Abstract:
An apparatus ( 100 ) for performing thermal cycles has a frame ( 131 - 134, 137, 147 ) enclosing a thermal chamber ( 110 ) laterally delimited by delimitation walls ( 103, 154   a,    154   b ) and configured so as to be delimited at the bottom by a reaction holder ( 104 ) carrying a plurality of reaction chambers ( 107 ) designed to receive chemical reaction substances. A lid ( 105 ), of transparent material, is fixed to the frame and delimits the thermal chamber at the top. A source of light radiation ( 165 ) is arranged outside the thermal chamber ( 110 ) facing the lid ( 115 ) and is configured to generate an excitation light radiation. A detector of light radiation ( 166 ) is arranged outside the thermal chamber facing the lid and is configured to collect a light radiation emitted in use by the reaction chambers ( 107 ). A processor ( 171 ) is connected to the detector of light radiation ( 166 ) and is configured to detect, in use, a feature of the light radiation emitted.

Description:
PRIOR RELATED APPLICATIONS 
       [0001]    This invention claims priority to Italian application MI2011A001893, filed on Oct. 19, 2011, 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 INVENTION 
       [0004]    The present invention relates to a diagnostic apparatus, in particular for performing thermo-cycling operations during an RT-PCR (reverse-transcription polymerase-chain reaction), with optical detection. 
       BACKGROUND OF THE INVENTION 
       [0005]    As is known, use of diagnostic apparatuses operating on small amounts of specimens is increasingly widespread since they advantageously improve the reliability of the assay, reduce the volume thereof, and thus reduce the time required for this activity, as well as the corresponding costs. 
         [0006]    Known devices basically comprise a solid substrate, immobilizing particular receptors, such as, for example, biomolecules (DNA, RNA, proteins, antigens, antibodies, haptens, sugars, etc.) or chemical species, or micro-organisms or parts thereof (bacteria, viruses, spores, cells, organelles, etc.). “Receptors” mean herein any member of a pair or multiple of elements that may bind together (binding pair) so that the receptor binds or reacts with, and thus detects, its own binding mate (or binding mates). Herein, receptors include traditional receptors, such as protein receptors and ligands, but also any element designed to interact or mate, such as, for example, lectins, carbohydrates, streptavidins, biotins, proteins, substrates, oligonucleotides, nucleic acids, porphyrins, metal ions, antibodies, antigens, and the like. 
         [0007]    According to the optical-detection technique, when these receptors are arranged in direct contact with a specimen to be analysed, the presence in this specimen of molecules able to mate or interact with the receptor activates specific markers, for example fluorescent markers, which, when excited with a light radiation at a first wavelength, emit light radiation having a second wavelength different from the first wavelength. 
         [0008]    Known fluorescence diagnostic devices comprise a compatible layer having a surface that is functionalized so as to form detection areas comprising receptors having the specific markers. 
         [0009]    There are many different ways for preparing tests that involve optical signals. For example, a common three-component binding assay uses a first immobilization, on a solid substrate, of an antibody that may mate with an antigen in a specimen solution. Binding with the antigen is then detected using a second antibody, which binds to a different epitope of the same antigen and has a fluorescent label attached thereto. Thus, the amount of fluorescence is correlated to the amount of the antigens in the specimen. 
         [0010]    Another solution comprises immobilization on the substrate or the use of a solution of an oligonucleotide probe that is then hybridized with complementary DNA or cDNA or mRNA in the specimen, and the double-strand nucleic acid may be detected with an intercalating dye, such as, for example, ethidium bromide. 
         [0011]    According to another solution, two fluorescent markers are brought into strict proximity in the assay, and quenching of a marker is measured in assays based upon fluorescence resonance energy transfer (FRET). 
         [0012]    Alternatively, binding of heavy metals with fluorophores may also be detected by means of fluorescent dyes. 
         [0013]    Various apparatuses have been proposed having an active or passive approach for detection of the optical signal, irrespective of the treatment performed on the assays and of the technique of generation of the optical signal. 
         [0014]    In these apparatuses, the light radiation is collected by a detector, such as, for example, a photodetector of a CCD (charge-coupled device) type or of a CMOS type sensitive to the wavelength of the emitted light radiation, in which the light intensity or its variation is a function of the amount of specific markers activated in the assay, and thus of the amount of detected molecules or biomolecules. 
         [0015]    In the case of analysis with qRT-PCR (quantitative reverse-transcriptase polymerase-chain reaction), the apparatus generates thermal cycles (for example, at 60° C., 72° C., and 90° C.) for amplification of the sought target molecule and immediately supplies an accurate quantitative estimate thereof. 
         [0016]    In order to perform correctly the process of amplification, the thermal cycles have to occur at controlled and uniform temperature within an area referred to hereinafter also as “reaction chamber”. For example,  FIG. 1   b  shows a portion of an apparatus  1  for performing diagnostic analyses including a reaction zone  2  having guides  3  wherein a holder  4  is inserted and has a heating device  6 . 
