Abstract:
The present invention relates to a detector for measuring fluorescence in a liquid sample and to devices for biochemical analyses comprising it, in particular to devices for performing analyses of real time PCR. The detector of the present invention has a series of advantages such as drastic simplification of the detection configuration, reduced costs, better performances due to the greater freedom in planning the optical configuration which allows dividing the detector itself into independent areas.

Description:
TECHNICAL FIELD OF THE INVENTION 
       [0001]    The present invention relates to a detector for measuring fluorescence in a liquid sample and to devices for biochemical analyses including it, in particular to devices for performing analyses of real time PCR. 
       STATE OF ART 
       [0002]    In the last years the spreading of molecular diagnostic tests based upon the amplification of nucleic acid sequences, and in particular the techniques using specifically planned fluorescent markers and enzymes, have allowed the quick and quantitative (or half-quantitative) analysis of nucleic acid sequences for a plurality of diagnostic applications, several thereof aimed at identifying pathogenic agents responsible for infective diseases, but even in other fields such as oncology and pharmacogenomics. 
         [0003]    The most widespread technology is the Real-Time PCR (rtPCR), however a series of other methods, based upon both temperature cycles or isothermal reactions, is much used. 
         [0004]    A great effort has been made to optimize the biochemical portion in this type of analysis, but the devices needed to perform the reactions and to acquire the results are still a factor limiting the spread of these analyses. In particular, the sizes, costs and skills required to use this type of instruments are still prohibitive. 
         [0005]    In the state of art analysis systems are described wherein several detectors are used, each one dedicated to one specific detection wavelength. However, these devices have several disadvantages such as for example the lack in availability on the market of multi-sensor cameras, cost and complexity in producing a detector including several single detectors. 
         [0006]    The patent application WO2012151358 describes a lighting apparatus comprising an array of light sources and a matrix of lenses coupled to a mechanical arm of the apparatus. 
       SUMMARY OF THE INVENTION 
       [0007]    The technical problem placed and solved by the present invention is then to provide a detector allowing to obviate the drawbacks mentioned above with reference to the known art. 
         [0008]    Such problem is solved by a detector according to claim  1  and by a device according to claim  9 . 
         [0009]    The present invention further provides the following advantages:
   a drastic simplification of the detection configuration, reduced costs, better performances due to the greater freedom in planning the optical configuration allowing to divide the detector itself into independent areas;   optimizing the performances of the reactions, by improving at the same time the system reliability;   improving considerably the instrument applicability by means of developing a design of the sample holder allowing the access to optics and electronics separated along different mechanical axes.   
 
         [0013]    Preferred features of the present invention are subject of the depending claims. 
         [0014]    Other advantages, features and use modes of the present invention will result evident from the following detailed description of some embodiments, shown by way of example and not for limitative purposes. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0015]    The figures of the enclosed drawings will be referred to, wherein: 
           [0016]      FIG. 1  shows an exploded view of a preferred embodiment of the detector according to the present invention; 
           [0017]      FIG. 2  shows an exploded view of a sample-holding cartridge used in a preferred embodiment of the device according to the present invention; 
           [0018]      FIG. 3  shows a perspective view of a motion mechanical system in the “open” configuration thereof used in a preferred embodiment of the device according to the present invention. 
           [0019]      FIG. 4  shows a perspective view of the same motion mechanical system of  FIG. 3  in the “closed” configuration thereof. 
           [0020]      FIG. 5  shows a perspective view of the pressure plate group used in the mechanical system of  FIGS. 4 and 5 . 
           [0021]      FIG. 6  shows a longitudinal section of the configuration of a preferred embodiment of the device according to the present invention; 
           [0022]      FIG. 7  shows an exploded view of a sample-holding cartridge used in a preferred embodiment of the device according to the present invention. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0023]    By firstly referring to  FIG. 1 , a detector for measuring fluorescence in a liquid sample according to a preferred embodiment of the invention is designated as a whole with  10 . 
