Abstract:
A method for real-time quantitative detection of single-type, target nucleic acid sequences amplified using a PCR in a microwell, comprising introducing in the microwell a sample comprising target nucleic acid sequences, magnetic primers, and labelling probes; performing an amplification cycle to form labelled amplicons; attracting the magnetic primers to a surface through a magnetic field to form a layer including labelled amplification products and free magnetic primers; and detecting the labelled amplification products in the layer with a surface-specific reading method.

Description:
BACKGROUND 
       [0001]    1. Technical Field 
         [0002]    The present disclosure relates to an integrated device and method for real time quantitative PCR (Polymerase Chain Reaction). 
         [0003]    2. Description of the Related Art 
         [0004]    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. 
         [0005]    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. 
         [0006]    Next, the DNA is amplified by an amplification reaction. Beforehand, it is denatured by thermal, biochemical or chemical processes. 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. 
         [0007]    The discussion herein is simplified by focusing on detection of DNA by PCR amplification as an example of a biological molecule that can be analyzed using the present device. However, the present device and method can be used for other chemical or biological tests. 
         [0008]    As indicated, PCR allows an initial amount of DNA strands, added to a reaction mix, to be multiplied. The amplification process includes basically three steps, including a denaturation step, where the DNA target, double-helix strands are separated into target single-helix strands by heating the mixture to a first high temperature, e.g., higher than 90° C.; an annealing step, where primers are annealed to the target single helix strands at a second, lower temperature, 50-65° C.; and an extension step, where new DNA strands, complementary to the DNA target strands, are synthesized at an intermediate temperature, e.g., at 70-85° C., forming double-helix amplification products, also called “amplicons”. 
         [0009]    Real time quantitative PCR is a widely used technique based on the real-time monitoring of PCR reaction during its progress, and can allow quantitation of initial target molecules based on amplification curves. This can be accomplished using fluorescent probes or dyes, such as ethidium bromide (EtBr), which intercalates into a double helix and then fluoresces. Thus, fluorescence increases when fluorescent probes or dyes bind with double-helix amplification products, and this increase in fluorescence can be monitored as the reaction proceeds and the level of amplification product exponentially increases. Labelled probes that hybridize to amplified sequences can be used in the same manner. The more initial target molecule present at the beginning of the reaction, the more signal will be obtained at a point t when the reaction is still in the exponential part of the amplification curve. 
         [0010]    Although consolidated instruments are widely available on market, the current trend is to develop new devices aimed to increase throughput and portability. Such devices may involve packed arrays of nanoliter wells able to perform highly parallel screening of pre-charged cartridges for point-of-care applications. 
         [0011]    Among the various aspects characterizing present real-time quantitative PCR (RTQ-PCR) instruments, such as reproducibility, parallelization, etc., one important aspect is related to the speed in executing tests. In fact, due to the inherent working methods of PCR, several cycles of amplification are necessary to detect product, each including switching the temperature between two or three different temperatures, as above discussed. Every cycle may take several seconds and tens of cycles are typically necessary to perform a test. In particular, when few initial DNA copies are available, the entire test can take one hour or more. 
         [0012]    This can be a limiting aspect, in particular for point-of-care applications, where speed is a very important consideration. 
         [0013]    This limitation may be clearly appreciated from the plots of  FIGS. 1 and 2 .  FIG. 1  plots an RTQ-PCR curve showing the increasing fluorescence during cycling. As may be seen, the curve has three different portions; a starting one, portion A, where fluorescence remains low (background level); a second portion, portion B, after a point called Threshold Cycle “CT”, where the curve grows exponentially for few cycles; and a final portion, portion C, where the curve saturates and no further amplification is seen. 
         [0014]    In portion A, background fluorescence is dominant. Background fluorescence is intrinsic in RTQ-PCR methods and cannot be avoided, nor subjected to strong reduction simply by optimizing detection methods. In fact background fluorescence is due to the presence of free fluorescent probes or fluorophores in the reaction vessel or well. Due to the initial high concentration of the free fluorescent probes, in this initial portion fluorescence due to amplification is negligible or not distinguishable from the intrinsic fluorescence. After the threshold cycle CT, however, the fluorescence curve strongly increases due to the exponential growth of amplification products. 
         [0015]    As shown in  FIG. 2  (from sabiosciences.com/pathway7.php), the threshold cycle CT depends, i.a., on the concentration of the starting target strands in the reaction vessel (indicated laterally to each curve). As visible, in case of very low concentrations (1-10 4 ), the Threshold Cycle CT may require several amplification cycles; thus the entire procedure requires long time. 
         [0016]    Thus, a need exists of providing a device and a method to reduce the time needed to perform RTQ-PCR. 