         [0017]    The area above the holder  4  forms a thermal chamber  10  facing light-emitter elements  11 , for example LEDs, and a detector  12 , for example a CCD detector. 
         [0018]    In addition, the apparatus  1  has a ventilating unit  8 , for example comprising a fan arranged underneath the reaction zone  2 . 
         [0019]    An electronic device (not shown) controls the supply of current to the heating device  6  and the activation of the ventilating unit  8  so as to obtain the desired thermal cycles. 
         [0020]    The holder  4  (see also  FIG. 2  where the holder is shown longitudinally, rotated by 90° with respect to  FIG. 1B ) is here formed by a moulded body, for example, of suitable biocompatible transparent plastic (for example, polycarbonate) and has a parallelepipedal shape having a bottom on which the heating device  6  is fixed. On the top side, the holder  4  has a series of chambers  7  open at the top and designed to contain the reagents and the reaction products. 
         [0021]    As shown in  FIGS. 2 and 3 , in the example, the heating device  6  is formed by a die, comprising a substrate  15  of semiconductor material having, on one side thereof, a coil  16  of conductive material, for example aluminium or other metal, which extends throughout the area of the chambers  7 . The coil  16  is connected to pads  17 . Other types of holder are, however, possible, for example without chambers  7 ; in this case the chambers are formed directly in the die on the back of the heating device. 
         [0022]    With the structure shown, it is not possible to obtain a high thermal uniformity in the reaction zone  2 . In fact, as shown in the graph of  FIG. 1   a , which illustrates the thermal profile in the reaction zone  2  along the axis Y, the temperature, which is close to the value TR of the surrounding environment at a distance from the holder  4 , increases rapidly to the heating value TH in proximity of the heating device  6 , and then drops gradually within the thermal chamber  10 , increasing until reaching a value close to the ambient temperature TR above the thermal chamber  10 . 
         [0023]    In particular, tests conducted by the applicant have shown that, because of dissipation, a thermal gradient exists, within the reaction zone  2 , of approximately 10-15° C. 
         [0024]    This is disadvantageous since it may jeopardize the correctness of execution of the reactions and thus of the obtained diagnostic result. 
         [0025]    Thus, what is needed in the art is a semiconductor based design that eliminates or at least substantially reduces the temperature differential, and provides a more uniform heating platform that can be used in various assays, especially amplification based assays and other temperature sensitive reactions. 
       SUMMARY OF THE INVENTION 
       [0026]    The aim of the present invention is to provide a diagnostic apparatus that provides a higher thermal uniformity in the reaction zone. 
         [0027]    According to the present invention, a diagnostic apparatus is provided, defined as follows: 
         [0028]    An apparatus for performing thermal cycles, comprising a frame; a thermal chamber laterally delimited by delimitation walls and configured to be downwardly delimited by a reaction support carrying a plurality of reaction chambers intended to accommodate reaction chemicals; a lid of a transparent material, upwardly delimiting the thermal chamber; a light radiation source, arranged externally to the thermal chamber so as to face the lid and configured to generate an excitation light radiation; a light radiation detector, arranged externally to the thermal chamber so as to face the lid and configured to collect light radiation emitted in use by the reaction chambers; and a processing element, coupled to the light radiation detector and configured to detect, in use, a feature of the emitted light radiation. 
         [0029]    In another embodiment, the invention is an apparatus for performing thermal cycles, comprising a parallelepipedal frame, a thermal chamber inside said frame and laterally delimited by side walls, downwardly delimited by a reaction support, and upwardly delimited by a transparent lid. The reaction support has a first heater facing said thermal chamber, and is configured to receive one or more reaction chambers thereon or therein. The lid has a second heater facing said thermal chamber. One or both of said lid and said reaction support is also outfitted with a thermal sensor. The thermal chamber also has air flow paths above and below it (and inside the frame), and one or more fans to draw air along said air flow paths. The thermal sensor(s), heaters and fan(s) are each operably coupled to a processor for controlling the heaters and fan(s) and thus controlling and providing a uniform temperature throughout the chamber. Also inside the frame is a light radiation source, arranged externally to the thermal chamber so as to face the lid and configured to generate an excitation light radiation, a light radiation detector, arranged externally to the thermal chamber so as to face the lid and configured to collect light radiation emitted in use by the reaction chambers, and a processing element, coupled to the light radiation detector and configured to detect, in use, an emitted light radiation. 
         [0030]    The following abbreviations are used herein: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 ABBREVIATION 
                 TERM 
               