         [0024]    Inside the liquid sample which will be positioned in the suitable reaction area of the detector, not shown in figure, the chemical and biochemical reactions of interest will take place, which will be detected in qualitative or quantitative way according to the optically detectable reaction. In the embodiment of  FIG. 1  the generated signal is of fluorescent type. 
         [0025]    The detector  1 , in the present example, comprises an optical sensor  1 , particularly of the CMOS or CCD type, connected by suitable mechanical means  2 ,  3  to the optical unit. 
         [0026]    The optical unit of the detector apart from comprising a light source for irradiating the liquid sample under examination comprises a plurality of optical filters  6  and of lenses  5 , in the present example there are  4  different filters  6  and lenses  5 , but according to other embodiments they could be  6 ,  8 ,  10 ,  20 , etc. The filters could have different wavelengths according to the type of analysis which will be performed. According to an embodiment one or more of said lenses  5  could be aspherical lenses. 
         [0027]    The detector further comprises a first element  4  which divides the optical unit into mechanically separated different areas (in this example into  4  areas), each one thereof houses a specific filter  6  and lens  5 , therefore the light passing through a determined area of the sample will be detected by a specific partition of the sensor  1 . 
         [0028]    As shown in  FIG. 1  the optical unit and the sensor are arranged perpendicularly to the longitudinal axis (A) corresponding to the plane wherein the reaction area of the liquid sample under examination is positioned. 
         [0029]    The arrangement described and visible in  FIG. 1  makes then possible to perform m independent measurements (wherein m is the number of areas wherein the sensor is partitioned) for each one of the n areas of the reaction of the sample under examination, equalling to a total of n×m independent determinations. Such result could be obtained even by using m independent sensors (each one with the dedicated optical group thereof), but such configuration has obvious economic and performance disadvantages, as it is preferable that the single sensible areas be as close as possible in order to maximize the overlapping of the ‘view cones’ and then the usable reaction area. 
         [0030]    Preferably the used optical sensor will be then of CMOS type and able to make ‘imaging’ that is constituted by a series of ‘pixel’ constituting the minimum sensible unit, the described result of the configuration is to have m separate images, each one thereof will have a 1/m resolution with respect to the total one of the sensor (with respect to the pixels analysed by the unit itself). In the detector according to the invention then preferably sensors will be used equipped with a resolution so that even a number of pixel 1/m is sufficient to do an imaging of the reaction areas. 
         [0031]    Still by making reference to the embodiment of  FIG. 1  the optical unit comprises even a window  8  faced towards the reaction area wherein the liquid sample under examination is placed, transparent at a defined wavelength. The detector  10  even comprises means  7  for fastening the optical unit, in particular such means  7  will comprise a base element  7  having elements for fastening to the transparent window  8 . 
         [0032]    A device for performing biochemical analyses is also subject of the present invention comprising a detector according to what herein described, in particular devices for performing analyses of real time PCR. 
         [0033]    By making now reference to  FIG. 2  a sample-holding cartridge is shown designated as a whole with  11  used in a preferred embodiment of the device according to the present invention. 
         [0034]    Such sample-holding cartridge  11  allows the independent and simultaneous detection of optical and electronic events which take place in the liquid sample under examination and it comprises:
   a plate  12 , or according to other embodiments a set of single smaller plates, preferably made of thermally conductive material, such as for example aluminium;   an optional frame  13  placed around said plate  12  for handling and providing the structures used to seal the plate after charging the reagent;   a cover  15  used for sealing the cartridge, made of a transparent material to allow the passage of optical source to the reaction which takes place in the sample;   an optional electrical interface  14 ,  16  for electrically connecting the cartridge to the elements existing in the device for electrical measurements and/or impedance. Such elements existing in the device could be the means for electrical measurements and/or impedance known to the person skilled in the art.   
 
         [0039]    More in details the electric interface is constituted by a connector  14  and an electronic board  16  having electrodes connected by means of suitable wire assembly (for example traces made of Cu or other metal lying above or inside the electronic board  16 ) and which end in the connector  14 . Therefore, the reactions in the sample under examination take place in electric contact with a suitable electronic board  16  which is in the device and which through impedance, voltage, amperage measurements performs the wished measurements. 