         [0017]    Numerous techniques and methods have been proposed to improve the efficiency of RTQ-PCR. 
         [0018]    EP2077336 discloses, e.g., a method for simultaneous quantitative analysis of multiple nucleic acid sequences in a single compartment, having the aim of increasing the number of different sequences that can be simultaneously detected. To this end, a number of surface-immobilized oligonucleotides probes, complementary to the multiple sequences to be detected, act as capture probes and are detected using a highly surface-specific readout device. Capture probes may be immobilized on paramagnetic beads attracted to a surface through a magnetic field. 
         [0019]    US2008305481 discloses methods and systems that use fluorescently encoded superparamagnetic microspheres for the immobilization of amplification products during the PCR process. Also this document is directed to allowing multiplex analysis of RTQ-PCR. 
         [0020]    Both known solutions are directed to increase multiplexing capability, by carrying out a single bead (or bead-by-bead) detection. Among various detection techniques, photodetection using Linear Lens Changer (LLC) or confocal detectors is taught. To discriminate against background fluorescence, the optical plane of the detector is set to be the surface where the beads are concentrated, thus improving the signal to noise ratio. 
         [0021]    For allowing detection of beads immobilized on the bottom surface of the vessel, the use of either very complex detection techniques or of relatively large beads is required. In fact, confocal detectors are not able to discriminate fluorescence in volumes smaller than their resolution and have a minimum resolution of about 1 μm. In other words, they collect all the fluorescence emitted a volume that may be approximated by cylinder having a base diameter and a height of 1 μm. 
         [0022]    This is not a problem for performing a bead-to-bead scanning, as suggested in a multiplexed detection, discussed in the above documents, since large beads may be used. 
         [0023]    However, this solution does not solve the problem of substantially reducing the Threshold Cycle CT in the detection of a single type of amplification products, where discrimination between different target products is made using different vessels arranged in big arrays, if so desired. 
         [0024]    In this case, in fact, the detection of a small amount of amplicons each time does not allow a reduction of the process time. 
         [0025]    On the other hand, if beads of smaller dimensions are used, also a volume of the liquid overlying the layer of immobilized amplicons would be detected by the confocal reader, since the latter is not able to detect volumes smaller than its resolution. As a result, the free floating non-activated fluorophores would be detected as well. In such a case, the reading would be again affected by the background fluorescence. 
         [0026]    The dependence of the thickness of layer immobilized on a surface of the vessel versus the bead dimensions and the obtainable gain are shown in  FIGS. 3   a ,  3   b , where  FIG. 3   b  shows the right-hand portion of the curve of  FIG. 3   a , in an enlarged scale. Line D (whose values are shown on the left) plots the layer thickness as a function of the beads diameter, while line E plots the cycle gain (that is the reduction in the number of cycles necessary to reach the Threshold Cycle C T  due to the reduction of dimensions in the beads). As is visible in the graph, the cycle gain increases considerably in case of smaller beads, but the simultaneous decrease of the thickness well below the confocal microscope resolution prevents the detection through a confocal detector. In other words, when a confocal detector is used, relatively big beads are be used, where the gain is low. 
       BRIEF SUMMARY 
       [0027]    Thus, the aim of the disclosure resides in devising a method and a device overcoming the limitation of the known solutions and allowing a rapid RTQ-PCR in an array of vessels. 
         [0028]    According to the present disclosure, there are provided a method and a device for real-time quantitative detection of single target nucleic acid sequences, as defined in claims  1  and  9 , respectively. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0029]    For the understanding of the present disclosure, preferred embodiments are now described, purely as a non-limitative example, with reference to the enclosed drawings, wherein: 
           [0030]      FIG. 1  shows the plot of the fluoresces vs. number of amplification cycles during a RTQ-PCR; 
           [0031]      FIG. 2  shows the plots of the threshold cycles vs. the number of performed cycles as a function of the number of starting target sequences present in the sample; 
           [0032]      FIG. 3   a  shows the plot of the layer thickness and obtained gain vs. bead diameter; 
           [0033]      FIG. 3   b  shows a portion of the plot of  FIG. 3   a , in an enlarged scale; 
           [0034]      FIG. 4  is a lateral view of a reaction microcell in a first step of the reaction; 
           [0035]      FIG. 5  is a lateral view of the reaction microcell of  FIG. 4  in a subsequent step of the reaction; 
           [0036]      FIG. 6  is a lateral view of a different reaction microcell in the same step as  FIG. 5 ; 
           [0037]      FIG. 7  shows another embodiment of the reaction microcell; 
           [0038]      FIG. 8  is a prospective view of a microcell; 
           [0039]      FIG. 9  shows a cross-section of a portion of the microcell of  FIG. 8 ; 
           [0040]      FIG. 10  is a prospective view of an array of microcells; 
           [0041]      FIG. 11  shows an implementation of an integrated confocal detector element; and 
           [0042]      FIG. 12  shows a device for detection of RTQ-PCR including the microcells of  FIG. 8  and the confocal detector elements of  FIG. 11 . 
       