               
                   
                   
               
             
             
               
                   
                 ABS 
                 Acrylonitrile butadiene styrene 
               
               
                   
                 CCD 
                 Charge-coupled device 
               
               
                   
                 cDNA 
                 Copy DNA 
               
               
                   
                 CMOS 
                 complementary-symmetry metal-oxide 
               
               
                   
                 DNA 
                 Deoxyribonucleic acid 
               
               
                   
                 FRET 
                 Fluorescence resonance energy transfer 
               
               
                   
                 GaN 
                 Gallium nitride 
               
               
                   
                 LED 
                 Light emitting diode 
               
               
                   
                 mRNA 
                 Messenger RNA 
               
               
                   
                 PCR 
                 Polymerase chain reaction 
               
               
                   
                 PVC 
                 Polyvinyl chloride 
               
               
                   
                 qRT-PCR 
                 Quantitiative real time PCR 
               
               
                   
                 RNA 
                 Ribonucleic acid 
               
               
                   
                   
               
             
          
         
       
     
         [0031]    The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise. 
         [0032]    The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated. 
         [0033]    The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive. 
         [0034]    The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim. 
         [0035]    The phrase “consisting of” is closed, and excludes all additional elements. 
         [0036]    The phrase “consisting essentially of” excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0037]    For a better understanding of the present invention, a preferred embodiment thereof is now described, purely by way of non-limiting example, with reference to the attached drawings, wherein: 
           [0038]      FIG. 1  displays (A) the thermal profile within a known diagnostic apparatus and (B) a side view of a part of the known diagnostic apparatus; 
           [0039]      FIG. 2  is a ghost side view of a holder used in the apparatus of  FIG. 1 ; 
           [0040]      FIG. 3  is a bottom view of the holder of  FIG. 2 ; 
           [0041]      FIG. 4  displays (A) the thermal profile within the reaction zone of the present diagnostic apparatus and (B) a side view of the reaction zone of the present diagnostic apparatus; 
           [0042]      FIG. 5  is a perspective bottom view of the lid in the reaction zone of  FIG. 4 ; 
           [0043]      FIG. 6  is an enlarged cross-section and perspective view of a portion of the lid; 
           [0044]      FIGS. 7A-7E  are different embodiments of the heater arranged on the lid of  FIG. 5 ; 
           [0045]      FIG. 8  is a cross-section of the present apparatus, taken along the plane of section VIII-VIII of  FIG. 9 ; 
           [0046]      FIG. 9  is a longitudinal section of the present apparatus, taken along plane IX-IX of  FIG. 8 , including an expanded view of the fan and fan compartment for detail. 
           [0047]      FIG. 10  is a block diagram of the present apparatus. 
       
