Patent Publication Number: US-2023160024-A1

Title: Microdot array having pcr-primers fixed in each microdot and method of forming the same on a substrate for gene based pathogen detection

Description:
BACKGROUND 
     Technical Field 
     The present disclosure relates to a Lab-on-Chip having microdots that contain selected gene primers, and in particular, to a method and apparatus for providing gene amplification based on primers positioned in microdots on a silicone substrate. 
     Description of the Related Art 
     Accurate and quick diagnosis is a key element in effective identification and treatment of infectious diseases. The continued advance of point-of-care (PoC) diagnostic technology holds the potential to revolutionize healthcare in terms of practices, cost and logistics, also removing the limit and the ambiguity of diagnoses based on clinical signs and results based on microbiological cultures. Lab-on-Chip (LoC) devices are pivotal to scale down laboratory processes, multiple stages of samples handling, trained clinicians, laboratory equipment, and financial investments, providing all of them in one single microchip. Sample processing like amplification PCR or LAMP, hybridization and detection, occurs entirely on-chip, limiting contamination and avoiding handling from expert technicians and making diagnosis more robust. 
     Pathogen detection typically is targeting two types of molecules: Proteins, like antibodies or antigens, and Nucleic Acids, such as DNA and RNA. While antibodies and antigens can be identified using enzyme-linked immunosorbent assay (ELISA), DNA detection is usually performed by identifying the presence of a specific target sequence associated with the pathogen, using amplification as Polymerase Chain Reaction (PCR), LAMP or qPCR, to increase the target strands initially present inside the sample. 
     This technique, often referred to as Nucleic Acid Amplification Testing (NAAT), is more prone to be integrated inside the Lab-on-Chip Platform, creating a sample-to-result device. 
     Detection of specific strands of DNA is performed with several techniques, ranging from the optical to the electrochemical. Many optical methods have been developed utilizing fluorescence dyes but also including Raman scattering and Plasmon resonance. Most of these techniques require several handling and washing steps in liquid or hybridization of probes on the spotted microarray. 
     Currently, a variety of Lab-on-Chip products are provided in the marketplace. |Lab-on-Chips are useful for various medical diagnostic purposes, among them gene amplification and other medical uses. Such current Lab-on-Chips require a number of cycles of amplification, hybridization, and washing, as well as a number of other sequences to take place in order determine the results of a PCR that has occurred within the reaction chamber. This can result in a high cost and in some cases, be a time-consuming process. In addition, the reaction chamber must be specially prepared and remain properly sterilized in order to ensure the PCR amplification process is properly carried out. While such Lab-on-Chip gene amplification products on the market today have certain uses, a product which is lower in cost, provides faster reaction times, and is more reliable would be beneficial. 
     BRIEF SUMMARY 
     Disclosed herein are a method and structure for detecting the presence of a target DNA or gene using RT-qPCR amplification of the gene being tested for. According to principles of the present disclosure, a substrate has a plurality of microdots formed thereon. Each microdot contains one or more primers for gene amplification. In one embodiment, a pair of primers that include forward and reverse primers are present, while in another embodiment, just one primer is present. In each embodiment, the primer will hybridize to a particular target gene to be tested. While having primers that are forward and reverse primers are beneficial, any other primer combination that results in gene amplification is acceptable for attaching to a target gene to be tested. The microdots are placed on the substrate, having the primers located within the microdots. After being placed on the substrate, they are annealed and stabilized so that the primers remain vital and available for use for an extended period of time, up to several months. 
     When the test is to be carried out, the substrate having the microdots containing the primers thereon is positioned in a housing. The housing has a fluid to be tested introduced therein covering the microdot array. While the fluid is present overlying the substrate, the amplification of the gene is carried out causing any gene within that particular fluid to extend and create increased length gene chains. The fluid also contains fluorophores which will be fixed into the gene as intercalating fluorophores in the gene when it continues to increase in size as it amplifies in the particular microdot. Concurrently with the RT-qPCR cycles being carried out, the microdots are subjected to an excitation energy, such as by applying a laser light, UV light, or other acceptable excitation energy that will cause the fluorophores to output light that are present within the gene. As the amplification cycles are carried out, fluorescent light will be emitted from the microdots on which the target gene is being hybridized. As the length of the target gene grows, it will increase the fluorescence intensity at those particular locations where it grows. For those microdots which output light, this will be sensed and provide an indication that the target gene for those particular microdots is present in the sample under test. 
     According to one embodiment, the disclosure provides for a substrate which has an array of microdots thereon, with various sets of microdots having primers therein that are fixed and viable in order to attach to a particular target gene that matches the specific primers of that microdot. This structure is placed in a storage location and maintained as active and available until used at a later time. If desired, for this embodiment, the substrate can be placed into a housing and held in storage. At a future time, testing of a gene sample can carried out by placing the sample in a fluid and applying the fluid to the substrate while gene amplification takes place. 
     According to another embodiment, after the amplification has occurred and the presence of the target gene has been sensed and the results provided, the sample fluid is removed, the substrate washed and a new fluid is placed on the microarray positioned on the substrate. The additional fluid performs a denature process of the amplified gene present on the microdots in order to make the microdots available to receive additional genes. This additional fluid also contains specific beacon probes that will attach to different types of the target gene, strands of which may be left on the microdots which hybridize the target gene. Accordingly, different variations of the particular gene can be sensed using the beacon probes. Each beacon probe will output a different color of light depending on the particular variation and its attachment to a microdot. 
     The present disclosure provides a substrate having an array of microdots that are stabilized thereon, each microdot containing a plurality of primers that will attach to and hybridize with a selected target gene. This provides the benefit that the substrate can be stored and shipped, and maintained in a ready position for use at a future date. It also provides the benefit that the excitation of the microdots is carried out concurrently with the amplification of the gene using the RT-qPCR process, thus sensing whether the target gene is present or not during the amplification process itself, so the results can be rapidly determined. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIG.  1    is a side view of a starting substrate for receiving microdots according to principles of the present disclosure. 
         FIG.  2    is a side view of the substrate of  FIG.  1    in the next step of the process in preparation to receive the microdots. 
         FIG.  3    is a side view of a plurality of microdots as deposited on the substrate according to a next step in the process of fabricating a microdot array according to the disclosure herein. 
         FIG.  4    is a top plan view of an array of microdots according to an embodiment disclosed herein. 
         FIG.  5 A  is an end view of the microdots having view of at least two primers enlarged as fixed therein. 
         FIG.  5 B  is an end view of the microdots having only one primer in each microdot. 
         FIG.  6    is an end view of a fluid under test being applied to the microdots. 
         FIG.  7    is an end view showing the gene amplification during the qPCR process. 
         FIG.  8    illustrates how fluorophores are present in a selected gene strand as the amplification occurs. 
         FIG.  9    is graph showing the level of fluorescence detection after a selected number of RT-qPCR amplification cycles. 
         FIG.  10    is a top plan view of fluorescence from a substrate having an array of microdots thereon according to the detected gene in the test fluid. 
         FIG.  11    is a graph showing the fluorescence intensity changing based on the number of RT-qPCR amplification cycles. 
         FIG.  12    is a flow chart of a method of carrying out gene amplification on the described microarray according to this disclosure. 
         FIG.  13    is a test pattern of an array of microdots having different gene primers therein. 
         FIG.  14    shows the results of repeated amplification cycles being carried out on a substrate prepared according to the present disclosure. 
         FIG.  15    is a flow chart of the process for carrying out sample testing according to the methods of the present disclosure. 
         FIG.  16    illustrates a further embodiment of the present disclosure. 
         FIG.  17    illustrates an additional step in the embodiment of  FIG.  16    in which a specific beacon probe is provided at selected locations in the microdot array. 
         FIG.  18    is a side elevation view of a sample fluid on a substrate prior to starting the PCR cycles according to one embodiment of the disclosure herein. 
         FIG.  19    is a side elevation view of  FIG.  18    after the PCR cycles are completed. 
         FIGS.  20 A to  20 E  are various views of the process of fabricating the substrate for a microdot array according to one embodiment of the disclosure herein. 
         FIGS.  21 A to  21 E  are various views of additional steps after  FIG.  20 D  of the process of fabricating the substrate for a microdot array according to one embodiment of the disclosure herein. 
         FIG.  22    is a side view of a completed sampling chamber in preparation for receiving sampling fluid according to one embodiment as described herein. 