    
    
     DETAILED DESCRIPTION 
       [0043]    According to one embodiment, paramagnetic or super-paramagnetic nanobeads derivatized with primers are used. After each amplification cycle or after a number of amplification cycles, a magnetic field is applied to a well containing the reaction liquid to confine and concentrate the nanobeads into a multilayer formed by overlaying nanobeads, and fluorescence is read in the concentration layer using a surface-specific, spatially discriminating detection technique, e.g., using a confocal microscope. 
         [0044]    In particular, the parameters of the method may be selected so that the multilayer has a thickness matched to the vertical resolution of the surface-specific reading device. Here, the term “matched” means the vertical resolution value ±10%. 
         [0045]    In addition, the parameters of the system may be selected so as to set the focal plane at a central region of the multilayer. Ideally, the focal plane is set at the median plane, that is at a height of h/2 with respect to the bottom wall, where h is the thickness of the multilayer, but also a distance in the range h/2±30% is acceptable. 
         [0046]    Since fluorescence is measured in the nanobead confinement region formed by a multilayer having a thickness comparable with the reading vertical resolution, where the bonded probes are much more concentrated than non-bonded probes, the background fluorescence is strongly reduced, allowing the threshold cycle CT to be reached with fewer thermal cycles. Thus, RTQ-PCR measurements can be greatly speeded up. 
         [0047]    Use of PCR primers linked to magnetic nanobeads has been already demonstrated in literature with the purpose of developing new assay methodology (see, e.g., Lermoa et al., “In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens”, Biosensors and Bioelectronics 22(9-10): 2010-2017 (2007); see also C. S. Jacobsen, “Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay”, Appl. Environ. Microbiol. 61(9): 3347-3352 (1995)). 
         [0048]    Further, although we have exemplified the system using primers bound to magnetic beads, other assay formats may be possible. For example, magnetic nanoparticles or other magnetic labels, such as ferrocene-based labels, single molecule magnets, and the like, can be developed and used to magnetize the PCR products in other ways. 
         [0049]    Since the nanobeads are magnetically confined in a multilayer having a preselectable thickness, beads having a diameter of a few nm may be used. For example, the nanobeads may have a diameter lower than 50 nm, preferably at most about 16 nm (e.g., less than 17 nm); more preferably between 5-10 nm, e.g., of about 5-6 nm, or even smaller (e.g., until 1-2 nm). Therefore, a very high concentration of the nanobeads in the multilayer is obtained, together with a very high compaction of activated fluorescent probes and the reduction of free space among the nanobeads. Consequently, also the number of non-activated fluorophores in the monitored region is also reduced to a negligible extent. Since all the activated fluorophores are contained in the detection region, their concentration increases, thus reducing the number of amplification cycles before Threshold Cycle CT is reached. 
         [0050]    When nanobeads smaller than 17 nm are used, it has been demonstrated that they may be obtained as single crystals with superparamagnetic characteristics (see, e.g., Cafer T. Yavuz et al. “Low-Field Magnetic Separation of monodisperse nanocrystals”, Science 314 (5801): 964-967 (2006), at page 966, left hand column, indicating a value of about 16 nm for iron oxide particles); to obtain nanobeads in the 17-50 nm range, it is necessary to include the single crystals in a matrix (e.g., polymeric), in a more complex and expensive way. 
         [0051]    For furthering the compaction effect and reducing the effect of non-activated fluorophores, according to a further aspect, lateral confinement of the nanobeads is also proposed. To this end, each cell has its own magnet arranged below the bottom surface of a respective vessel and having a smaller diameter than the bottom surface of the well. Feasibility of obtaining containment of superparamagnetic material at low magnetic field has been demonstrated, e.g., in Cafer T. et al. (2006). 
         [0052]    These and other aspects of the present system and method will be disclosed in detail hereinafter. 
         [0053]      FIG. 4  shows a reaction microcell  1  for performing RTQ-PCR. Here the microcell  1  has a microwell  2  including lateral walls  3  and bottom wall  4 . A magnet  5  extends in proximity of the bottom wall  4 , here under the bottom wall  4 , although the magnet could also be positioned in the bottom wall or over the bottom wall; in the latter case, a protective film (not shown) may cover the magnet  5  so that the materials do not interfere with the assay. 
         [0054]    An objective  6 , in particular a confocal objective, is arranged over the microwell  2  and is connected to a processing unit  8 . 
         [0055]    A power supply  7  is connected to the magnet  5  to activate it and generate a magnetic field in a detection step. 
         [0056]    The microcell  1  is configured to perform RTQ-PCR and multi-layer fluorescence detection, as described hereinbelow. 
         [0057]    In detail, initially a solution  10  is introduced in the microwell  2 , for example by injection. The solution  10  comprises target double helix sequences (schematized at  11 ); magnetic primers  12  (that is, as shown in the enlarged detail, primers  13  immobilized on magnetic nanobeads  14 ); DNA polymerase (not shown), free fluorescent probes  15  and free dNTPs  16 , in a per se known manner. 
