Abstract:
A microreactor includes: a substrate ( 2; 102; 202 ) made of semiconductor material; a plurality of wells ( 5; 105; 205 ) separated by walls ( 6; 106; 206 ) in the substrate ( 2; 102; 202 ); a dielectric structure ( 7; 107; 207   a,    207   b ) coating at least the top of the walls ( 6; 106; 206 ); a cap ( 3; 103; 203 ), bonded to the substrate ( 2; 102; 202 ) and defining a chamber ( 10; 110; 210 ) above the wells ( 5; 105; 205 ); and a biasing structure ( 2, 8, 13;   102, 108, 113; 202, 208 a,  208 b,  213 ), configured for setting up a voltage (VB) between the substrate ( 2; 102; 202 ) and the chamber ( 10; 110; 210 ).

Description:
PRIOR RELATED APPLICATIONS 
       [0001]    This application claims priority to Italian Patent Application No. TO2013A000264 of Mar. 29, 2013 and incorporated by reference in its entirety herein. 
       FEDERALLY SPONSORED RESEARCH STATEMENT 
       [0002]    Not applicable. 
       REFERENCE TO MICROFICHE APPENDIX 
       [0003]    Not applicable. 
       FIELD OF THE DISCLOSURE 
       [0004]    The present disclosure relates to a microreactor and to a method for loading a liquid into the microreactor. 
       BACKGROUND OF THE DISCLOSURE 
       [0005]    As is known, some types of microreactors, such as microreactors for biochemical analyses, comprise arrays of wells for receiving small volumes of reagents in the form of liquids or gels, especially water-based ones, and/or given volumes of specimens to be analyzed. Microreactors of this type respond, amongst other things, to the widespread need of increasing the level of parallelism in the execution of analysis procedures, for example for diagnostic or experimental purposes. Each well may be prepared with different reagents, and hence various analytical procedures may be conducted simultaneously on a single biological sample. 
         [0006]    In a typical application, the wells are prepared for performing reactions of amplification of nucleic acids, for example by PCR (Polymerase Chain Reaction). In addition to the mixture of reagents necessary for amplification, loaded in each well are respective detector sequences, which comprise single stranded oligonucleotides capable of binding to corresponding DNA or RNA sequences that may be present in the biological sample. This way, each well may be dedicated to recognition of a specific target sequence (for example, corresponding to a specific pathogenic agent). 
         [0007]    Handling of small volumes of biological sample may, however, create difficulties, particularly in loading of the wells. All the wells must receive a sufficient amount of biological sample. Once loading has been carried out, moreover, the contents of each well must be kept segregated from those of the other wells during the reactions in order to prevent any contamination that might jeopardize the outcome of the processes. 
         [0008]    The biological sample may be introduced into the wells manually, using pipettes. In this case, the microreactors are initially uncovered to enable access to the wells, and are closed only subsequently. Manual loading presents evident limits both owing to the impossibility of treating very small volumes (a few microliters) and because contaminations are relatively likely to occur. 
         [0009]    In other microreactors, the specimen is loaded in a common reservoir and distributed to the wells via microchannels, in which the fluid advances by capillary action. In this case, very small volumes of fluid can be treated. But, problems may arise owing to the formation of bubbles in the microchannels, thus impeding flow and reducing the amount of available liquid. 
         [0010]    When capillary forces are involved, air bubbles may easily remain trapped during loading. The geometry and the affinity of the specimen with the material forming the microchannels may produce highly unstable menisci. The edges of the menisci may join up in given conditions, and the air bubbles may be trapped inside the liquid. A single air bubble may occupy a relatively wide portion of the microchannels and prevent an adequate volume of specimen from reaching one or more wells. Analysis may be impaired because important process parameters, such as volume, equilibrium of the reagents, pressure and temperature, are affected by the presence of bubbles. 