         FIG.  23    is an isometric view of a cartridge having a plurality of sampling chambers according to one embodiment as disclosed herein. 
         FIG.  24    is a graph showing the heating temperature of the chamber of  FIG.  22    over time according to one embodiment. 
         FIG.  25    is an enlarged view of a portion of the cycle of  FIG.  24   . 
         FIG.  26    is a further enlarged view of the heating of the chamber of  FIG.  22    over time according to one embodiment. 
         FIG.  27    is an enlarged view of a portion of the graph shown in  FIG.  26   . 
     
    
    
     DETAILED DESCRIPTION 
       FIG.  1    illustrates an apparatus  100  having a support base  102 , a substrate  104 , and a gene fixing substrate  108  overlying an intermediate substrate  106 . According to one embodiment, the support member  102  can be any acceptable member to rigidly hold the substrate  104  in place during the preparation process of the microarray substrate. This can be a large metal plate, a large wafer, such as a silicon wafer, or other structure which is capable of supporting other layers during the manufacturing process. The substrates  102  and  104  can be a base glass substrate, a silicon substrate, or any acceptable substrate having sufficient strength to support its own weight, and that of other layers on top of it. According to one embodiment, the layer  104  is a printed circuit board type structure, such as a PCB having alternating layers of fiberglass and conductors and insulators. In other embodiments, the layer  104  is plastic, glass or other low cost substrate. The layer  106  is preferably a silicon layer, whether polysilicon, single crystal silicon, or other layer, that can be easily adhered to the substrate  104  and is also compatible to receive the gene fixing layer  108 . The gene fixing layer  108  can be any material that is compatible with having gene material fixed thereon, which may include single crystal silicon, glass, TEOS layer, silicon Nitride, polymers, metal oxide nanostructured layer metal(0) nanostructured layer, quartz, or other material that can have a high degree of purity, as well as receive various anchoring layers for connection to gene structures. The thickness and footprint area of the layers  102 ,  104 ,  106  and  108  are not drawn to scale. In most embodiments, layer  102  will be the largest in area and the thickest. 
     Each reference to gene structures, gene, genes, an amplicon, DNA, RNA or the like, includes within its meaning any genetic material and reference to any of these elements in the specification and claims should be interpreted broadly to include a few strands of a genetic material, part of a gene, part or all of a DNA strand, part or all of an RNA strand, part or all of a chromosome or any form of genetic material. 
     The material selected for the layer  108 , and in some embodiments layers  106  and  104 , is based at least in part on the layer material being able to assist in recognizing and sensing the change in the optical properties of the gene material as amplified. A single crystal silicon, quartz, polycrystalline silicon, a silicon based compound, such as silicon nitride or silicon oxide and other materials that improve the ability to detect changes in the optical properties of the materials positioned thereon are preferred for one or more of the layers  104 ,  106  and  108 . 
       FIG.  2    illustrates the microarray apparatus  100  after a preparation sequence of steps has been carried out. According to one embodiment, the upper layer  108  is subjected to cleaning and activation processes (such as O 2  plasma treatment, oxidizing H2O:H2O2:NH4OH solution, etc.), thus providing a hydroxyl enhanced layer on top of the substrate  108 . After this, a chemical reaction process is carried out in order to provide a reactive termination groups coating  110  on the substrate  108  surface. In one embodiment, a TEOS layer is formed on top of the substrate  108 . The TEOS layer can provide a crosslinking for silicon polymers in order to provide a stable and highly adhesive attaching surface for structures later to be applied to the substrate  108 . There are various acceptable methods known in the art for forming the TEOS surface and the reactive termination group coating. These include using vapor phase deposition of epoxy-silane, followed by the appropriate heating cycles. The use of epoxy silane as an adhesion promotor is known in the art, and any acceptable technique for forming a silane adhesive promoter, including an epoxy silane adhesion promoter, may be carried out. The adhesion promotors  110  are placed on the substrate  108 , either as a blanket layer or at selected locations which are to receive the microdots as described subsequently herein. Alternatively, instead of using the epoxy O-ring with silane for the anchor layer, a general-X reactive termination for primer anchoring can be used. 
       FIG.  3    is a side view of an end of the substrate illustrating that microdots  112  have been applied to the substrate  108 . The microdots  112  are applied after the appropriate preparation by the epoxy silane adhesion layer has been applied (layer  110 ), preferably at specific locations in order to anchor the microdots solidly at the desired location in order to form the microdot array. 
     The microdots  112  are prepared and placed according to the following method. According to the methods as disclosed herein the microdots  112  could be composed by a pairs of primers forward (FW) and reverse (Rev) at various FW/Rev ratio ranging from 90:10 to 10:90, or by a single primer. 
     The first embodiment will be used for the detection of double stranded DNA or RNA, genes such as bacteria ( Mycobacterium tuberculosis , sepsis bacteria, etc.), fungi ( Candida albicans , etc.), protozoans, DNA-virus (hepatitis B virus) and virus-dsRNA (such as Rotavirus, Infectious bursal disease virus, etc.), while the single primer embodiment in used for the detection of pathogen Virus-ssRNA such as SARS cov-2 with a gene single strand RNA. 
     A primer is a short single strand of a DNA sequence that is used in a RT-qPCR technique. In the RT-qPCR method, a pair of primers is used to hybridize with a specific strands of genome dsDNA and therefore defines the region at which a gene will be amplified. In a standard RT-qPCR process the gene is first denatured by thermal process to form single strands DNA (denaturation process). Selected primers that are custom to have a particular target DNA adhere to them (annealing process) and then the amplification process mediated by enzyme polymerase can take place (extension process). Usually the RT-qPCR experiments are properly designed to have the same temperature for annealing and for the extension process, in order to have a twostep RT-qPCR reaction. The specificity of amplification reaction is mediated by the primers. For example, a pair of forward and reverse primers are used which are customized to a particular target locus to allow for amplification of a particular target gene DNA strand, but will not hybridize other gene strands. Thus, there are known primers which can match to a particular type of pathogen gene (DNA or RNA), for example, a pair of primers FW and Rev (at various molar ratio from 90:10 to 10:90), that can match to and hybridize a specific gene double strand DNA of bacteria (such as  A. streptococcus , A.  Staphylococcus ) or DNA virus (such as Hepatitis B virus, etc.). Regarding the pathogen RNA virus with single strand such as COVID 19 (also known as SARS-CoV-2) or double strands gene such as Rotavirus, two embodiments can be used and here discloses:
         1) Reverse transcriptase on fluid: pathogen gene RNA is firstly reverse transcribed to cDNA, then the pair primers into the microdots matched to the cDNA amplicons are extended in real time mode generating strands complementary to the extended probes.   2) Surface reverse transcriptase: a) for single strand RNA virus, the whole gene is recognized by a microdot containing a specific single primer anchored at surface, then the reverse transcriptase amplification at surface is carried out; b) for double-strand RNA gene virus, the whole gene is recognized by a pair of specific FW/Rev primers on microdots, than the reverse transcriptase PCR at surface is carried out.       

     Other pathogens for which primers are known include the PAN1, PAN2, and other potential target viruses that might be present in the sample taken. There will be, of course, future pathogens, including viruses, bacteria, fungi, protozoa and other organic material for which primers either currently exist or can easily be constructed in order to perform the RT-qPCR technique. 
     According to the methods as disclosed herein, a pair of specific primers (at various molar ratio) or a single primer for growing the target DNA to be tested, are mixed into a so-called printing-fluid. It is composed of various components such as saline buffer (phosphate, Tris, etc.) and low amounts of additives (from 1 to 10% of DMSO, glycerol, surfactants etc.), to obtain proper fluid characteristics (such as density, viscosity, polarity, etc.), to obtain uniform microdots at the surface. They are then made stable in the fluid for future linking to a target DNA when it becomes available. The fluid is then placed onto the substrate  108  as microdots as disclosed herein. In this particular example, the fluid is placed on the substrate  108  after the appropriate anchoring layers have been formed on the substrate  108  in order to fix and stabilize the microdots containing the viable primers. 