         [0058]    For example, the fluorescent probes  15  may be Sybr Green or other commercially available fluorescently labelled probes (e.g., molecular beacons, scorpions, lightup probes, etc.); the nanobeads  14  are of paramagnetic or superparamagnetic material such as nanocrystals of iron, magnetite (Fe 3 O 4 ), maghemite (γFe 2 O 3 ), so as to be easily magnetized in presence of a magnetic field and attracted by the magnet  5  to the bottom wall  4 , but get back to the non-magnetized state, where they are free to move in the solution  10 , as soon as the magnetic field is removed. As indicated, the nanobeads have a diameter smaller than 50 nm, preferably 5-10 nm, e.g., 5-6 nm. 
         [0059]    Then, an amplification cycle is performed, in a per se known manner, including a denaturation step at a first, higher temperature, to obtain single strands from the target, double helix sequences; an annealing step, at a lower temperature, to allow annealing of the magnetic primers  12  to the single strands; and an extension step, at an intermediate temperature, where DNA polymerase syntheses new strands from the dNTPs  16 , to obtain amplification products, shown schematically at  20  and including at some point fluorescent probes  15 , as visible in the enlarged detail. 
         [0060]    Then, the magnet  5  is energized, to generate a magnetic field, as schematically shown in  FIG. 5 . As indicated, the parameters of the magnetic field are selected so that the multilayer  22  has a thickness matched to the vertical resolution (also called “axial resolution” or “optical slice thickness”) of the objective  6 . For example, a magnetic field of about 1 Tesla is generated. 
         [0061]    In addition, as discussed above, the system is designed so that the focal plane of the objective  6  is set at a central region of the multilayer  22 . As a consequence, all the magnetic primers  12  are attracted toward the bottom wall  4  of the microwell  2 . As above explained, due to the dimensions of the nanobeads  14 , and the concentration thereof in the solution, all the nanobeads  12 , either tied to respective amplicons or not yet bound, are attracted to the magnet and form a multilayer  22  of beads. During this step, the objective  6  reads the florescence in the multilayer  22  and generates a corresponding electrical signal S. 
         [0062]    The electrical signal S generated by the objective  6  is sent to the processing unit  8 , for standard processing. 
         [0063]    As soon as the magnet  5  is deenergized, the nanobeads  14  are free to move away from the bottom wall  4  and, by virtue of the Brownian motion, are free to float away and to spread in the liquid  10  in the microwell  2 . 
         [0064]    The above cycle is repeated more times, until the fluorescence exceeds the threshold C T  and the processing unit may generate the analysis results, usually providing the concentration of target molecules in the sample. 
         [0065]    In the embodiments of  FIGS. 6 and 7 , lateral confinement is also performed. In detail, in both embodiments, the magnet  5  has a smaller diameter than the bottom wall  4 , for example ⅕ to 1/10. By applying a sufficiently strong magnetic field, for example, of about 1T, the nanobeads  14  feel a strong holding force that causes them to stack, to form a multilayer having a width about the same as the magnet dimension. This effect may be increased by providing a recess in the bottom wall  4 , see, e.g.,  FIG. 6  at  25 , although this is not necessary (as shown in  FIG. 7 ). 
         [0066]      FIGS. 8 and 9  show another implementation of the microwell  2 . Here, the lateral walls  3  are defined by a well body  29 , of plastics, e.g., of polycarbonate, and are cylindrical. The bottom wall  4  is formed in a base body  30  different from the well body  29 , and closes microwell  2  at the bottom. The base body  30  comprises a substrate  31  of semiconductor material, e.g., silicon, covered by a dielectric layer  32 , e.g., of silicon dioxide. 
         [0067]    A nucleus region  33 , of ferromagnetic material such as iron, nickel, cobalt and alloys thereof, is embedded in the dielectric layer  32 , in a central position of the base of the microwell  2 . A coil  34  of conductive material, e.g., a metal such as aluminum, winds around the ferromagnetic region  33 , at a distance therefrom, within the same dielectric layer  32 . Coil  34  and nucleus region  33  form the magnet  5 . Here, it is the width of the nucleus region  33  that determines the lateral confinement of the multilayer  22 . In this manner, a microtiter plate having a magnetic base for each well can be made using standard and inexpensive semiconductor manufacturing techniques. 
         [0068]    For example, the microcell  1  may have the following dimensions: diameter and height of the microwell  2 : 300 μm; thickness of the nucleus region  33 : 1-2 μm; thickness of the coil  34 : 1-2 μm; thickness of the dielectric layer  32 : slightly higher than the nucleus region and coil, just to cover them, e.g., 2-2.5 μm; inner radius of the coil  34 : 250 μm; outer radius of the coil  34 : 400 μm; width W of the aluminum strip forming the coil  34 : 1.5 μm; distance L between the turns of the coil  34 ; number N of turns of the coil  34 :  50 ; permeability Km of the nucleus region  33 :  103 . With such a microwell  2 , by feeding the coil  34  with a current i of 10 mA, it is possible to generate a magnetic field BN of: 
         [0000]    
       
