemical microreactors are advantageously used for biological tests. 
     BACKGROUND OF THE INVENTION 
     Typical procedures for analyzing biological materials, such as nucleic acid, involve a variety of operations starting from raw material. These operations may include various degrees of cell purification, lysis, amplification or purification, and analysis of the resulting amplified or purified product. 
     As an example, in DNA-based blood tests the samples are often purified by filtration, centrifugation or by electrophoresis so as to eliminate all the non-nucleated cells. Then, the remaining white blood cells are lysed using chemical, thermal or biochemical means in order to liberate the DNA to be analyzed. 
     Next, the DNA is denatured by thermal, biochemical or chemical processes and amplified by an amplification reaction, such as PCR (polymerase chain reaction), LCR (ligase chain reaction), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), and the like. The amplification step allows the operator to avoid purification of the DNA being studied because the amplified product greatly exceeds the starting DNA in the sample. 
     The procedures are similar if RNA is to be analyzed, but more emphasis is placed on purification or other means to protect the labile RNA molecule. RNA is usually copied into DNA (cDNA) and then the analysis proceeds as described for DNA. 
     Finally, the amplification product undergoes some type of analysis, usually based on sequence or size or some combination thereof. In an analysis by hybridization, for example, the amplified DNA is passed over a plurality of detectors made up of individual oligonucleotide probe fragments that are anchored, for example, on electrodes. If the amplified DNA strands are complementary to the probes, stable bonds will be formed between them and the hybridized probes can be read by observation by a wide variety of means, including optical, electrical, mechanical, magnetic or thermal means. 
     Other biological molecules are analyzed in a similar way, but typically molecule purification is substituted for amplification and detection methods vary according to the molecule being detected. For example, a common diagnostic involves the detection of a specific protein by binding to its antibody or by a specific enzymatic reaction. Lipids, carbohydrates, drugs and small molecules from biological fluids are processed in similar ways. 
     The discussion herein has been simplified by focusing on nucleic acid analysis, in particular DNA amplification, as an example of a biological molecule that can be analyzed using the devices of the invention. However, as described above, the invention can be used for any chemical or biological test. 
     The steps of nucleic acid analysis described above are currently performed using different devices, each of which presides over one part of the process. The use of separate devices decreases efficiency and increases cost, in part because of the required sample transfer between the devices. Another contributor to inefficiencies are the large sample sizes, required to accommodate sample loss between devices and instrument limitations. Most importantly, expensive, qualified operators are required to perform the analysis. For these reasons a fully integrated micro-device would be preferred. 
     Integrated microreactors of semiconductor material are already known. For example, publication EP1161985 (corresponding to U.S. Pat. No. 6,710,311 et seq) describes a microreactor and the respective manufacturing process suitable for making an integrated DNA-amplification microreactor. 
     According to this process, a substrate of monocrystalline silicon is etched in TMAH to form a plurality of thin channels; then an epitaxial layer is grown on top of the substrate and of the channels. The epitaxial layer closes at the top the buried channels and forms, together with the substrate, a semiconductor body. 
     The surface of the semiconductor body is then covered with an insulating layer; heating and sensing elements are formed on the insulating layer; inlet and outlet apertures are formed through the insulating layer and the semiconductor body and connect the surface of the structure so obtained with the buried channels. Then, a covering structure accommodating an inlet and an outlet reservoir is formed or bonded on the structure accommodating the buried channels. 
     The above solution has proven satisfactory, but does not allow separation of the samples because the channels are connected in parallel through the common input and outlet reservoirs. However, in some applications there is need for separating the channels from each other and from the outside environment, both for preventing evaporation and for preventing cross-contamination between channels. 
     Therefore, the aim of the present invention is to provide a microreactor and a manufacturing process overcoming the drawbacks of the known solution. 
     SUMMARY OF THE INVENTION 
     According to the present invention, there are provided a chemical microreactor and its manufacturing process, as defined, respectively, in claim  1  and claim  11 . 
     For a better understanding of the present invention, two preferred embodiments thereof are now described, simply as non-limiting examples, with reference to the attached drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1 and 2  show respectively a cross-section and a top view of a first wafer incorporating a part of a microreactor during a manufacturing step. 
         FIGS. 3 and 4  are a cross-section and a top view of a second wafer of the microreactor according to a first embodiment of the present microreactor. 
         FIG. 5  is a cross-section of the second wafer during a subsequent manufacturing step. 
         FIG. 6  is a cross-section through a composite wafer obtained by bonding the first and second wafers in a final manufacturing step. 
         FIG. 7  is a cross-section of the microreactor in use. 
         FIGS. 8 and 9  are cross-sections of a first wafer incorporating a part of a microreactor according to a second embodiment. 
         FIGS. 10 and 11  are respectively a top view and a cross-section through a composite wafer obtained by bonding the first with a second wafer in a final manufacturing step according to a second embodiment. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Hereinbelow, a first embodiment of the invention will be described with reference to  FIGS. 1 to 7 . The various layers and regions are not in scale, for better representation. 