     The microdots can be formed on the substrate  108  using the starting fluid by any acceptable technique. One technique that is acceptable is to feed the fluid into a jet printer-type of structure, such as the type used for inkjet printers, the spraying of fine aerosols, or other microprinting devices. A type of inkjet printer will be selected which uses a pressure change diaphragm, a piezoelectric layer, or other pressure change member, in order to output the fluid. While it is known in inkjet printing that some types of inkjet printers use a heater which boils the fluid and causes the fluid to be ejected at a high temperature, such inkjet printers will likely destroy the DNA in the microdot and thus will not be used. Accordingly, an inkjet printer which operates at low temperature and to eject the fluid at a cool lower temperature, below 30° C., is preferred. Thus, a temperature is selected for the ejection of the fluid which will maintain the DNA&#39;s viability for linking to a target DNA. A number of such low temperature printers are known in the art today of which piezoelectric printers are one class and any of those available in the art are acceptable. 
     Another acceptable technique is to place the fluid into a pipette and drop the fluid onto the substrate  108  to form the individual microdots. Yet another acceptable technique is to use a solid glass rod, without a lumen, and to extract a drop from the fluid using the surface friction between the fluid and the solid glass rod to pick it up and then deposit the microdot onto the substrate  108  at the desired location. Any other of the many acceptable techniques may be used in order to transfer the fluid containing the desired primers from the fluid reservoir to place them in small microdots on the substrate  108 . 
     As shown in  FIG.  4   , an array  114  of microdots are placed on the substrate  108 . Among the group of microdots  112  are particular microdots that contain specific primer for a selected target pathogen gene (DNA or RNA). In the embodiment shown in  FIGS.  4  and  5   , each of microdots  112 ( a ),  112 ( b ),  112 ( c ) and  112 ( d ) contain a different pair of primers that will each adhere to different target genes. 
     Viewing  FIG.  5 A , it is shown that specific FW-primer (a) and Rev-primer  116 ( b ) within the microdot  112 ( a ) are custom for specific double stranded DNA genes. If there is DNA in the specific gene within the sample fluid  122  that contains strands that would bond with the particular primers that had been placed in microdot  112 ( a ), it will amplify on primers  116 ( a ) and  116 ( b ). However, there will be no growth in microdots  112 ( b ),  112 ( c ), or  112 ( d ). 
     Similarly in  FIG.  5 B , it is shown that a single primer  116  within the microdot  112 ( x ) is custom for a specific single stranded RNA gene. If there is the specific gene within the sample fluid  122  of the strand-RNA that would bond with the particular primers that had been placed in microdot  112 ( a ), it will amplify on primers  116 ,  118  or other single primer placed therein. In one embodiment, a single primer is used for single-strand RNA genome pathogen (SARS COV2). Thus, it is possible to provide the gene amplification using a single primer, a pair of primers, three or more primers or other combination of primers that are available for use at the time the invention is practiced. 
     Each of the microdots has been selectively provided with a pair of FW-Rev-primers (at various FW/Rev ratios) or a single-primer that will hybridize with each strand of the specific gene. Within the array  114  of the microdots  112 , each microdot will hybridize to a different gene target sample. For example, the microdots  112 ( a ) will hybridize to a different type of target gene than microdots  112 ( b ) and microdots  112 ( c ) will hybridize to a different target gene. 
     As noted,  FIG.  5    is a side view of the substrate  108  in which the different types of DNA primers from each microdot  112  are easily viewable. Each of the microdots  112 ( a ),  112 ( b ),  112 ( c ), and  112 ( d ) are illustrated having different DNA primers  116 ( a ) and  116 ( b ) for forward and reverse primers that are specific to that particular microdot. Another microdot will have different forward and reverse primers  118 ( a ) and  118 ( b ) which are shown associated with the microdot  112 ( b ). The primers  116 ( a ) and  116 ( b ) are shown greatly enlarged and extending further from the microdot  112 ( a ) than they are with respect to the scale of the microdots and are enlarged in order to better illustrate the ability to adhere to the gene DNA target. 
       FIG.  6    shows the next step in which the substrate  108  has been placed in a housing  120 . Namely, the substrate  108  can be placed in cartridge that contains various open chambers that can act as the housing  120  hold the substrate  108  in the bottom and receive a sample containing fluid  122 . Over the fluid  122  a sealing layer  200  is provided to maintain the integrity of the sample fluid  122 . The sealing layer  200  can be mineral oil, wax or other material that is less dense than the sample fluid  122 . The substrate  108  can be placed in the housing  120  at a desired time, for example, well prior to introduction of the sample fluid  122 . The substrate  108  can be placed in a cartridge that comprises housing  120  right after the microdots are placed thereon, as explained later herein. The substrate  108  is removed from the support  102  when it is placed in the housing  120  since the support used during the manufacturing process is no longer needed. The substrates  104  and  106  can remain with the substrate  108  and be placed in the housing  120  in one embodiment, and can also be separated from the substrate  108  so that only it is placed in the housing  120 . 
     As shown in  FIG.  7   , after the sample fluid  122  containing the gene target to be tested in place in the housing  120 , a number of RT-qPCR amplification cycles are carried out. The carrying out of RT-qPCR amplification cycles on a fluid containing a gene target to be amplified is well known in the art as described in many publications and the details need not be provided here. In summary, the amplification includes a number of heating and cooling cycles under selected conditions to cause DNA in the fluid  122  to grow and attach to the primers present in the microdots as it grows. According to this disclosure, the primers are fixed in the microdots  112  at a prior time, several days, weeks or months before being exposed to the gene target in the fluid  122 . Namely, primers  116 ( a ) and  116 ( b ) that are present in the microdots  112  and have exposed linking sites at the surface of the microdot. The gene target will be not amplified on the other microdots  112 ( b ),  112 ( c ) and  112 ( d ) because the gene target for the primers within those microdots is not present in the fluid  122 . The different types of primers are drawn as different shapes, such large zig-zags, curls, square lines, or tight zig-zags for microdots  112 ( a ),  112 ( b ),  112 ( c ) and  112 ( d ) respectively for illustration purposes only to show that they are specific to hybridize with only the gene target for which they are custom fit and not with other genes. 
     As shown in  FIG.  8   , fluorophores  130 , as SYBR Green, SYBR Gold, etc., are present in the fluid  122  and enter the double strand amplicon as it grows. In some embodiments, the double strand amplicon can be a gene structure that is a double stand amplification of genetic material. It can be, for example, DNA, RNA, or some other PCR amplified gene material. The fluorophores can be present as intercalating fluorophores in some embodiments. Alternatively, the fluorophores can be present in the double strand amplicon in different structures or modes. They can be present linked to the amplicon by an affinity to part of the structure, by coupling to an available site, or by other techniques. Thus, the fluorophores can become associated with the gene material as it is amplified by any acceptable method, whether by intercalating, coupling to the gene by attaching to available sites on the structure or any acceptable technique. This will provide an optical signal for each of the amplified strands that has been amplified from a respective microdot  112 . 
     As the amplification occurs, an increasing number of fluorophores  130  enter the growing double strand amplicon structure  124 ( a ), increasing the light that will be given off when the microdot is subjected to excitation energy. The fluid  122 , together with each of the microdots  112 , are subjected to an excitation energy while the amplification process is occurring in order to cause the target DNA to fluoresce if it is present on the microdot. For example, an argon laser, UV light, or other light source can be applied as an excitation energy to the microdots  112  in order to cause the fluorophores present in the target DNA to emit light. As illustrated in  FIG.  8   , various fluorophores are placed into the amplified DNA structure  124 ( a ) as it grows so that they are present within the double strand amplicon structure. When the microdot  112  is subjected to an excitation signal, such as a laser light, UV light, LED Light or other source, then the fluorophores  130  will fluoresce providing a visible response if the target DNA  124  has grown at the particular microdot. 
