Patent Document

RELATED APPLICATION 
       [0001]    This application is related to, and claims the priority benefit of, U.S. Provisional Patent Application Ser. No. 61/482,971, filed May 5, 2011, the contents of which are hereby incorporated into the present disclosure in their entirety. 
     
    
     FEDERALLY SPONSORED RESEARCH 
       [0002]    This invention was made with government support under 59-1935-8-850 awarded by USDA. The government has certain rights in the invention. 
     
    
     BACKGROUND 
       [0003]    Reliable water monitoring and control systems are becoming increasingly important to ensure reliable water quality for the public and also for monitoring and control of industrial run off. The development of a modular water monitoring and control system would provide a standard operational platform that can be easily modified and employed across numerous applications. 
         [0004]    In addition, detecting deoxyribonucleic acid (DNA) biomarkers is an important feature in many areas dealing with biomolecules. In particular, detection schemes directed to both qualitative (i.e., detecting the presence of one or more DNA biomarkers), and quantitative (i.e., where an absolute or relative amount of DNA is present within a sample) is important. Furthermore, detecting one or more changes or differences in DNA sequences, such as single nucleotide polymorphisms is also desirable. 
         [0005]    Deoxyribonucleic acid (DNA) microarray detection is a widely used molecular biological technique. Equivalent in size to a standard laboratory slide, these microarrays include “printed” chains of the constituent molecules of DNA which include Adenine, Thymine, Cytosine, and Guanine. The ordering of molecules in these chains, or probes, determines what target strains of DNA they will bind to. One common mode of operation involves a fluorophore and a quencher molecule being attached at either end of the probes. Tension in the probe causes it to form into a “hairpin” loop, positioning the fluorophore and quencher molecules beside each other. Excitation of the microarray by a laser will cause no fluorescence when no target DNA is present due to fluorescence resonance energy transfer (FRET) between the fluorophore and quencher, as known to a person of ordinary skill in the art. However, when a target strand of DNA is bound to the probe, the hairpin loop is forced open separating the fluorophore and quencher molecules. When excited by laser light, the fluorescence occurs due to the lack of FRET caused by the separation of the fluorophore and quencher molecules. While highly successful, this method has some drawbacks, especially when trying to fully automate such a process. Light will be emitted only when an undamaged fluorophore is present. Should no light be emitted under excitation, lack of emitted light may mean either the target DNA is not present and light is not being emitted due to the action of the quencher molecule, or target DNA is present but no light is being emitted due to damage occurring to the fluorophore. There is inherent risk in this method of obtaining false negatives, limiting its application in fully automated systems. 
         [0006]    Therefore, a system for detecting biomolecular structures such as DNA biomarkers in an automated or semiautomated fashion is needed. 
       SUMMARY 
       [0007]    The present disclosure includes disclosure of systems and methods for detecting deoxyribonucleic acid (DNA) biomarkers. In at least one embodiment, such a system is configured to monitor and control standard parameters (temperature, pH, free chlorine, redox potential, TDS, turbidity), via an array of sensors. In at least one embodiment, such a system may be configured to provide online data logging and remote control. In at least one embodiment, such a system may be configured to perform automated microbiological testing using a DNA hybridization based optical detection sensor, wherein the sensor is configured to provide automated sample collection, primer and buffer addition, thermocycling, and fluorescence detection via laser excitation and a linear CCD. 