         [0011]    The aim of the present disclosure is to provide a microreactor and a method for loading a liquid into a microreactor that will enable the limitations described to be overcome. 
       SUMMARY OF THE DISCLOSURE 
       [0012]    According to the present disclosure, a microreactor and a method for loading a liquid into the microreactor are provided. 
         [0013]    The microreactor includes a substrate made of semiconductor material; a plurality of wells separated by walls in the substrate; a dielectric structure coating at least the top of the walls; a cap, bonded to the substrate and defining a chamber above the wells; and a biasing structure, configured for setting up a voltage between the substrate and the chamber. This structure allows fluid in the chamber above the wells to enter the wells when voltage is applied through the electrowetting phenomena. Bubbles are avoided and all wells can be simultaneously filled. 
     
    
     
       DESCRIPTION OF DRAWINGS 
         [0014]    For a better understanding of the disclosure, some embodiments thereof will now be described, purely by way of non-limiting example and with reference to the attached drawings, wherein: 
           [0015]      FIG. 1  is a simplified top plan view of a microreactor according to one embodiment of the present disclosure; 
           [0016]      FIG. 2  is a cross section of the microreactor of  FIG. 1 , taken along the line II-II of  FIG. 1 ; 
           [0017]      FIGS. 3 and 4  show the view of  FIG. 2  in successive steps during loading of a liquid; 
           [0018]      FIG. 5  is a cross section of a microreactor according to a different embodiment of the disclosure; 
           [0019]      FIG. 6  is a simplified top plan view of a microreactor according to a further embodiment of the present disclosure; 
           [0020]      FIG. 7  is a cross section of the microreactor of  FIG. 6 , taken along the line VII-VII of  FIG. 6 ; 
           [0021]      FIG. 8  is a simplified block diagram of a cartridge for biochemical analyses incorporating a microreactor according to one embodiment of the present disclosure; and 
           [0022]      FIG. 9  is a simplified block diagram of an apparatus for analysis that uses the cartridge of  FIG. 8 . 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    In  FIGS. 1 and 2 , a microreactor according to one embodiment of the present disclosure is designated as a whole by the reference number  1 . The microreactor  1  may be, for example, of the type used for biochemical analyses, particularly for reactions of amplification of nucleic acids and identification of specific target sequences. It is understood, however, that the disclosure is not limited to this type of application and may advantageously be exploited for providing microreactors of any kind. 
         [0024]    The microreactor  1  comprises a semiconductor substrate  2 , for example doped monocrystalline silicon, and a cap  3  bonded to a top face  2   a  of the substrate  2  through a bonding layer  4 . By “top face” is meant, here and in what follows, a face of the substrate  2  that, in use, is to face upwards, such that the cap  3  prevents exit of the fluid that has been loaded into the microreactor  1 . 
         [0025]    The substrate  2  contains a plurality of wells  5 , arranged in arrays and separated from one another by walls  6 . The top of the walls  6  (i.e., in practice the top face  2   a  of the substrate  2 ) and the bottom of the wells  5  are coated by dielectric regions  7 , whereas vertical faces of the walls  6  are exposed (e.g., uncoated). 
         [0026]    The wells  5  may have, for example, a rectangular or circular shape. In one embodiment, the wells  5  have dimensions such that the surface tension of the fluid to be loaded into the microreactor  1 , particularly the surface tension of the water for samples of biological fluids, is sufficient to prevent spontaneous filling of the wells  5  themselves. For example, the wells  5  have a square cross section with a side of approximately 800 μm and have a depth of approximately 400 μm. 
         [0027]    The substrate  2  is moreover provided with a first biasing electrode  8 , which, in one embodiment, is provided on a bottom face  2   b.    