       FIG.  9    is a graph that shows the change in fluorescence intensity as RT-qPCR cycles are carried out on the fluid under test. In particular,  FIG.  9    shows the fluorescence intensity output of two microdots, the first line  140  showing the change in fluorescence intensity output for a target probe for the specific pathogen SARS-CoV-2 virus as additional RT-qPCR cycles are carried out. Line  142  shows the increase in fluorescence intensity for a microdot having primers that bond to gene PAN1 virus as RT-qPCR is carried out. In particular, as shown in  FIG.  9   , while the fluid  122  is present overlying the microdots on the substrate  108 , the RT-qPCR cycles are carried out which results in growth and hybridization to those primers which match to the target material present in the sample fluid  122 . As illustrated in  FIG.  7   , microdot  112 ( a ) has gene molecules attached to the primers causing them to grow. As they grow, the number of fluorophores  130  within the amplicons  124 ( a ) will increase in number and the fluorescence intensity will increase. The excitation energy, whether in the form of a laser, UV light, or other excitation energy, is concurrently applied to the liquid  128  and the microdots  112  while the RT-qPCR cycles are being carried out. In the example shown in the graph of  FIG.  9   , at approximately 30 cycles, the number of fluorophores within the DNA  124 ( a ) have increased to the point that fluorescence intensity begins to be visible. As the cycles continue, the target DNA  124 ( a ) has grown larger so that at approximately 40 cycles the fluorescence intensity has reached to be approximately 200 a.u. for the gene target G1 for the microdot under consideration, line  140 . With respect to the microdot that hybridizes to the gene G2, the fluorescence intensity is in the range of approximately 100 after 40 cycles, line  142 . Since the excitation energy is applied to the to the sample fluid  122  while the RT-qPCR cycles are carried out, the transition from no fluorescence to a high level of fluorescence can be recognized and sensed. According to one embodiment, each RT-qPCR cycle takes in the range of one minute, though in some embodiments, the RT-qPCR cycles may be several minutes in length for one cycle. In the situation in which a single RT-qPCR cycle takes approximately one minute, this means that after about 30 minutes of the test being conducted, 30 cycles will be completed and the results will be known whether or not the target DNA is present in the sample under test as present in the sample fluid  122 . 
     The present disclosure has the benefit that the excitation energy is applied to the microdot  112  concurrently with the cycles being carried out and while the sample fluid  122  is present covering the microdots. This provides the advantage that as soon as fluorescence begins, this can be sensed and results provided to the user of the test. It also provides the benefit that the amount of gene target present can be approximated based on the number of cycles required for the fluorescence to start and its overall ending intensity. If the fluorescence intensity climbs quickly, to a high value, for example, over 200 a.u., this provides an indication that there is a high amount of the gene target in the sample fluid  122 . It also has the benefit that as soon as the target DNA is detected as being present, the test can end rather than continuing for an extended period of time. 
       FIG.  10    is a top side view of the substrate  108  as shown in  FIG.  4    after the RT-qPCR cycles have been carried out and illustrating with a circle the microdots  112  which have a high fluorescence intensity. As can be seen viewing  FIG.  10   , the array  114  of microdots has a number of microdots which are outputting light, the amount of light being output indicated by the size of the diameter of the particular microdots  112 . The intensity of fluorescence signal is related to the amount of gene target present that has been amplified in the sample. 
     This level of fluorescence as shown by  FIG.  10    would be similar to that shown in  FIG.  9    in which line  140  corresponds to the light output by microdot  112 ( a ) and line  142  corresponds to microdot  112 ( b ). The microdots not illuminated in  FIG.  10    are those for which the specific gene target for those particular microdots is not present in the fluid  122  which is being tested. In particular, each microdot  112  as shown in the  FIG.  4    is still present on the substrate  108 , however, the majority of the microdots  112  did not have the specific gene target for which the primers were selected to hybridize with present in the fluid under test. Accordingly, the gene target of the sample  122  did not attach to those microdots and after a number of RT-qPCR cycles were carried out, the microdots did not output fluorescent light and remained dark as line  144  in picture  9 . Therefore, it can be reported that the target DNA for which those particular microdots had been selected to test for was not present in the sample  128 . 
       FIG.  11    is another example of the fluorescence intensity changing on a substrate  108  as RT-qPCR cycles are carried and excitation energy as applied to the entire array  114  of microdots. In the example of  FIG.  11   , all microdots  112  are each excited, each one outputting a light signal, the group of light signals labeled generally  150 , in which several individual microdots are outputting fluorescent light because the target DNA for many microdots  112  is present in the sample under test. The flat line  144  indicates that no light is being emitted by those microdots for which the gene target is not present. Accordingly, even after more than 40 cycles, the microdots  112  for which the gene target is not present for which their primers have been selected to hybridize with still remain dark, indicating that the target gene for those microdots is not present in this sample under test. According to a preferred embodiment, each substrate  108  will contain a plurality, usually in excess of a dozen microdots, many containing the same primers for hybridizing with the same target DNA. Accordingly, if the target gene is present it would be expected that all of the microdots that have the same primers that will hybridize with the particular DNA under test will increase in fluorescence intensity at about the same time, thus providing duplicative testing sites and a higher degree of assurance regarding the validity of the test, whether a positive or a negative for the DNA under test. 
       FIG.  12    illustrates one flowchart according to a particular method to carry out the process of constructing and using a substrate  108  according to the principles of the present disclosure. In a first step  160 , a substrate has the surface treated with a silicon oxide plasma, or alternatively, the substrate  108  is subjected to a plasma in the presence of oxygen gas, therefore creating an active silicon oxide upper surface. According to an alternative embodiment, the plasma may be carried out in the presence of a nitrogen gas, thus creating a nitride surface, for example, a silicon nitride upper surface. Depending on the material for the microdots, either a silicon oxide or a silicon nitride surface is preferred, other substrates can be used, such as glass, plastic, polymers etc. According to the next step, a vapor phase deposition is carried out. According to one embodiment, epoxy-silane is formed using a vapor phase silanization process in order to provide a strong adherence of material to the upper substrate  108 , as illustrated in step  162 . After the creation of an adhesive later, whether by the vapor phase deposition of an epoxy-silane material or other adhesion layer, the microdots having the primers (pairs at various molar ratios or a single primer) are placed on the substrate  108  in step  164 . The microdots  112  are anchored to the substrate  108 , and then the appropriate annealing is carried out in order to stabilize the microdots  112  on the substrate  108 . Once stabilized, the primers in the microdots remain active for potential bonding at a later time that might be several weeks or months away. After step  164 , the substrate  108 , having a microdot array  114  thereon comprised of a number of microdots  112 , can be placed in storage for a long period of time, several months, or perhaps several years. When desired, the substrate  108  can then be sent to a number of different locations, such as hospitals, laboratories, testing sites, or even in the field at various doctors&#39; offices, or drive-by health clinics. The substrate  108  has been properly stabilized so that the microdots  112  remain vital and available for DNA amplification when they are subjected to a test sample. The various microdots may remain stable for several months, using the appropriate sealing and annealing techniques as is known in the art. 
     At this stage in the process, the substrate  108  in the cartridge that contains housing  120  will be in the hands of many healthcare professionals at numerous locations awaiting use for samples under test. At some period of time, after step  164 , the substrates  108  will be placed in a proper housing, such as a cassette, a clip, or some type of receiving surface that contains a housing  120  so that it can receive a fluid sample  122 . The substrates  108  can be placed in the housing  120  prior to being shipped to the various medical testing labs, or can be shipped as substantially flat substrates  108  and can be placed in the housing  120  just prior to the test being carried out. As shown in step  166 , the substrates are placed in a housing  120  that has a place for a reservoir at some point after being properly manufactured and stabilized. 
     In step  168 , a fluid to be tested is applied to the substrate  108  while in the housing  120 . The step  168  can be carried out at any time in the future, depending on when the samples are collected and it is desired to conduct a test. After the fluid under test, in the example shown, fluid  122 , has been applied to the substrate  108 , then surface amplification takes place using qPCR with intercalating fluorophores as shown in step  170 . In one embodiment (specific for DNA gene pathogen) the particular PCR process that is carried out is Amplification using Surface Real Time qPCR. The surface amplification provides the amplification of the DNA gene present in the fluid  122  to attach to the surface of the particular microdot having a pair of primers that matches the target gene, but not to any other microdots. 
     In a second embodiment (specific for single and double stranded RNA gene pathogens) a reverse transcriptase reaction needs to be performed, before beginning the Surface Real Time qPCR. 
     If the gene under test contains the specific sequences that match with the primers of a particular microdot  112 , then the number of fluorophores  130  will increase as the amplification reaction DNA chain connected to the respective microdot becomes longer. As the amplification cycles are carried out, concurrently an excitation energy is applied to the microdots  112  as shown in step  170 . In particular, as illustrated in step  172 , the excitation energy is applied to the microdots as the RT-qPCR cycles are being conducted, and the microdots which have primers that match with the target DNA present in the sample under test  122  will begin to increase in the light intensity output, as illustrated in  FIGS.  9  and  11   . In particular, step  172  shows the RT-qPCR cycles being carried out while there is sensing of an increase of the output of fluorescent light in the sample under test. The number of RT-qPCR cycles carried out can vary depending on the target DNA and the type of fluid being used. In some embodiments, the number of RT-qPCR cycles may be in the range of 30 to 40, while in other embodiments, the number of RT-qPCR cycles may exceed a few hundred, again depending on the type of DNA being tested for, the material of the microdots, and the fluid  122  being tested. 