     Initially, process steps are carried out similar to those above described for the known process. Accordingly,  FIG. 1 , a first wafer  1  of monocrystalline silicon is etched in TMAH to form a plurality of channels  3 . To this end, a grid-like mask is used, e.g. as disclosed in EP1193214 (corresponding to US2002045244 and U.S. Pat. No. 6,770,471) or as disclosed in copending patent application “Integrated chemical microreactor with large area channels and manufacturing process thereof” filed on the same date. 
     Then, a structural layer is grown on top of the channels. The structural layer closes the top the channels  3  and forms a substrate  2  of semiconductor material with buried channels. The surface  4  of the substrate  2  is then covered with a first oxide layer; heating elements  10  of polycrystalline silicon are formed thereon; a second oxide layer is deposited and forms, with the first oxide layer, a first insulating layer  5 ; contact regions  11  (and related metal lines) are formed in contact with the heating elements  10 ; a second insulating layer  13  is deposited, for example of TEOS, defining an upper surface  12  of the first wafer  1 . 
     Then, inlet apertures  14   a  and outlet apertures  14   b  are etched. The apertures  14   a  and  14   b  extend from the upper surface  12  through the second insulating layer  13 , the first insulating layer  5  and the substrate  2  as far as the channels  3  and are substantially aligned with the longitudinal ends thereof. This is visible in  FIG. 2 , wherein channels  3  are drawn with dashed lines. In the shown example, one inlet aperture  14   a  and one outlet aperture  14   b  is formed for each channel  3 . In the alternative, two or more channels  3  may share the same inlet and outlet apertures  14   a ,  14   b , if parallel processing in a part of channels  3  is desired. 
     In the meantime, beforehand or subsequently, a second wafer  15  of glass is treated to form reservoirs ( FIGS. 3 and 4 ). In detail, the second wafer  15 , formed by a glass sheet  18  having a surface  19 , is subjected to a lithographic process, in a per se known manner, to define an inlet opening  16   a  and an outlet opening  16   b  intended to be aligned with the inlet and outlet apertures  14   a ,  14   b  and to form inlet/outlet reservoirs. 
     Then,  FIG. 5 , a bonding layer  20  is applied on surface  19  of the glass sheet  18 . For example, the bonding layer  20  is made of dry resist, with a thickness of 10-30 μm, and may be the product known by the commercial name “Riston® YieldMaster®” by Du Pont, that can be laminated in thin layers, or the resist sold by the firm Tokyo Ohka Kogyo Co., Ltd. 
     Subsequently,  FIG. 6 , the second wafer  15  is turned upside down and put on the first wafer  1 , with the bonding layer  20  in contact with the surface  12  of the first layer; then the sandwich including the first wafer  1 , the bonding layer  20  and the second wafer  15  is treated to cause bonding of the bonding layer  20  to the first wafer  1 , thereby obtaining multiple wafer  21 . 
     For example, bonding may be carried out at a temperature of 140-180° C., preferably 160° C.; at a force of 5-9 kN, preferably 7 kN (for wafers having a diameter of 6″) and in a vacuum or low pressure condition of 5×10 −7  to 5×10 −6  bar, preferably 10 −6  bar. 
     In this way, the channels  3  are not connected to the inlet and outlet openings  16   a ,  16   b  forming inlet and outlet reservoirs, but are separated therefrom and from the outside environment by the bonding layer  20  that now acts as a sealing layer; thereby the channels are kept at the low pressure condition that existed during bonding. 
     After dicing the multiple wafer  21  into single microreactors  22 ,  FIG. 7 , the inlet opening  16   a  is closed by a plug  25 . The plug  25  is e.g. formed by applying a drop of liquid thermosetting material that is subsequently hardened by heat. 
     In the alternative, the plug  25  may be applied only when the microreactor  22  is used, and may comprise a preformed plug  25  already connected to a syringe  26  of the retractable type. Preferably, the plug  25  is of a resilient material that is able to be punctured by the syringe  26  and to close the puncture passage after removal of the syringe, without forming shavings. For example, the plug  25  may be made of PVC including a softener, of the type used for biomedical applications. 
     In use, when liquid is to be inserted in a specific channel  3 , a syringe  26  is inserted through the plug  25 , perforates the bonding layer  20  and injects the mixture or mixtures to be treated in the selected channel (or channels)  3 . Injection of the liquid to be treated is favored by the presence of low pressure (vacuum). 
     The syringe  26  is then removed and the plug  25  closes to as to ensure a complete isolation of the channel(s)  3  containing the injected liquid with respect to the environment during thermal cycling or other provided treatment. 
     At the completion of the treatment, the liquid is extracted by perforating the bonding layer  20  at the outlet reservoir  16   b ; for example, another syringe may be used to aspirate the liquid, or a plunger may break the bonding layer  20  at the outlet reservoir  16   b  and a pressure be exerted from the inlet reservoir  16   a.    