    
    
     DESCRIPTION OF THE INVENTION 
       [0048]      FIG. 4B  shows a portion of an apparatus  100  for performing diagnostic analyses in order to detect the presence and amount of target molecules using the qRT-PCR technique referred to above. In particular,  FIG. 4B  shows a reaction zone  102  including guides  103  for a holder  104  similar to the one described with reference to  FIGS. 1-2 . In particular, the holder  104  has a first heating device  106  formed in a die  105  and forms a series of reaction chambers  107  (shown in ghost view and open on the top side) designed to contain the reagents and the reaction products and/or test-tubes. Alternatively, the first heating device  106  may be molded directly on the bottom or on a side of the holder  104 . 
         [0049]    The area above the holder  104  here forms a thermal chamber  110  closed at the top by a lid  115 . The lid  115 , of a generally rectangular shape, is transparent to the light of emitter elements  165  ( FIG. 8 ) and to the fluorescent light emitted during the reaction so as to enable collection via a detector  166  (also visible in  FIG. 8 ), arranged above the thermal chamber  110 . In particular, the lid  115  may be glass, polycarbonate, polyacrylate, GaN or other transparent material to enable passage of the radiation emitted during the reaction, guaranteeing thermal tightness of the underlying reaction zone  102 . 
         [0050]    A second heating device  116  is arranged on the lid  115 , on the inside surface facing the holder  104  (see also  FIG. 5 ), and is shown in greater detail in  FIGS. 5-7 . 
         [0051]    In particular, the second heating device  116  is formed by a conductive path, for example a metal layer, such as Al, Cu or Mo, of large thickness, optimized for a high current capacity (e.g., 2-3 μm able to carry a current comprised between 0.5 A and 2 A) and may have different shapes, shown e.g. in  FIGS. 7A-7E . The conductive path generally has a peripheral portion  116   a  ( FIGS. 5 ,  7 A- 7 E) extending in a direction parallel to three sides of the lid  115  and is connected to pads  117 . The peripheral portion  116   a  may possibly be connected to a plurality of intermediate portions  116   b , parallel to and/or crossing one another ( FIG. 5 ,  7 A- 7 C) and passing through the area of the lid  115 , preferably so as to lie in the space between one reaction chamber  107  and another, so as not to interfere with the radiation emitted during PCR. In turn, both the peripheral portion  116   a  and the intermediate portions  116   b  may be formed completely or partially as coils, as represented in the enlarged detail of  FIG. 5 . 
         [0052]    In order to facilitate initial setup and driving, the first and second heating devices  106 ,  116  may have the same nominal value of resistance (for example 15 to 24 Ω). 
         [0053]    Furthermore, a temperature sensor  120  may be fixed on the lid  115  ( FIG. 5 ), comprises a separate die connected to its own pads  121 , and able to measure the temperature in the thermal chamber  110  and to supply a signal that may be used for a temperature feedback control. 
         [0054]    The pads  117 ,  121  of the second heating device  116  and of the sensor  120  are connected to a connector  122 , in turn connected to a control unit of the apparatus  100 , as described hereinafter with reference to  FIG. 10 . 
         [0055]    As shown in  FIG. 6 , the second heating device  116  may be covered by a passivation layer  123  forming a shielding for the metal paths  116   a ,  116   b . For example, the passivation layer  123  may be silicon oxide or silicon nitride with a thickness of approximately 1 μm, and has the function of eliminating possible reflections of the light emitted during the reaction caused by the lid  115 , in addition to protecting the metal paths of the second heating device  116 . 
         [0056]    In this way (see  FIG. 4B ), the area comprising the thermal chamber  110  and the holder  104  up to the first heating device  106  may be kept at a substantially uniform temperature, as represented in  FIG. 4A , thanks to the heating supplied from below by the first heating device  106  and from above by the second heating device  116 . 
         [0057]    The lid  115  may be manufactured from a plate, for example of glass, having a much greater area than the lid  115 , printed with the metal paths  116   a ,  116   b  (at the same time forming a number of heating devices  116  for a plurality of lids  115 ) and covered with the passivation layer  123 . The glass plate may then be scored to separate the individual lids  115 ; the temperature sensor  120  and the connector  122  are mounted on each lid  115  via e.g., conductive paste, glue, or other means, and the lid  115  is mounted on the apparatus  100 . All the steps are preferably of a dry type so as to prevent any surface roughness. 