     In a third embodiment (for a single and double stranded RNA gene pathogen) a direct surface reverse transcriptase is carried out. In this case the ssRNA gene is recognized by a single primer on microdots and the surface reverse transcriptase is carried out. Similarly, the dsRNA gene is recognized by the pair of specific primer and then the surface reverse transcriptase is carried out. 
       FIG.  13    illustrates a substrate  108  having thereon an array  114  of microdots  112 . In the example of  FIG.  13   , the array  114  has been specifically constructed in order to facilitate and improve the sensing of a particular target DNA. The type of hatching in each microdot of  FIG.  13    indicates the purpose of the microdot, with hatching for a positive test, negative or other. 
     In the example of  FIG.  13   , the substrate  108  contains four alignment sets  180  of microdots that are in each of the corner of the substrate  108 . In particular, alignment sets  180  are positioned in each of the corners of the substrate  108 . These alignment sets  180  contain two different types of primers in the respective microdots  112 . In particular, the alignment arrays  180  will include a microdot  112 ( p ) which is a RT-PCR control positive and microdot  112 ( n ) which is a RT-PCR control negative. In this particular example, microdot  112 ( p ) is always positive and shows fluorescence output, thus, microdot  112 ( p ) will output light in all circumstances; if microdot  112 ( p ) is dark, then the substrate  108  is discarded as not valid because this is an indication that a global error has occurred, either in the test or in the construction of the completed substrate  108 . The positive microdots  112 ( p ) also assist with alignment during the test, particularly if all the other dots are dark. Thus, the positive microdots will act as alignment markers when the sensing is being carried out to ensure that the substrate and cartridge are properly positioned in the testing apparatus. 
     The rest of the array is composed by various microdots  112 ( t ) with target primers specific for pathogen genes (such as hepatitis, SARS-CoV-2) and microdots  112 ( vt ) for variants recognition. The microdots  112 ( t ) and  112 ( vt ) will fluoresce if the pathogen or its variants are found to be present in the sample, but will remain dark and not fluoresce if the specific target gene is not present in the sample. Microdots  112 ( vt ),  112 ( vo )  112 ( vn ) may also be present on the substrate  108 , but are not shown in  FIG.  13    for simplicity. 
     Microdot  112 ( n ) has primers that will not react with the target gene to which microdot  112 ( t ) is positive. Namely, microdot  112 ( n ) contains an opposite type of primers that will prevent growth of the type of gene which microdot  112 ( t ) is testing for and seeking for the target. Accordingly, microdot  112 ( n ) will remain dark and show a negative test at all times if the test is a valid test. If microdot  112 ( n ) emits light the test is considered invalid. Microdot  112 ( o ) is provided to test for additional types of pathogen genes, which might be present in the sample and related microdots  112 ( vo ) for variants of the additional types of genes being tested for will also be present in the microarray. 
     For example, it is known that it is common for other pathogen microorganisms such as PAN1 or PAN2 to be present in the same environment in which gene virus such as SARS-CoV-2 is present. Accordingly, the presence or absence of another gene, which is distinctly different from the target under test, but often found with it, is also beneficial to provide an indication whether or not the test is a true positive test or a false positive test, or on the other hand, is a true negative test or a false negative test. In one embodiment, a set of orientation microdots  182  are at the top and bottom of the substrate  108 . The orientation microdots  182  are provided at a preset location where the specific alignment and makeup of the microdots at the edges of the substrate  108  are known. As can be appreciated, in some embodiments, the substrate  108  will have only one or two alignment sets  180  of the microdots and may or may not have orientation sets  182  of the microdots. Alternatively, the substrate  108  may have multiple alignment sets at various locations throughout the array instead of only at the corners. 
     Viewing  FIG.  13   , it can be seen that throughout the entire array  114  there are a variety of different microdots having different primers therein (designed to recognize the pathogen and its variants) that are prepared in advance to hybridize with different target DNAs that may or may not be present in the fluid  122  under test. 
       FIG.  14    illustrates the results of a test being carried out on the substrate  108  of  FIG.  13    in which a positive sample of SARS-CoV-2 is present in the fluid  122  and amplification cycles in excess of 30 have been carried out. As can be seen in  FIG.  14   , each of the control microdots  112 ( p ) are illuminated, indicating that the test has been properly carried out with respect to amplification. It would be expected that each of the control positive microdots  112 ( p ) would be illuminated in the instance of the target DNA not being present. Accordingly, the illumination of microdots  112 ( p ) indicates that the amplification has properly occurred and that the sample has been properly tested in order to determine whether or not the target DNA is present. It can also be seen in this particular instance that the microdots  112 ( t ) that contain the primers for the target gene, which for this example is SARS-CoV-2, are also illuminated. This indicates that each of the microdots  112 ( t ) has hybridized and grown the target gene corresponding to pathogen species being present. 
     Similarly, in the particular sample under test, PAN1 was shown as present in the other sample being tested, as indicated by the microdots  112 ( o ) also being illuminated. 
     As can be seen in  FIG.  14    each location in which a negative microdot  112 ( n ) is present, no light is output and the substrate  108  is dark at that location, as shown in  FIG.  14   . This illustrates that the test has been properly carried out with respect to the microdots  112 ( n ) continuing to show a negative value and not turning to positive through the amplification cycles. 
       FIG.  15    illustrates a further embodiment and additional steps being carried out on the substrate  108  and the sample under test of the fluid  122  containing the target gene. As illustrated in  FIG.  15   , each of the initial steps is the same as  FIG.  12   , namely the surface is prepared in step  160  in which a plasma treatment of either oxygen or nitrogen is carried out. After this, the surface has an adhesion layer applied, for example, with an epoxy-silane vapor phase deposition in step  162  which is followed by applying the microdots  112  having the primers contained therein after which the primers are properly stabilized. In step  166  the substrate  108  is placed in a housing, and then in step  168 , the fluid is then applied to be tested. Surface amplification is conducted as previously described in step  170  and then the signal acquisition is carried out in step  172 . 
     If the test indicates that the target gene is present in step  172 , then it is possible to carry out a further test of the target gene under consideration. Specifically, in the further embodiment of additional testing, after the target gene has been fully hybridized and a large strand of DNA of the target material has attached to the microdot  112 ( t ) and  112 ( vt ), then further testing can be carried on that particular target gene strand. 
     According to the embodiment of  FIG.  15   , the fluid  122  under test is washed away and removed from the substrate  108 . The target gene strands  124 ( a ) and  124 ( b ) remain coupled to the respective microdots. As previously indicated, the fluid  122  under test was present during the entire amplification process and also present during the application of excitation energy to the microdots in order to cause them to fluoresce in the presence of the fluorophores being absorbed into the target gene being built during the amplification process. In some instances, the mutation vt of the target gene might not be sufficiently close to the target gene to cause a reaction on either microdots  112 ( t ) or  112 ( vt ). It is preferred in some embodiments to insert specific primers at different locations or in different microdots  112  in order to sense for yet unrecognized variations of the target gene t for which microdots  112 ( t ) and  112 ( vt ) have been tuned. After the test results show positive, the fluid  122  is removed and the microdot array  114  is subjected to a new fluid which denatures at least one of the primer, whether forward or reverse of the microdots that contain the grown gene material  124 . In particular, during the denaturation process, as is known, some of the grown gene  124  will be separated and the different strands of DNA  124 ( b ) will be removed from the microdots in order to free up the exposed ends of the various primers and different portions of the DNA  124  which has been hybridized and grown during the amplification process. After this, in step  174 , a pair of specific beacon probes (properly designed to recognized the specific mutation and wild-type) labelled with different colors (i.e., Cy5 and Cy3) are introduced into the fluid. 