         [0008]    The present disclosure includes disclosure of methods for detecting DNA biomarkers. In at least one embodiment, such a method comprises the steps of loading a volume of amplification reagents into an automated detection device; entering at least one control parameter into the automated detection device; loading a sample into the detection device; mixing the sample with the amplification reagents to create a reaction volume; conducting at least one thermal cycle on the reaction volume; hybridizing the reaction volume to the at least one dual-fluorescent oligonucleotide probe; detecting a fluorescence emission, wherein the at least one dual-fluorescent oligonucleotide probe hybridized to the reaction volume is excited by a laser and emits a fluorescence detected by an emission detector; logging data from the fluorescence emission; analyzing the data from the fluorescence emission; automatically cleaning the automated detection device; and conducting a verification test, wherein at least one dual-fluorescent oligonucleotide probe is excited by a laser and emits a fluorescence detected by an emission detector. In an aspect of at least one embodiment of the present disclosure, the foregoing steps are repeated at least one time. In at least one embodiment of the present disclosure, a method for detecting DNA biomarkers comprises a step of conducting a second verification test prior to loading a sample. In at least one embodiment of the present disclosure, a method for detecting DNA biomarkers utilizes a reaction volume of at least 100 μl. In at least one embodiment of the present disclosure, a method for detecting DNA biomarkers utilizes red or green fluorescence. In at least one embodiment of the present disclosure, in a method for detecting DNA biomarkers a step of automatically cleaning an automated detection device occurs concurrently with a step of conducting at least one thermal cycle on a reaction volume and a step of detecting a fluorescence emission. In at least one embodiment of the present disclosure, a method for detecting DNA biomarkers comprises a step of holding a reaction volume at a detection temperature to hybridize the reaction volume to at least one dual-fluorescent oligonucleotide probe. In at least one embodiment of the present disclosure, a method for detecting DNA biomarkers comprises a step of automatically cleaning the automated detection device comprising three discrete cleaning cycles. 
         [0009]    The present disclosure includes disclosure of an automated DNA detection device. In at least one embodiment, such an automated DNA detection device comprises top clamp having an optical aperture; a microarray slide connected below the top clamp, the microarray slide comprising at least one dual-labeled fluorescent oligonucleotide probe, wherein the optical aperture of the top clamp allows for a fluorescence emission of at least one dual-labeled fluorescent oligonucleotide probe and emission detection by an emission detector; a reaction chamber connected to the microarray slide, the reaction chamber comprising a reaction volume; a thermoelectric module connected to the reaction chamber, wherein the thermoelectric module is capable of heating or cooling the reaction volume; a water block connected to the thermoelectric module, wherein the water block and the thermoelectric module operate to perform at least one thermal cycle; a fluidic system in communication with the water block, thermoelectric module, reaction chamber, laser, and emission detector, wherein the fluidic system comprises at least one reservoir, waste chamber, cooling system, valve, pump, and sensor operably connected to one another to control the flow of at least one fluid through the fluidic system, wherein at least one sensor can detect the flow of at least one fluid within the fluidic system and provide at least one feedback communication to the emission detector; and a bottom clamp operably connected to the top clamp to secure the microarray slide, reaction chamber, thermoelectric module, water block, and fluidic system to one another. In at least one embodiment of the present disclosure, an automated DNA detection device comprises a fluidic system comprising three reservoirs. In at least one embodiment of the present disclosure, an automated DNA detection device comprises at least one feedback communication comprising at least one of the following: a quality control communication, a self-cleaning communication, and/or a probe verification communication. In at least one embodiment of the present disclosure, an automated DNA detection device is reusable. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0010]      FIG. 1  shows an optical detection scheme according to at least one embodiment of the present disclosure utilizing detection probes and a linear charged coupled device (CCD) array. 
           [0011]      FIG. 2  shows a graph which depicts conversion of raw barcode data via image analysis to graphical representation of the barcode data. 
           [0012]      FIG. 3  shows a schematic of at least one embodiment of the system according to the present disclosure that utilizes a specialized microarray that includes dual-labeled fluorescent oligonucleotide probes, which fluoresces red in the absence of sequence-specific binding using fluorescence resonance energy transfer (FRET), and fluoresces green in the presence of sequence-specific binding (due to the disruption of FRET). 
           [0013]      FIG. 4  depicts a schematic of a dual-labeled fluorescent oligonucleotide probe as described in  FIG. 3 . 
           [0014]      FIG. 5  shows a schematic of at least one embodiment according to the present disclosure of the assembled detection unit used in the system. 
           [0015]      FIG. 6  shows a top view of the detection unit of  FIG. 5 . 
           [0016]      FIG. 7  shows an exploded view of the detection unit of  FIGS. 5 and 6  with various internal components broken away. 
           [0017]      FIG. 8  shows passage of light to the CCD under excitation in the detection unit of  FIGS. 5-7 . 
           [0018]      FIG. 9  depicts a graph of temperature vs. time representing a thermal response of a thermal chamber of the system during detection of four genes common to  E. coli  O157:H7. 
           [0019]      FIG. 10  depicts polymerase chain reaction (PCR) products for one of the genes referred to in  FIG. 9  (namely hylc). 