         [0040]    With respect to the cartridges of the state of art, such as for example the plates of the devices to perform Real-Time PCR measurements offering the possibility of performing optical or electronic measurements, there are not cartridge modules allowing to perform independently both types of analyses on the same device. The herein represented possibility of using different axes regarding the cartridge to perform measurements or handling of different nature (in the herein represented example optical measurements according to an axis and electronic measurements according to another axis perpendicular thereto) one can extend to include additional for example mechanical and thermal interactions, wherein the same motion and heat quantity is extracted from the cartridge (the pressure plate and the heating element described subsequently in the specific embodiments). 
         [0041]    Examples of using electronic measurements include the electronic detection of hybridization events of surface proteins of determined cells (for example, tumour cells, bacteria) with antibodies immobilized on electrodes placed inside ‘small wells’ existing in the cartridge. The impedance variation on the electrode given by the cellular mass linked to the same can be detected and it designates the presence in the sample of the cellular species which one wants to detect. 
         [0042]    By making now reference to  FIGS. 3 and 4  a motion mechanical systems is shown, designated as whole with  20  respectively in the ‘open’ configuration thereof, wherein the cartridge is inserted or removed and in the ‘closed’ configuration thereof, wherein the analysis is performed. 
         [0043]    The system  20  has essentially a layout developing longitudinally with respect to the device, therefore the opening and closing mechanism takes place by means of moving vertically the closing lock. This allows inserting into the mechanism a pressure plate  27  which in the closing step exerts a predetermined pressure on the upper face of the cartridge (for example by means of the presence of springs in the motion mechanism). 
         [0044]    The arrangement is so that the upper lock can be raised to open the device (that is the analysis instrument) and allow the insertion of the sample holder, whereas by lowering the upper portion the closure of the device is obtained, and to cause the pressure plate  27  to exert onto the sample holder a pressure predetermined for example by the springs or by other equivalent mechanism. 
         [0045]    This differentiates from the metallic masks used in the instruments of real time PCR of the state of art, as these have only the purpose of heating the upper portion of the sample holder (in that case a plate) to avoid condensations, but not exerting a significant pressure, as they are simply rested. 
         [0046]    The use of this system and in particular of the pressure plate  27 , and of a geometry spreading vertically as described, apart from this effect (the pressure plate too is a a temperature which can be controlled by means of suitable circuit) has different advantages thereamong:
   improving the thermal exchange with the cartridge housing, when this takes place by thermal conduction with the sample holder (almost all cases);   guaranteeing an optimum electric contact for example among the bump contacts on the lower surface of the sample holder and of the pogo pin lying in the housing;   improving the mechanical sealings when the reaction volumes are closed, for example, by stoppers or other methods subjected to pressure from inside of the reaction volume;   masking spurious lights which could reach the sensor;   transferring kinetic energy to the cartridge itself, for example to break vesicles filled up with reagents in a suitable moment of the analysis reactions, transferring fluids from a region of the cartridge to another one and sealing reaction volumes, in case even by using graduated ‘pins’ on the face of the pressure plate directed towards the cartridge itself and by segmenting the total motion of the pressure plate in a series of separated passages.   
 
         [0052]    According to the embodiment represented schematically in  FIGS. 3 and 4  the mechanical system  20  comprises a first slide frame  21  integral with the device. To this first frame a second mobile frame  22  is connected by means of a slide mechanism (with tracks). 
         [0053]    On the mobile frame  22  means ( 23   a ,  23   b ,  24 ) are fastened to exert the pressure on the pressure plate (and then on the cartridge) in the ‘closed’ configuration. 
         [0054]    According to the embodiment represented in figure said means for exerting the pressure on the pressure plate comprises first  23   a  and second elements  23   b  spaced apart therebetween by springs. Said second elements  23  engage onto the screws  24  which belong to the pressure plate group. The screws  24  push then downwards the framework  25  and consequently the pressure plate  27  towards the cartridge, which engages in the housing  28 . 