     According to a different embodiment, the bonding/sealing layer is applied to the semiconductor wafer and an auxiliary hole is provided to create the vacuum inside the channels during bonding, as shown in  FIGS. 8-10 , wherein the first wafer has been represented in a very schematic way. 
     In detail,  FIG. 8 , a first wafer  1  is subjected to the same manufacturing steps described above with reference to  FIG. 1 . Thus, the first wafer  1  is etched to form channels  3 ; a structural layer is grown to form a substrate  2  of semiconductor material; insulating layers  5 ,  13 , and heating elements  10  and contacts  11  (none shown, please refer to  FIG. 1 ) are formed. 
     Then the inlet and outlet apertures  14   a ,  14   b  are etched. According to the second embodiment, simultaneously with the inlet and outlet apertures  14   a ,  14   b , at least one hole  30  is formed for each channel  3 , intermediate to the inlet and outlet apertures  14   a ,  14   b . In case of more channels  3  connected to same inlet/outlet apertures  14   a ,  14   b , a single hole  30  may be sufficient. 
     Then,  FIG. 9 , a bonding layer  31  is formed on a surface  32  of wafer  1 . Preferably, the bonding layer  31  is dry resist which is laminated onto the surface  32 . For example, the bonding layer  31  may be of the same material as bonding layer  20  of  FIGS. 5-7  and have the same thickness (10-30 μm). 
     Thereafter, the bonding layer  31  is lithographically defined to form connection openings  33  over the holes  30  (see also  FIG. 10 ). Preferably, one connection opening  33  is formed for each hole  30 , as shown in the drawings; in case of parallel connected channels  3 , a connection opening  33  is in common to more holes  30  and/or more channels  3 . 
     Thereby, the inlet/outlet apertures  14   a ,  14   b  are upwardly closed by the bonding layer  31 , but the channels  3  are connected to the outside environment by the holes  30  and the connection openings  33 . 
     Then,  FIG. 11 , the first wafer  1  is bonded to a second wafer  15  formed by a glass sheet  18  wherein, previously, an inlet opening  16   a  and an outlet opening  16   b  have been formed, analogously to what has been described with reference to  FIGS. 3 and 4 . Also here, the input and output openings  16   a ,  16   b  are designed so as to be aligned to the inlet and outlet apertures  14   a ,  14   b.    
     Bonding may be carried out as before described, that is at a temperature of 140-180° C., preferably 160° C.; at a force of 5-9 kN, preferably 7 kN and in a vacuum or low pressure condition of 5×10 −7  to 5×10 −6  bar, preferably 10 −6  bar. Thus, during bonding, the channels  3  are maintained at low pressure by virtue of the holes  30  and the connection openings  33 . 
     Thereby, a multiple wafer  35  is obtained, wherein the input and output openings  16   a ,  16   b  are closed upwardly by the bonding layer  31  and the holes  30  are upwardly closed by the glass sheet  18 . However, the channels are buried inside the monolithic structure of the first wafer. As used herein “buried channel” is defined as a channel or chamber that is buried inside of a single monolithic support, as opposed to a channel or chamber that is made by welding or otherwise bonding two supports with a channel or two half channels together. Of course, other components may be welded or otherwise attached to the monolithic support, as required for the complete integrated device. 
     Therefore, also here, the channels  3  are sealed from the outside environment by the bonding layer  31  and are kept at the low pressure condition existing during bonding. 
     In use, analogously to the above, the mixture or mixtures is inserted in the selected channel (or channels)  3  in a very simple way, by virtue of the vacuum condition in the channel(s)  3  by simply perforating the bonding layer  31  with a syringe at the input opening  16   a . Furthermore, a plug  25  may be provided to seal the channel(s)  3  after perforation. 
     By virtue of the described reactor and process, the finished microreactor  22  has channels  3  sealed from the outside, and allows separation of the material accommodated in the channels from the external environment. Furthermore the microreactor  22  is able to avoid any interference and contamination by the environment as well as by adjacent channels. 
     The manufacturing process is straightforward and employs steps that are common the manufacture of microreactors of this type; thus the resulting device is simple and cheap. 
     The separated channels described herein may be combined in an integrated device with any other components required for the application of interest. For example, the separated channels may be combined with one or more of the following: micropump, pretreatment channel, lysis chamber, detection chamber including detection means, capillary electrophoresis channel, and the like (see especially, Italian patent application TO2002A000808 filed on Sep. 17, 2002, publication nos. EP1400600, filed on Sep. 17, 2003 and US2004132059 filed on Sep. 16, 2003, in the name of the same applicant). The heaters may be integral, or may be provided by the platform into which the disposable microreactor wafer is inserted. The overall design of the complete device will be dictated by the application, and need not be detailed herein. 
     It is clear that numerous variations and modifications may be made to the process and to the microreactor described and illustrated herein, all falling within the scope of the invention, as defined in the attached claims.