         
           
             
               B 
               N 
             
             = 
             
               
                 ∑ 
                 
                   n 
                   = 
                   1 
                 
                 N 
               
                
               
                   
               
                
               
                 
                   
                     
                       μ 
                       0 
                     
                      
                     i 
                   
                   
                     2 
                      
                     
                         
                     
                      
                     
                       R 
                       n 
                     
                   
                 
                  
                 
                   K 
                   m 
                 
               
             
           
         
       
       
         
           
             
               B 
               N 
             
             ∼ 
             
               1 
                
               
                   
               
                
               T 
             
           
         
       
     
         [0069]    wherein μ 0  is vacuum permeability and R n  is the radius of each turn of the coil  34 , whose maximum value, in case, is limited by the magnetic saturation of the material of the nucleus region  33  (iron, nickel, cobalt and alloys). 
         [0070]    Microwell  2  may be part of a well array  40  having a common base body  45  (of semiconductor material, analogous to body  30  of  FIG. 9 ) and a common wall body  46  (analogous to well body  29  of  FIG. 9 ), as shown in  FIG. 10 , wherein the microwells  2  may be configured to detect a same target product or different microwells  2  may detect different targets. 
         [0071]    The well array  40  may be part of an integrated device for performing RTQ-PCR, as shown in  FIG. 11 . Here, the device  50  comprise the well array  40 , a power source  41 , common for all the microcells  2  and connected to the well array  40 ; a detector array  42 , formed by a plurality of confocal optical detectors  43 , arranged above the well array  40 ; and a processing unit  44 , for controlling the various steps of the RTQ-PCR and the energization of the magnets  5 . In case, the power supply  41  and the processing unit  44  may be integrated in the substrate  31  (not shown in  11 ) of the common base body  45  of semiconductor material. 
         [0072]    The detector array  42  may be implemented by the confocal optical detector disclosed in Italian Patent application T02011A000298, filed on 1 Apr. 2011 in the name of the same applicant, that is characterized by its small overall dimensions, and thus can be used in the system  50 . 
         [0073]    As shown in  FIG. 12 , each detector  43  comprises a light source  51 , a first lens  52 , an optoelectronic sensor  53 , a pinhole  54 , and a second lens  55 , arranged in succession and vertically aligned. 
         [0074]    Operatively, when the fluorescent probes  15  ( FIG. 3 ) are illuminated by a first optical beam B 1  generated by the light source  51 , they generate a second optical beam B 2  at a generally different wavelength. 
         [0075]    Thus, the optical beams B 1  and B 2  propagate along detector axes H. 
         [0076]    The optoelectronic sensor  53 , the pinhole  54 , and the second lens  55  may be formed within a same dielectric region  66 , transparent for the used wavelengths, for example, silicon oxide SiO 2  to define a monolithic body. The second lens  55  may be of antimony trioxide Sb 2 O 3  or TiO 2 . 