     In one particular example, the SARS-CoV-2 virus has a number of variants that may exist, some of which are known as the UK variant, the Brazil variant, or additional mutations or variants that may occur over time. After a determination of a positive SARS-CoV-2 test has been conducted, it may afterward be desirable to determine the particular mutation or variant of the type of SARS-CoV-2 virus present that has been tested. The microdots  112 ( t ) of the primer in the first test are prepared to show a positive result with any type of variant of the SARS-CoV-2 virus present. Therefore, the microdots  112 ( vt ) that include the sequence region of mutation will be interrogated by the pair of beacon probes. In step  174 , further testing is carried out with specific beacon probes having a specific DNA attached thereto which will bond only to the target DNA for specific variants. The specific beacon probes each have a different color to fluoresce under excitation. If the particular variant of the beacon probe is present in the sample, it will bond to where some strands of the gene mutation were or remain present. Accordingly, once the beacon probes are introduced, the beacons will hybridize with the extended gene or DNA strand, giving a fluorescence signal. The signal ratio Cy5-mut/Cy3-wt will indicate the presence/absence of mutation. 
       FIG.  16    illustrates the carrying out of steps  174  and  176  of the flowchart of  FIG.  15   . In particular, a new fluid  187  is introduced into the housing  120  after the fluid under test  122  has been removed and the substrate  108  has been properly washed and annealed and treated in preparation for the next step. The fluid  187  includes a number of specific beacon probes of different mutation, each of which will adhere to particular variants of the target gene under test. The fluid  187  will carry out a denature process in which some of the hybridized DNA  124 ( b ) and/or some of the hybridized DNA  124 ( a ) is removed from the microdot (see  FIG.  7    in which the DNAs  124 ( a ) and  124 ( b ) have grown onto the microdot  112 ( a )). In particular, some or all of the hybridized DNA  124 ( a ) and  124 ( b ) will be removed from the microdot  112 ( a ), or in the example of  FIG.  13   , microdot  112 ( t ) for the microdot for the target material. After the microdots  112 ( t ) are hybridized and then denatured, this leaves open sites on the microdot  112 ( a ) that are receptive to particular types of beacon probes for the variant to be tested. 
     As shown in  FIG.  17   , at this stage, a plurality of beacon probes, that correspond to the different variants (and its related wild-type) of the virus under test, are present in the fluid  187 . The beacon probes Cy5-mut-190 will attach to the microdot  112 ( a ) in the event the particular variant under test corresponds to the type of microdot being sensed by the beacon probe  189 . 
     Thus, within the fluid  187  will be a number of different beacon probes to test for different variants of the target gene. Those beacon probes which correspond to the variants that have been found will attach to and begin to fluoresce the signal of Cy5, but some of Cy3-wt- 190  will hybridize and the signal Cy3 will be also recorded. Then the presence/absence of mutation will be declared by the Cy5/Cy3 signal ratio. 
       FIGS.  18 - 27    illustrates one embodiment in which the microdot array as shown and described with respect to  FIGS.  1 - 7    can be constructed within a cartridge and prepared for use in the field. 
       FIG.  18    shows a chamber  190  which contains a sample fluid  122  according to the principles as described herein. The sample fluid  122  can correspond to the sample fluid  122  shown in  FIGS.  6  and  7   , or a different prepared sample fluid, according to any method acceptable in the art. In the embodiment shown, the sample fluid is positioned on top of a sealing layer  200  which has been previously prepared to receive the sample  122 , as will be described herein. Above the sealing layer  200  and the sample fluid  122  may be any ambient environment, for example, open air, a nitrogen chamber, an inert argon atmosphere, or a sterile environment which permits the introduction of the sample fluid  122  into the chamber  190 . 
     The chamber  190  also includes a substrate  104 . According to one embodiment, the substrate  104  is the same substrate that was prepared for use and accessed as explained in  FIGS.  1 - 3   . It may also be a different type of substrate, depending on the type of sealing layer  200 , or the type of housing  120  that will be used. According to one embodiment, the substrate  104  is a silicon substrate. It can have prepared thereon the layers  106  and  108  as previously described herein and be configured to receive the various microdots  112  as described and shown in  FIGS.  4 - 5 B . In this embodiment, the substrate  104  includes a circular recess  204 . In the center of the circular recess is a ridge  206 . Accordingly, the recess tool is in the form of a donut, with the ridge  206  extending up through the hole of what can be considered the donut shape. The ridge  204  can have other shapes besides circular, for example, rectangular, oblong, diamond or other embodiments. A pattern which has recesses in a closed loop around a center ridge  206  is preferred. Under the substrate  104  is a heater  202 . A layer  107  overlies the substrate  104  outside of the recess and the ridge  206 . 
     The sealing layer  200  has a recess in the sealing layer overlying the substrate. This recess is located in a central region of the sealing layer. The sealing layer has a first height in the central region and a second, greater height in a peripheral region of the sealing layer. A recess  204  is positioned in the upper surface of the substrate, the recess being positioned below the recess in the sealing layer. The recess in the sealing layer is overlying at least a portion of the recess in the substrate. The ridge  206  in the upper surface of the substrate is positioned adjacent to the recess in the upper surface of the substrate and the recess in the sealing layer is overlying the ridge  206  in the substrate. The upper surface of the ridge  206  is in the same plane as an upper surface of the substrate at locations outside of the recess in the substrate, which includes locations positioned below the region of the sealing layer having the second, greater height. 
     Also shown in the embodiment of  FIG.  18   , one example of the relative dimensions of the chamber  190  is provided. According to one embodiment, the recess is in the range of 50 μm to 250 μm deep, with a depth of 75-250 μm preferred. The ridge therefore has a height in the range of 52-250 μm, and preferably 150 μm above the bottom of the recess tool. On the left-hand side of the graph in  FIG.  18    are the relative micron dimensions of one example embodiment, with the zero height being aligned exactly with the top of the ridge  206 . Accordingly, as can be seen, the total thickness of substrate  104  is somewhat greater than 500 μm, for example, in the range of 600-700 μm. As can be appreciated, in some embodiments the thickness of the substrate  104  can be in the range of 400-3000 μm, with a thickness of approximately 600 μm preferred. 
       FIG.  19    illustrates a subsequent time after the substrate  104  has been heated, which has caused the sealing fluid  200  to modify its shape and the sample fluid  122  has moved to be in physical contact with the substrate  104 . In this position, the sample  122  has been heated to an initial temperature, and is prepared to undergo a number of PCR cycles in order to detect the presence of a desired target gene within the sample fluid  122 . In the embodiment shown in  FIG.  19   , a sample time of 12 seconds has passed from when the heater  202  heated the chamber  190  and caused the sample fluid  122  to descend to be in physical contact with the substrate  104 . Overlying the substrate  104  will be a large number of microdots  112  as previously described with respect to  FIGS.  1 - 10   . These microdots can be physically formed on the substrate  104 , as described with respect to  FIGS.  1 - 5 B  with the various layers  106  and  108  overlying the substrate  104  in the manner previously described. Therefore, in one embodiment, the microdots  112  are formed by placing them onto the properly prepared substrate  104  with the intervening layers. The microdots may therefore be both in the recess  204  and on top of the ridge  206 . These are not shown in  FIGS.  18  and  19    for simplicity&#39;s sake. It will be appreciated, however, that a plurality of microdots  112  can be in the recess tool as well as a plurality of microdots on top of the ridge  206 . 
     In an alternative embodiment, the microdots  112  may be formed on a thin substrate which is fitted and positioned to sit on top of the substrate  104 . In this alternative embodiment, a custom prepared substrate is provided, on which the microdots  112  are positioned. This custom substrate is then placed on top of the substrate  104  and properly positioned in the recess and, in some instances, on top of the ridge  206  having the microdots positioned to be physically in contact with the sample fluid  122 . As previously noted, for simplicity of illustration, the microdots  112  are not shown in  FIGS.  18  and  19   . 
     The method of making the chamber  190  will now be described with respect to this particular embodiment.  FIG.  20 A  shows a starting sequence for the formation of the chamber  190  according to a particular embodiment. The substrate  104  is provided overlying on which a buffer layer  107  is formed. The buffer layer  107  may be an oxide layer that is grown on the silicon substrate  104  using a thermal process in an oxygen environment. It may also be a deposited nitride layer, a deposited oxynitride layer, with alternating layers, a silicon carbide layer or other layer  107  which properly buffers the substrate  104  and prepares it to receive other structures. A metal heating layer is then deposited onto the buffer layer  107 , in order to form the heater  202 . In a preferred embodiment, the metal layer is aluminum, although in other embodiments it may be comprised of titanium, nickel, copper, or other acceptable material in order to perform the heating function of the PCR process to be later carried out. The deposited metal is patterned and etched to provide a heater and also the sensor definition. As will be appreciated, the method of forming a heater as well as an appropriate thermal center to determine the temperature of the heater and the silicon substrate  104  is known in the art and therefore the details of which are not described herein. 