         [0028]    The cap  3  is bonded to the top face  2   a  of the substrate  2  and is arranged for covering the wells  5 . Inside, the cap  3  has a cavity that defines a chamber  10  together with the substrate  2 . In greater detail, the chamber  10  extends over the entire array of the wells  5  and is accessible from outside through an inlet opening  11  for loading a fluid to be processed, for example a biological sample. The chamber  10  is shaped in such a way that the fluid introduced through the inlet opening  11  distributes over the top of all the wells  5 . In some embodiments, the volume of the chamber  10  is smaller than or substantially equal to the sum of the volumes of all the wells  5  (for example, 20 μl). 
         [0029]    The cap  3  moreover has biasing openings  12  configured to enable introduction of one or more needle-shaped second electrodes  13  within the chamber  10 , while preventing exit of the fluid. For this purpose, the biasing openings  12  have a cross section of a size such as to prevent spontaneous rising of the fluid by capillary action. For example, the cross section of the biasing openings  12  has dimensions smaller than the dimensions of the wells  5 . The openings  12  enable the exit of any air present in the chamber  10  during introduction of liquid into the chamber  10  itself. 
         [0030]    In one embodiment, the biasing openings  12  are arranged in positions corresponding to walls  6  or crossings between walls  6  that delimit adjacent wells. 
         [0031]    The microreactor  1  exploits the electrowetting phenomenon. 
         [0032]    When a volume of liquid  14 , such as a biological sample in the example described, is loaded into the microreactor  1  through the inlet opening  11 , the chamber  10  is filled. However, the liquid  14  does not manage to penetrate into the wells  5  as a result of the surface tension and of the dimensions of the wells  5  themselves. 
         [0033]    Through the biasing openings  12 , one or more second electrodes  13  are hence brought into contact with the liquid  14  inside the chamber  10 . Using an electric power-supply source  15 , the first biasing electrode  8  and the second biasing electrodes  13 , a biasing voltage VB is applied between the substrate  2  and the liquid  14 . In practice, the entire substrate  2  functions as a biasing electrode. 
         [0034]    The liquid  14  and the substrate  2  are initially insulated both by the dielectric regions  7  that are located at the top of the walls  6 , and by the air contained in the wells  5 . In general, in a condition where a liquid and an electrode are separated by an insulating region and are subjected to a voltage, the forces that act within the liquid cause wettability to increase. In the example described, in practice, the biasing voltage VB causes the surface tension of the liquid  14  to be overcome, and brings about an interruption in continuity. It should be noted that the arrangement of the second electrodes  13  above walls  6  or crossings between walls  6  favors division of the volume of liquid  14 . 
         [0035]    The liquid  14  thus begins to penetrate into the wells  5 , filling of which is started, as shown in  FIG. 3 . When the liquid  14  comes into contact with the exposed faces of the walls  6 , its conditions are modified because the electrical insulation from the substrate  2  ceases locally. However, the portion of liquid  14  inside the wells  5  is subjected to capillary forces that start to prevail, thus filling the wells. 
         [0036]    Once the surface tension of the liquid  14  has been overcome, the wells  5  are filled as a result of the capillary forces. In this way, the initial volume of liquid is divided into a plurality of drops that occupy respective wells  5  and are separated from one another, as shown in  FIG. 4 . 
         [0037]    The capillary forces are exploited only in order to complete the filling of the wells, and there is no risk of formation of bubbles in the liquid. 
         [0038]    Once the liquid  14  has been distributed in the wells  5 , the electrical-supply source  15  is turned off or disconnected from the microreactor  1  in order to remove the biasing voltage VB and prevent waste of energy. 
         [0039]    In addition, the chamber  10  may be filled with oil once the wells are loaded to prevent evaporation of the liquid  14  during the reactions, which may comprise thermal cycles, as in the example of nucleic-acid amplification by PCR. The openings  11 ,  12  may be sealed, for example with wax, before performing the reactions envisaged for the microreactor  1 , but this may not be needed with an oil cap. 
         [0040]    The microreactor described advantageously enables handling of extremely small volumes of liquid (a few microliters or even fractions of microliters) and introduction of the liquid into the wells from the chamber without any need for mechanical parts and preventing the formation of bubbles. 