         [0058]    In order to optimize the heating and cooling steps provided for performing the reaction, for example PCR or qRT-PCR, the shown apparatus  100  has a double cooling circuit, as shown in detail in  FIGS. 8 and 9 , which represent two sections in mutually perpendicular planes. 
         [0059]    In detail, the apparatus  100  has a frame defining a closed structure, of a parallelepipedal shape, including a first and a second delimitation walls  131 ,  132  on the transverse sides ( FIG. 8 ), a first and a second columns  133 ,  134 , on the longitudinal sides ( FIG. 9 ), a bottom  147 , and a roof  137 . In this way, the walls  131 ,  132 , the columns  133 ,  134  (in particular with the walls  154   a ,  154   b  described hereinafter), the lid  115  and the holder  104  delimit the thermal chamber  110  where the temperature is uniform in a vertical direction (direction Y in  FIG. 4B ). 
         [0060]    Light-emitter elements  165 , for example LEDs, and a detector  166 , for example a CCD detector, are arranged above the thermal chamber  110 , within the frame of apparatus  100 . 
         [0061]    The delimitation walls  131 ,  132  define the guides  103  and, together with the columns  133 ,  134 , form resting structures for the lid  105 . 
         [0062]    Advantageously, the portions  131   a ,  132   a  of the delimitation walls  131 ,  132  that form the guides  103 , are formed by rails that may be removed from the rest of the delimitation walls (and are fixed, for example, with screws or other releasable constraint means, such as a ledge they can slide onto or even just a friction fit) so that they may be replaced at each reaction (disposable rails). The rails  131   a ,  132   a  are of isothermal material, such as ABS or PVC so as to favor maintenance of a uniform temperature within the reaction zone  102 . Further, since the rails can be removed after each reaction and disposed of, this serves to prevent even accidental contamination of the reagents within the chambers in successive analyses. 
         [0063]    The columns  133 ,  134  define internally a double path for the cooling air, including an inlet path  135 , a pair of cooling paths  145 ,  155 , and an outlet, common, path  136 . In detail, as shown in  FIG. 9 , the inlet path  135  is formed by an inlet opening  140  on the bottom  147  of the first column  133 , by a first grid  141  above the inlet opening  140 , and by an inlet chamber  142  above the first grid  141 . The first grid  141 , as the other grids indicated hereinafter, is formed for example by a mesh or a porous filter so as to prevent suction or in general inlet of dust, in particular particles of large dimensions. Immediately under the reaction zone  102  ( FIG. 8 ), towards the inside of the apparatus  100 , the first column  133  has a first plurality of openings  143  (see also  FIG. 8 , only one visible in  FIG. 9 ) where a part of the air is caused to flow under the reaction zone  102  so as to lap the holder  104  at the bottom (thus forming the first cooling path  145 ). In order to guide the air along the first cooling path  145  ( FIG. 9 ), a deflector  144  extends underneath the reaction zone  102  between the first and second columns  133 ,  134 , in a longitudinal direction with respect to the apparatus  100 . 
         [0064]    The second column  134  has, above the end of the deflector  144 , a second plurality of openings  148  (only one whereof is visible in  FIG. 9 ) where the air in the first cooling path  145  is drawn, by a first fan  149  arranged in a first fan compartment  147 , towards an outlet chamber  158  arranged in the second column  134 , along the outlet path  136 . 
         [0065]    In addition, part of the air flowing through the reaction zone  102  ( FIG. 8 ) is deflected so as to travel over the holder  104  at the top (second cooling path  155 ). To this end, the inlet chamber  142  is connected at the top to a rear area  151  (behind the holder  104  and closed by a sliding door  153 ) and then to a third plurality of openings  152  (see also  FIG. 8 ) formed in a first top wall  154   a  and opening onto the thermal chamber  110 . A second fan  156  in a second fan compartment  157  in the second column  134  draws the air along the second cooling path  155  and through a fourth plurality of openings  153  (only one visible in  FIG. 9 ) formed in a second top wall  154   b  near the top end of the second column  134 . As may be seen in  FIG. 9 , in particular in the enlarged detail, the second fan  156  is arranged at a higher level with respect to the first fan  149 , but is offset laterally with respect thereto. Here, the first fan  149  is not aligned longitudinally to the first cooling path  145  but is shifted laterally. Alternatively, the second fan  149  may be offset with respect to the second cooling path  155 . 