     As shown in  FIG.  20 B , the substrate  104  is flipped over, to place the heater  202  on the bottom and present a top surface having the layer  107  exposed to receive additional process steps. A mask  210  is patterned and etched onto the buffer layer  107  overlying the substrate  104 . The mask  210  has a pattern formed therein of the desired shape of the recess tool and the ridge  206 . In the embodiment shown, as can be seen in  FIG.  20 C , the recess tool will be in a circular shape, with the ridge  206  in a circular shape, accordingly a recess forming mask  210  is formed having that shape. With the mask  210  in place, an anisotropic etching takes place in order to form the recess tool, and leave the ridge  206 . The etching carried out is selective to etch silicon of substrate  104  and to not etch the buffer layer  107 . As can be appreciated, prior to carrying out the etching of the silicon, a selective etch is used to etch layer  107  as shown in  FIG.  20 B  followed by the anisotropic etch of the silicon  104  in order to create the recess tool and leaving the ridge  206  as shown in  FIG.  20 D .  FIG.  20 E  shows a top side view of the substrate  104  in which the buffer layer  107  can be seen on the top, the recess tool for is a circular recess and the ridge  206  in the central portion thereof. 
     This etching can be carried out with the dry silica and anisotropic edge to a depth of about 150 μm according to a preferred embodiment. As previously described, the depth of the etch may be in the range of 50 μm-250 or 300 μm the depth being selected based on the microdots  112  to be placed therein. The microdots  112  may have different heights, depending upon the primers  116  to be placed therein. Accordingly, the depth of the recess tool for may take into account the diameter and height of the microdots  112  to be placed therein. In addition, if the recess tool is going to receive an additional substrate to be placed therein it may be etched deeper in order to account for the height of additional substrate which includes the microdots  112  placed thereon. 
     As seen in  FIG.  21 A , after the recess tool has been etched, the buffer layer  107  is restored across the entire top of the substrate  104 . In the embodiment in which the substrate  107  is a thermal oxide, this can be achieved by heating the substrate  104  in an oxygen-containing atmosphere to thermally grow an oxide layer uniformly on the exposed surface of the substrate  104  to achieve the buffer layer  107 . Alternatively, a nitrogen layer, carbide layer or additional buffer layer can be deposited on top of the wafer  104 . This can be done with the previous layer  107  in place and add additional thickness to those portions where the layer  107  is already present, and provide a layer which is somewhat thinner in the recess tool for which it has been provided. Alternatively, the entire buffer layer  107  can be removed and an entirely new buffer layer  107  can be provided. According to one embodiment, the buffer layer  107  is 0.5 μm thick and is provided by a CVD deposition of the PETEOS layer. 
     As shown in  FIG.  21 B , an isolation layer  212  is provided over the buffer layer  107 . The isolation layer  212  can be any acceptable photoresist, in one preferred embodiment, it is an SINR dry film layer of approximate 50 μm in thickness. Any other acceptable photoresist or other polymer layer may be used for the isolation layer  212 . As shown in  FIG.  1 C , the isolation layer  212  covers all portions of the substrate  104  except the recess  204  and the ridge  206 . The buffer layer  107  remains on these locations in order to provide a mask stop for the etching of layer  212 . Namely, the material selected for layer  212  will be selectively etchable with respect to the buffer layer  107 , and therefore, the isolation layer  212  can be pattern etched to have a desired shape with the buffer layer  107  acting as an etch stop. 
     As shown in  FIG.  21 D , housing  120  is then constructed onto the substrate  104 . The housing  120  can be a cartridge of the type shown in  FIG.  23    and described later herein. In this embodiment, the substrate has a size in which 6, 10, 12 or more recess and ridge combinations are formed therein. Namely, a relatively large substrate  104  is provided in which a plurality of recesses  204  and ridges  206  are formed at the same time during the same preparation process. After this, the housing  120  is placed on top of the substrate  104  in order to provide a plurality of chambers  190  as shown in  FIG.  23   . Alternatively, the housing  120  can be an individual housing that is custom for only a single ridge  206  and is prepared to receive just a single sample. The housing  120  is coupled by an adhesive  214 , which may be a polycarbonate glue, or other acceptable adhesive. In a preferred embodiment, a substrate  104  having a plurality of recesses  204  formed therein is glued by an adhesive to a large housing  120  which has the appropriate chambers which will be aligned with the recesses  204  in order to form a cartridge that can receive multiple samples at the same time. 
     In one embodiment, the housing  120  is a polycarbonate member which is been previously formed in a different process using techniques well known in the art. An adhesive which is acceptable for gluing a silicon substrate  104  to polycarbonate can be used. In one embodiment, the adhesive has a high adherence to the buffer layer  107  and therefore the housing  120  is attached directly to the buffer layer  107 . In alternative embodiments, additional preparation layers may be provided in order to properly affix the housing  120  to the substrate  104  in order to create the cartridge as shown in  FIG.  23   . 
     The final chamber  190  is shown in  FIG.  22    prior to receiving the sealing material  200 . In one embodiment, the layer  107  has been etched away from the substrate  104  in order to leave the silicon as the exposed surface to receive, at a later time, the sample fluid  122  as shown and described with respect to  FIGS.  6 ,  18  and  19   . In one embodiment, the layer  107  is removed just prior to the housing  120  being affixed to the substrate  104 . In other embodiments, the layer  107  is not etched away and remains present, while in another embodiment, a portion of layer  107  is etched away to leave the silicon with the native oxide on the exposed surface. The uppermost surface of substrate  104  can also be prepared as shown and described with respect to  FIGS.  1  and  2    in order to receive the microdots  112  as shown and described with respect to  FIG.  3   . This final preparation and providing of the microdots  112  can be done prior to the substrate  104  being attached to the housing  120 , which is preferred, but it can be done after it has been attached if desired. 
     As previously stated, the sealing layer  200  has a recess in the sealing layer overlying the substrate. This recess is located in a central region of the sealing layer. The sealing layer has a first height in the central region and a second, greater height in a peripheral region of the sealing layer. A recess  204  is positioned in the upper surface of the substrate, the recess being positioned below the recess in the sealing layer. The recess in the sealing layer is overlying at least a portion of the recess in the substrate. The ridge  206  in the upper surface of the substrate is positioned adjacent to the recess in the upper surface of the substrate and the recess in the sealing layer is overlying the ridge  206  in the substrate. The upper surface of the ridge  206  is in the same plane as an upper surface of the substrate at locations outside of the recess in the substrate, which includes locations positioned below the region of the sealing layer having the second, greater height. The microdots  112  are placed onto the substrate  104  at an appropriate time in the fabrication process. According to one embodiment, they are placed on the substrate  104  after the steps of  FIG.  22    in which the housing  120  has been fully formed and the cartridge is completed. According to another embodiment, the microdots  112  are placed on the substrate  104  just prior to the step as shown in  FIG.  21 D , before the attachment of the housing  120 . 
     In yet another alternative embodiment, after the cartridge is fully formed as shown in  FIG.  22   , an additional substrate is placed into the recess tool and on the ridge  206  having the microdots  112  formed thereon. In this alternative embodiment, the microdots  112  are formed on a substrate as shown in  FIGS.  1 - 5 B  following the process steps as described therein. After the cartridge having the housing  120  is prepared, then the separately prepared substrate can be placed in each of the individual chambers  190 . Accordingly, there are a number of acceptable techniques by which the microdots  112  may be positioned in each chamber  190 . Additional sequence steps and fabrication techniques may also be used in order to form the microdots  120  within each chamber  190 . 