           [0020]      FIG. 11  shows a schematic of at least one embodiment according to the present disclosure of a fluidic system that can be used with the detection unit of  FIGS. 5-7  and utilizes thermocouples (and other sensor systems) to control the temperature of the detection chamber, as well as fluidics controls, valves, pumps, and sensors to control the buffers (and other fluids) in the system. 
           [0021]      FIG. 12  shows the schematic of the fluidic system of  FIG. 11  depicting the flow of fluids during the mixing cycle. 
           [0022]      FIG. 13  shows the schematic of the fluidic system of  FIG. 11  depicting the flow of fluids during the first feed line cleaning cycle. 
           [0023]      FIG. 14  shows the schematic of the fluidic system of  FIG. 11  depicting the flow of fluids during the second chamber cleaning cycle. 
       
    
    
     DETAILED DESCRIPTION 
       [0024]    For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and described in the following written specification. It is understood that no limitation to the scope of the invention is thereby intended. It is further understood that the present invention includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles of the invention as would normally occur to one of ordinary skill in the art to which this invention pertains. 
         [0025]    A novel system has been developed to enable the detection of deoxyribonucleic acid (DNA) biomarkers in an automated, or a semi-automated process, and operated by non-technical (i.e. non-molecular biologists) personnel. The system can be used to detect DNA biomarker(s). It should be appreciated that a system according to the present disclosure can be used for both (a) qualitative detection, meaning that the system simply detects the presence of one or more DNA biomarkers, and (b) quantitative detection, where the absolute or relative amount of DNA is present within a sample. Furthermore, a detection probe system according to the present disclosure can be used to detect one or more changes or differences in DNA sequence, such as single nucleotide polymorphisms (SNPs). 
         [0026]    A system according to the present disclosure provides the capability to screen samples within the food processing industry to allow for a rapid, qualitative screening of consumable materials for evidence of pathogenic microorganisms. The purpose of such a system is to screen for DNA sequences that are unique to specific pathogens and provide a warning to a user if evidence of a pathogen is present, based on the detection of these DNA sequences. A schematic of an exemplary embodiment of the system designed for optical emission and detection of DNA sequences is depicted in  FIG. 1 . The system in  FIG. 1  includes a detection device which is designed to reduce the costs associated with 2-dimensional imaging. The system advantageously utilizes a linear charge coupled device (CCD) array  105  that is, in at least one embodiment, perpendicular to the lines  137  created by the placement of the detection device, similar to a barcode reader. The “raw barcode” data collected from the system can be converted via image analysis into a graphical representation, an example of which is demonstrated by the graph in  FIG. 2 . 
         [0027]    Noteworthy functions of a DNA screening system according to the present disclosure are to (1) amplify the gene region of interest using common thermocycling methods (e.g. polymerase chain reaction (PCR)), and (2) detect a color-change in the fluorescence of the capture probe, all within a single reaction chamber. The system uses a fluorescence-based color change, rather than known methods of detecting presence/absence of fluorescence commonly used in DNA microarray technology, to detect the DNA biomarkers, which significantly reduces the risk of false negative detection in samples. These functions are achieved by generating a specialized microarray that includes dual-labeled fluorescent oligonucleotide probes such as that shown in  FIG. 4 . As shown in  FIGS. 3 and 4 , the microarray fluoresces red  422  in the absence of sequence-specific binding, using fluorescence resonance energy transfer (FRET), and fluoresces green  421  in the presence of sequence-specific binding (due to the disruption of FRET). Furthermore, at least one embodiment of the present disclosure is designed to utilize quality controls inherent to the detection probe system that can be verified before, during, and after a sample is analyzed, which provides the system with more rigorous detection parameters than that common to DNA microarray technology, and can be employed to check the integrity of the system after a sample is analyzed and determine if the system can be used to analyze another sample (without changing the probe system). 