         [0041]    According to the embodiment illustrated in  FIG. 5 , a microreactor  100  comprises a substrate  102  made of semiconductor material, in which wells  105  are provided, and a cap  103 , bonded to the substrate  102  through a bonding layer  104  and having a cavity that defines a chamber  110  above the plurality of wells  105 . 
         [0042]    Dielectric regions  107  coat the bottom of the wells  105  and the top of walls  106  that separate the wells from one another, while lateral surfaces of the walls  106  are exposed. The substrate  102  is provided with a first biasing electrode  118  on a face opposite to the wells  105 . 
         [0043]    The cap  103  has an inlet opening  111  for introduction of a liquid  114  and venting openings (not shown) to enable exit of air from the chamber  110 . The cap  103  is moreover provided with a second biasing electrode  113  that extends along side walls of the cavity defining the chamber  110 , as far as a margin adjacent to the substrate  102 . In this way, the second biasing electrode  113  is in contact with a liquid introduced into the chamber  110  also when the liquid penetrates into the wells  105  and the level starts to decrease. The substrate  102  defines a second electrode  118  that enables the liquid to penetrate into the wells  105  by electrowetting. 
         [0044]      FIGS. 6 and 7  illustrate a further embodiment of the disclosure. In this case, a microreactor  200  comprises a substrate  202  made of semiconductor material and a cap  203  bonded to the substrate  202  through a bonding layer (not shown). 
         [0045]    Provided in the substrate  202  are wells  205  arranged in arrays and separated by walls  206 . A first dielectric layer  207   a  and a second dielectric layer  207   b,  made for example of silicon oxide, coat the lateral surface and the top of the walls  206 , as likewise the bottom of the wells  205 . 
         [0046]    First biasing electrodes  208   a,    208   b  are located, respectively, on the bottom of the wells  205  and on a face of the substrate  202  opposite to the wells  205 . The first biasing electrodes  208   a  are housed in openings of the first dielectric layer  207   a  and are hence in electrical contact with the substrate  202 . The second dielectric layer  207   b  coats the first biasing electrodes  208   a.    
         [0047]    A second biasing electrode  213 , in the form of a grid of conductive material, for example copper or aluminium, is incorporated between the first dielectric layer  207   a  and the second dielectric layer  207   b  and extends on the top of the walls  206  that divide the wells  205  from one another. Portions of the second biasing electrode  213  hence surround each well  205 . 
         [0048]    The first biasing electrodes  208   a,    208   b  and the second biasing electrode  213  are thus electrically insulated from one another. 
         [0049]    The cap  203  has a cavity that defines a chamber  210  above the wells  205 . The cap  203  moreover has an inlet opening  211  for enabling introduction of a liquid  214  into the chamber  210  and venting openings  212  for enabling exit of the air from the chamber  210 . 
         [0050]    An electrical-supply source  215  enables applying a biasing voltage VB between the first electrodes  208   a,    208   b  and the second electrode  213 . 
         [0051]    The electrical field that is set up in the liquid  214  as a result of the biasing voltage VB between the first electrodes  208   a,    208   b  and the second electrode  213  modifies the angle of contact with the surface of the second dielectric layer  207   b,  with which the liquid  214  is in contact, modifying the wettability. Once the liquid  214  has overcome the surface tension, it penetrates into the wells  205  and distributes therein dividing up. 
         [0052]    With reference to  FIG. 8 , a cartridge  300  for biochemical analyses, in particular analyses of nucleic acids, comprises a support  301 , for example a printed-circuit board, which houses a sample-preparation structure  302  and a microreactor according to one embodiment of the disclosure. In the example of  FIG. 8 , in particular, the support  301  houses the microreactor  200  described previously with reference to  FIGS. 6 and 7 . Moreover, the cartridge  300  comprises a heater  303  and a temperature sensor  305 , arranged on the support  301  and thermally coupled to the microreactor  200 . An interface  306  enables electrical connection of the biasing electrodes  208   a,    208   b,    213 , of the heater  303 , and of the temperature sensor  305  with an external analysis apparatus. In one embodiment, the support  301  is a semiconductor chip and defines the substrate in which the wells of the microreactor  200  are provided. 