       FIG.  23    shows a completed cartridge  127  having the housing  120  and the substrate  104  combined with each other. As can be seen, each chamber  190  has a recess tool therein with the ridge  206  and a film  212  which provides a reception location for the sealing layer  200  which can receive the sample fluid  122 . Accordingly, after the cartridge  127  is prepared, a sealing layer, such as wax, oil, or other appropriate sealing material, will be placed therein. In one embodiment, the sealing layer is wax, which has a density that will be less than the density of the sample fluid  122  to be received therein. The sealing layer  200  is appropriately shaped as shown in  FIG.  18    and remains with the indent therein in preparation to receive the sample fluid  122 . In the embodiment in which the sealing layer  200  is wax, the cartridge  127  can be fully completed with the recess in the sealing layer  200  prepared to receive a sample fluid  122  at some time in the future. Accordingly, the structure shown in  FIG.  18    is completed as a final product, except that the sample fluid  122  has not yet been placed into each of the chambers  190 . A large number of cartridges  127  can be prepared and shipped to various locations for future in-field use. The cartridges  127  are in a stable condition, with each of the microdots  112  properly sealed and protected. Namely, as can be seen viewing  FIG.  18   , the sealing layer  200  will completely cover and fully protect and isolate each of the microdots  112 . This will ensure that the microdots  112  cannot receive any contamination or material. Since the sealing layer  200  fully encases the microdots  112 , they can be assured of remaining clean and isolated until the PCR process is started. Thus, the cartridges  127  can be shipped to any number of different customers at different locations and be assured of remaining in a contamination-free condition waiting for use when a sample fluid  122  will be introduced therein at a later time. 
       FIGS.  24 - 27    illustrate the introduction of the sample fluid  122  into physical contact with the microdots  112  and the movement of the sealing layer  200  to be on top of, and fully encase the sample fluid  122 . As seen in  FIG.  22   , the heating coil  202  is heated up in order to apply a high temperature to the substrate  104 . In the graph shown in  FIGS.  24 - 27   , the horizontal axis is time and the vertical axis is temperature, in degrees centigrade. In the embodiment shown, the sample fluid is water-based and therefore the acceptable temperature will generally be in the range of 60-95° C. The selected temperature will be somewhat below the boiling temperature of the sample fluid. If the sample fluid is water-based, then temperature somewhat below 100° C. is preferred. If the sample fluid  122  is alcohol-based, then a temperature somewhat below 75° C. is preferred, since most alcohols boil in the range of 75-80° C. In addition, the temperature range is then selected to be within the parameters for which the gene is amplified. Most genetic material is amplified by heating cycles that vary between 50° C. and 100° C., and in some situations, heating cycles that vary between 60° C. and 95° C. Accordingly, the maximum temperature to which the sample fluid  122  will be heated will be based upon boiling temperature of the sample fluid, with the temperature selected to be sufficiently below the boiling temperature of the sample fluid that there is no danger of the sample boiling and also lower than the temperature at which the gene material will be degraded or destroyed. The boundary of the lower temperature will be something sufficiently low to create a PCR cycle for amplification of the gene material, however, not so low as to harden the sample fluid  122 . In addition, in most embodiments, it will be desired to keep the sealing fluid  220  in the liquid range. Accordingly, for a wax that has a melting temperature range of approximate 55° C., a lower temperature of approximately 60° C. is selected. 
     Viewing  FIG.  24   , the heating of the heating coil  202  is shown by line  222  over time of approximately 2000 seconds. At time zero, the heating of the coil advances from room temperature, generally in the range of 22-27° C., and is rapidly advanced in less than a few seconds to approximately 95° C. The temperature stays elevated at approximately 95° C. for a plateau time, shown by the plateau  224 . The length of the time for the plateau  224  is sufficiently long to ensure that the sealing material  200  has fully melted, that the sample fluid  122  has transitioned from on top of the sealing fluid  200  as shown in  FIG.  18    to be in contact with the microdots  112  on top of the substrate  104  as shown in  FIG.  19   . In addition to the length of the time, the plateau  224  is sufficiently long to ensure good movement of the fluid  122  in order to provide for adequate mixing of the sample gene for presentation to the microdots  112 . After a selected plateau time  222 , preferably in the range of 5-50 seconds, then the PCR cycle begins by having repeated cycles  226  to a low temperature and back up to a high temperature. The repeating of the heating cycles  226  is carried out until the PCR process has been sufficiently completed to amplify any target material within the sample fluid  122  which may be present. 
       FIG.  25    illustrates one example  230  of a heating pattern having a number of PCR heating cycles  226  according to one embodiment. According to this embodiment, the sample temperature is shown by the solid line and the temperature of the heating coil  202  is shown by the dashed line. At time 0, the heating coil  202  starts to increase in temperature from ambient temperature which will generally be in the range of 20-25° C. and begins to heat up the substrate  104 , which in turn heats up the sample fluid  122  as represented by the solid line. Silicon is highly thermally conductive and therefore when the substrate  104  is comprised of silicon, the temperature of sample fluid  122  will be approximately equal to the temperature of the heating coil  202 . As shown in the graph  230 , the temperature of the coil  202  will rise sharply as shown by the vertical line  222  until it reaches a plateau  224 . For plateau time  224  of approximately 150 seconds the temperature of the heating coil  202  is approximately equal to the temperature of the sample fluid  122 . After a selected time period, for example, 150-200 seconds, the first PCR cycle begins by a rapid drop in the temperature of the heating coil  202  is shown by line  228 . As the temperature of the heating coil  202  reduces, shortly thereafter the temperature of the sample  122  will also decrease, as shown by solid line  229 . Because the sample fluid  122  has more mass and a higher specific heat, it will not cool quite so quickly as the coil  202  and therefore will lag somewhat in its temperature decline. After a brief time period, for example, in the range of 8-12 seconds, the temperature of the sample fluid  229  will be approximately equal to the temperature of the heating coil  228 . Then, the next stage of the PCR heating cycle begins with the coil  202  rapidly increasing towards the maximum temperature of about 95° C. The temperature of the heating coil  202  will be approximately equal to the temperature of the sample fluid  122 , as shown in this embodiment, reaching the first peak as shown in the graph of  230 . The cycle begins again, with the temperature of the heating coil  202  decreasing rapidly as shown by the dashed line  228  and then the sample fluid following to also decrease as shown by the solid line  229 . The cycle  226  is repeated a number of times in order to provide the PCR amplification as is previously described herein. 
       FIG.  26    is an enlarged scale of a graph  240  which also shows thereon the melting temperature of the sealing material  200 , in this case, wax. In addition, in the embodiment of  FIG.  26   , the temperature relationship between the heater  202  and the sample fluid  122  is slightly different. In the heating cycle  240 , a wax that has a melting temperature of approximately 56° C. is used as shown by line  239 . As the heater  202  heats up as shown by dashed line  228 , the wax melts when the heater reaches slightly over 56° C. In addition, the sample  128  is slightly cooler than the heater  202  itself as can be seen by the separation between the line  228  and line  229 , the sample temperature line. After the temperature of the heater  202  has reached a plateau and is held there for a selected period of time to ensure that the sample has reached a constant, stable temperature, than the heating and cooling cycles of the PCR amplification process begin. In this embodiment, the temperature of the sample closely matches the temperature of the heating coil  202  as the heating coil cools, and decreases in temperature towards approximately 60° C. Then, after a selected period time such as 10-20 seconds, the temperature of the heating coil begins to rise as shown by the dotted line graph  228  and the temperature of the sample fluid  122  follows closely with a rise in its temperature. This pattern continues for a number of cycles, in order to carry out the PCR amplification process. 
       FIG.  27    is an enlarged view of the section shown in  FIG.  26   . This view more clearly shows in detail the transition of the materials at different temperatures as the heater  202  increases in temperature. As can be seen, the heater  202  heats up rapidly following line  228  advancing from ambient temperature, for example, about 22° C. and rapidly climbing over about a 15-22° C. towards its maximum temperature of approximately 93° C. The wax remains solid while the heating coil  228  is below 55° C. but a few seconds after the coil  202  passes through 55° C., the sample fluid  228 , along with the wax  200  also reach the melting temperature. The wax is shown by line  224  and the wax transitions to liquid wax at a time shown by vertical line  232 , which is approximately 7.5 seconds after the heating cycle begins. There is therefore some slight delay from when the heater  202  reaches the melting temperature of wax until the substrate  104 , the wax  200  and the sample  122  also reach the melting temperature of wax. This is based on the thermal properties of the substrate  104 , the wax, the thickness of the wax, the volume of the sample  122 , and other factors that affect the thermal properties of the wax  200 . The wax remains in the liquid form as long as its temperature is above the melting temperature of wax shown which is generally in the range of 55° to 57° C. This occurs generally within the first 10 seconds of the heating cycle shown in  FIG.  27    and the wax remains in the liquid form during the entire PCR heating cycles. 
     The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. 
     These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.