         [0066]    In both cases, the air coming from the first and second cooling paths  145 ,  155  is sucked into the outlet chamber  158  and discharged through an outlet opening  159  in the bottom  147  and through a second grid  160 . 
         [0067]    The apparatus  100  has an architecture that is represented in the block diagram of  FIG. 10 . In detail, the detector  166 , as a function of the light radiation collected, generates a first electrical signal supplied to a signal-processing unit  170 , which, on the basis of the first received electrical signal, supplies to a processing unit  171  a second electrical signal indicating the reaction that takes place in the chambers  107 , in qualitative and/or quantitative terms, according to the implemented reaction and the protocol. 
         [0068]    The processing unit  171 , in addition to supplying the outside world, through one or more input/output units  172 , with the information required, supervises thermal control of the reaction. To this end, it is connected to a temperature-control unit  175 , which, through own driving circuits  176 , controls actuation of the first and second heating devices  106 ,  116 , supplying the currents necessary for their operation (and thus operating as current source), as well as controlling actuation of the first and second fans  149 ,  156 . The temperature-control unit  175  is moreover connected to the temperature sensor  120  to receive the information on the temperature within the reaction zone  102  so as to control execution of the envisaged thermal cycles in a precise way. 
         [0069]    A power-supply unit  177  provides the power supplies requested by the various units of the apparatus  100 . 
         [0070]    Prior to the reaction, the rear door  153  is opened by sliding it upwards and rendering accessible the rear compartment  151  and the area that is to receive the holder  104 . Then, after the possible assembly of the guide portions  131   a ,  132   a  in the apparatus  100 , the holder  104 , with the first heating device  106 , is inserted in the rear compartment  151 , until it comes to stop against a detent arranged near the wall of the first column  133  ( FIG. 9 ). Then, after closing of the door  153 , the diagnostic program may be started, under the control of the central processing unit  171 , which then, based on the light radiation emitted, supplies the requested diagnostic result. 
         [0071]    The shown apparatus  100  is able to maintain a uniform temperature within the reaction zone  102 , and in particular within the chambers  107 , by virtue of the presence of two heaters (first and second heating devices  106 ,  116 ) arranged on the two sides of the holder  104 , which create an air cushion and reduce the thermal gradient between the top part and the bottom part of the holder  104 . 
         [0072]    The presence of two heaters  106 ,  116 , that may be governed and controlled in an independent way above and under the holder  104 , enables setting of the boundary conditions, thus rendering the system independent of the external/environmental variations and shielding the inside. 
         [0073]    The presence of two cooling paths  145 ,  155 , which generate two independent flows of air that lap the holder  104  at both its top and at the bottom and are controlled independently, enables precise thermal cycles to be performed, with high thermal uniformity, thanks also to the presence of the temperature sensor  120  on the lid  115 . 
         [0074]    The fact that the guides  103  are made as disposable rails  131   a ,  132   a  favors a high uniformity and prevents any contamination, as explained above. 
         [0075]    The lid  115  of transparent material, positioned at a certain distance from the chambers  107  where the reaction takes place, provides a thermal discontinuity with the surrounding environment, without interfering with the optical monitoring of the reaction and the collection of the emitted light radiation. 
         [0076]    Finally, it is clear that modifications and variations may be made to the apparatus described and illustrated herein, without thereby departing from the scope of the present invention, as defined in the attached claims. 
         [0077]    For example, as indicated, the holder  104  may be different, and likewise the members designed to provide the double cooling path may be made in a way different from the represented one. Similarly additional thermal sensors can advantageously be provided, e.g., below the reaction zone, in addition to the one above.