         [0053]    The sample-preparation structure comprises a lysis chamber  310 , a biofilter  311 , an elution chamber  312 , a waste chamber  313  and a microfluidic actuation circuit  315 , for example including a micropump and microfluidic valves and not described in detail herein. 
         [0054]    The lysis chamber  310  contains reagents that enable a preliminary preparation of the biological sample to be carried out. In particular, in the lysis chamber the nucleated cells present in the biological sample to be analyzed are broken up, and the DNA strands of the nuclei are extracted. The lysis chamber  310  is fluidly coupled to the biofilter  311  and to the waste chamber  313 . Once the sample has been prepared, the microfluidic actuation circuit  315  displaces the sample itself to the waste chamber  313  through the biofilter  311 . The biofilter  311  comprises a chamber having a surface made of silicon oxide, to which the DNA strands extracted from the cells remain anchored as they travel towards the waste chamber  313 . The remaining material, instead, is not withheld and is collected in the waste chamber  313 . 
         [0055]    The elution chamber  312  contains known solutions for flushing the biofilter  311 , which enable release of the anchorage of the DNA strands to the silicon-oxide surface. The solution is displaced by the microfluidic actuation circuit  315  from the elution chamber  312  towards the microreactor  301  through the biofilter  311 . The DNA strands are thus released and transferred in solution to the microreactor  200 , the wells  205  of which (here not shown) contain respective combinations of reagents for recognition of specific nucleotide sequences. In particular, the wells  205  may contain respective specific target nucleotide sequences or DNA probes capable of binding to complementary sequences that may be present in the sample. 
         [0056]    For carrying out a process of analysis, the cartridge  300  is inserted into an analysis apparatus  350 , illustrated in  FIG. 9 . The analysis apparatus  350  comprises a processing unit  351 , a reader device  352 , configured to receive the cartridge  300 , a power supply  353 , and a cooling unit  354 , both of which are controlled by the processing unit  352 . The cooling unit  354  may comprise, for example, a Peltier module or a fan and is thermally coupled to the microreactor  200  (not shown in  FIG. 9 ) of the cartridge  300  loaded in the reader device  352 . 
         [0057]    The processing unit  351  is configured to control the electrodes  208   a,    208   b,    213  (not shown) for causing a sample to penetrate into the wells  205  from the chamber  210  (which are not shown). 
         [0058]    The heater  303  and the temperature sensor  305  (neither of which is shown) of the cartridge  300  are respectively coupled to the power supply  353  and to the processing unit  351  through the interface  306  (not shown). The processing unit  351  controls the heater  303  and the cooling unit  354  on the basis of the temperature detected by the temperature sensor  305  for subjecting the microreactor  200  and the solutions contained therein to a plurality of thermal cycles according to a programmed temperature profile. The thermal cycles, in one embodiment, enable reactions of DNA amplification to be performed, in particular according to a PCR protocol. 
         [0059]    The reader device  352 , for example of an optical type, is configured for detecting the presence of target nucleotide sequences on the basis of the properties of the reaction results present in the wells of the microreactor  200 . 
         [0060]    Finally, it is evident that modifications and variations may be made to the microreactor and to the method described, without thereby departing from the scope of the present disclosure, as defined in the annexed claims. In particular, we have shown a single chamber over a plurality of wells, and such a device is appropriate for doing several different analyses of a single sample. However, a plurality of chambers, each covering a plurality of wells can easily be provided and thus allowing for the simultaneous analyses of more than one sample in the same cartridge.