         [0055]      FIG. 5  shows in details the pressure plate group. The pressure plate group positioned in the example of  FIGS. 3 and 4  in the centre of the system, it has a number of holes equal by position and sizes to the reaction volumes in the immediately underneath sample holder, and rested upon the lower portion in the housing thereof. The pressure plate group comprises a pressure plate  27  integral to the frame thereof (framework)  25 , and then to the upper portion of the system. The frame of the pressure plate  25  being fastened mechanically to the pressure plate  27  transmits the motion and the closing pressure to the pressure plate itself. Preferably, the shape of the pressure plate group will be suitable to enter a housing placed in case on the cartridge, wherein the thermally active portion forms a recess with respect to the supporting frame of the cartridge. 
         [0056]    The system optionally could comprise a releasing/hooking mechanism, such as for example those available on the market, which once having closed the mechanism keeps it in position and it can be activated to release the mechanism in the opening step.  FIG. 6  shows in details the configuration of another embodiment of the device mainly providing the overturning of the arrangement of some portions of the instrument and in particular:
       the sample lighting takes place from the bottom upwards (the optical group is then below the sample holder);   the geometry of the optical group remains unchanged, all elements being in relative identical positions with respect to the previously described embodiments;   the heating element, exemplified as aluminium plate  12 , instead, is ‘above’ the sample holder  11  and the heating (and/or cooling) surface contacts the upper surface of the sample holder  11 .       
 
         [0060]    As to the ‘vertical’ mechanical motion, two variants could be provided, the first one wherein the heating element is ‘mobile’ with respect to the structure and pushes against the sample holder, the second one wherein the sample holder, rested on an adequate mechanism with a function of releasing and graduating the pressure (for example springs), is pushed from the bottom against the heating element. 
         [0061]      FIG. 7  describes in details an embodiment of the sample holder  11  which can be used in the device according to the present invention. The sample holder could be entirely made of the same material or, alternatively, of a thermally conductive portion (for example aluminium) and with suitable properties (for example, reflectivity, its own fluorescence), in turn incorporated in the frame  13  allowing the handling and mechanical interfacing thereof. The sample holder could provide through holes crossing it from one side to the other one. 
         [0062]    By still referring to  FIG. 7  the device could comprise even an optical window  40  positioned on the lower face of the sample holder (for example by gluing, adhering, or mechanical locking). The optical window  40  could be integral with the frame for example by moulding in a single piece. The main feature of the optical window  40  will be that of being transparent at the wavelengths of interest. The device could further comprise even an adequate mechanism for closing the upper face of the sample holder, for example by means of an adhesive and thermo-conductive film  41 . Advantageously through this element the heat flows, heating/cooling the sample holder, but not the light (which instead arrives from the bottom). 
         [0063]    This configuration has additional advantages shown hereinafter:
   a. Typically when the closing of the reaction volumes takes place on the same side of optical reading, this creates criticalities due to the not-perfect compromise between adhesion property and sealing against pressure in the volume heated with the properties of optical transparency.   b. Furthermore if the liquid sample is on the ‘bottom’ of the reaction volume with respect to the sensor, the film or closing stopper becomes the coldest point in said volume wherein condensate is generated under the form of small drops. This forces to integrate an additional heating mechanism.   c. The reaction volume in the described configuration is in direct contact with the optical window (gravity makes that one rests thereupon), by producing two advantages: on one side the intensity of the signal increases as the volume emitting the signal is not on the ‘bottom’ of the small well from the sensor point of view, and the closing process with the optical window takes place during production and it is not responsibility of the single operator after having charged the sample. Then the formation of condensate is not possible.   
 
         [0067]    The present invention has been sofar described by referring to some preferred embodiments. It is to be meant that other embodiments belonging to the same inventive core may exist, as defined by the protective scope of the herebelow reported claims.