         [0077]    A simulation performed by the applicant shows the gain obtainable with the use of nanobeads arranged in a multilayer. In the simulation, a microwell  2  has been used having a radius and a height of 0.5 mm, thus a chamber volume of about 393 nL, a coil  34  with an outer diameter of 0.5 mm, a ferromagnetic region  33  with radius of 0.087 mm. The simulation was performed for beads of 10 nm setting a primer concentration of 100 nM and assuming a single primer for each nanobead  14 ; this sets a binding capacity of 3000 prime/μm 2  of bead surface. In case of 2D confinement (thus so as to have a multilayer  22  of 1 μm of thickness but no lateral confinement, for the same values, beads of 333 nm are used. 
         [0078]    The simulation gave the results shown in Table 1, wherein the concentration gain G C  represents the gain of the ratio activated probes vs. non-activated probes in the confinement layer with respect to beads dispersed in the solution (no confinement). The high value of the concentration gain G C  for the 3D-confinement demonstrates that only a very small fraction of the free probes are contained in the confinement layer  22 . The table also shows the cycle gain G CY  (that is the reduction of cycles to reach the Threshold cycles) obtainable with 3D- and 2D-confinement. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 SIMULATION RESULTS 
               
             
          
           
               
                   
                 3D confinement 
                 2D confinement 
               
               
                   
                   
               
             
          
           
               
                   
                 Bead diameter 
                 10 nm 
                 330 nm 
               
               
                   
                 Layer thickness 
                  1 μm 
                  1 μm 
               
             
          
           
               
                   
                 Gain G C   
                 34868 
                 986 
               
               
                   
                 Gain G CY   
                 15.1 
                 10.0 
               
               
                   
                   
               
             
          
         
       
     
         [0079]    The advantages of the present method and device are clear from the above. In particular, it is emphasized that the described embodiments afford a very fast and sensitive RTQ-PCR, that is well reproducible, compatible with high throughput. The device may be manufactured with very small dimensions, depending mainly on the number of cells (vessels) in the array, each microwell  2  being able to perform a quick and reliable analysis with very small volumes of solutions, of a few nanoliters. 
         [0080]    By virtue of the small dimensions, it is also possible to obtain rapid thermal ramps, thus further decreasing the analysis time; and the system may be manufactured using integrated circuit planar manufacturing technology, and thus shares its advantages of low cost, high reproducibility and reliability, small dimensions. 
         [0081]    Finally, it is clear that numerous variations and modifications may be made to the described and illustrated herein, all falling within the scope of the invention as defined in the attached claims. 
         [0082]    For example, the microwell  2  may have its own bottom wall and the magnet may be formed in a separate body, including the substrate  31  and the dielectric layer  32 . The separate body or base may fit under the wells, thus bringing the magnetic coils into the desired position, beneath each well. 
         [0083]    In addition, if the Threshold Cycle CT is known, the method may comprise performing a number of amplification cycles without fluorescence reading, and performing reading only when approaching the Threshold Cycle CT. 
         [0084]    The following references are each incorporated herein in their entirety for all purposes:
   Lermoa et al., “In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens”, Biosensors and Bioelectronics 22(9-10): 2010-2017 (2007).   C. S. Jacobsen, “Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay”, Appl. Environ. Microbiol. 61(9): 3347-3352 (1995).   Cafer T. Yavuz et al. “Low-Field Magnetic Separation of monodisperse nanocrystals”, Science 314 (5801): 964-967 (2006).   EP2077336   US2008305481   US20120248347