         [0028]    A detection device according to at least one embodiment of the present disclosure is shown in  FIGS. 5-7 .  FIG. 5  shows a perspective view and  FIG. 6  shows a top view of a detection device embodiment.  FIG. 7  shows an exploded view of the detection device embodiment depicted in  FIGS. 5 and 6 . Such a detection device comprises a top clamp  510  and a bottom clamp  514  that operably holds together the components of the detection device. The top clamp  510  contains an optical aperture  511 , which provides a window to allow for laser-based excitation  104  and CCD-based detection  105 ,  805  of the oligonucleotide probes on a microarray  108 ,  708 . As shown in  FIG. 7 , a DNA microarray slide  708  (that contains the dual-labeled fluoro-probes as shown in  FIG. 4 ) is sandwiched between a top clamp cushion  715  positioned under the top clamp  510  and a sealing gasket  718  positioned above a reaction chamber  513 . The reaction chamber  513  is where test samples, primers, and/or buffers (e.g., 100 μl in volume) can be introduced. The reaction chamber  513  is configured to provide sufficient volume to perform multiplex PCR (amplifying more than one target DNA sequence at a time). 
         [0029]    As indicated by  FIG. 1 , such a detection device contains inlet  101  and outlet  102  ports on the reaction chamber  513 , which are sealed using valves  1223 ,  1225 ,  1229  (2 and 3 way Pinch Valve parts available from Biochem Fluidics, such as part numbers 075P2NC12-02S, 075P3MP12-02S, 100PD3MP12-02S). 
         [0030]    The reaction chamber  513  may be capable of quickly heating or cooling for PCR-based amplification of the pathogenic gene templates, and can achieve temperatures that open or close the FRET-probe(s) as shown in  FIG. 3 . The heating and cooling functions of the detection device are achieved using thermoelectric (Peltier) modules  109 ,  509 , which are located between a water block  520  and the reaction chamber  513 , as shown in  FIG. 5 . The thermoelectric Peltier modules  509  and water block  520  (Peltier Heaters (40 mm×40 mm) and (20 mm×20 mm) and Water Block parts available at Custom Thermoelectric, such as part numbers 12711-5L31-05C, 03111-9L31-04CG, and WBA-1.62-0.55-CU-01) are used to perform thermocycling for cell lysis, PCR, and detection. The thermoelectric module  509  is driven via a solidstate relay connected to a DO port on a C Series 9274 module. The temperature of the system is controlled by integrated thermocouples (and other sensor systems, including biosensors)  1131  at the microscope slide surface, which provides precise temperature control of the reaction chamber  513  for real-time temperature readings. The thermocouples  1131  provide a feedback loop by providing heating/cooling signals to the Peltier modules  109 ,  509  and reaction chamber temperatures. This provides the basic I/O for the development of a control software system and supporting circuit board control system. Temperature control is implemented in LabView using a gain scheduling PI controller. 
         [0031]      FIG. 11  shows a schematic of an exemplary fluidic system according to at least one embodiment of the present disclosure. As depicted in  FIG. 11 , the system uses fluidics controls, valves  1123 ,  1125 ,  1129 , pumps  1130  (Dosing pump parts available from Biochem Fluidics, such as part number 120SP1220-5TV), and sensors  1131  (Tubing fluid sensors available from Newark Components, such as part number 47P7966) to precisely control the buffers (and other fluids) in the system. The fluidic system also contains a set of reservoirs  1122 A-C each connected to a valve  1123 A-C that can be turned “on” or “off”. The valves  1123 A-C are connected to the P1 position of a valve  1125 A. When in the P2 position, the valve  1125 A is connected to an air line  1124 A. The valve  1125 A is connected to another valve  1125 B. The valve  1125 B is connected to the reagent reservoir  1134  via the P1 position and another valve  1125 C via the P2 position. Valve  1125 C is connected to the reagent dosing point  1132  through the P2 position and the sample dosing point  1133  through the P1 position. The reagent reservoir  1134  is connected to a sensor  1131 D. The sample dosing point  1133  is connected to another sensor  1131 C. Both sensors  1131 C and D are connected to Sensor  1131 B, which is connected to a valve  1125 D. When valve  1125 D is in the P2 position, it is connected to the reaction chamber  1113 . When valve  1125 D is in the P1 position, it is connected to the P1 position of valve  1125 E. The P2 position of valve  1125 E is connected to sensor  1131 A, which is connected to the reaction chamber  1113 . Valve  1125 E is also connected to a pump  1130 B and a valve  1123 D. Valve  1123 D is connected to the waste chamber  1136 . Pump  1130 B is connected to valve  1125 F. Valve  1125 F is connected via the P2 position to valve  1129 A and to an air line  1124 B via the P1 position. Pump  1130 A is connected to valve  1125 F, and valve  1125 F is connected via the P2 position to valve  1129 B and via the P1 position to an air line  1124 C. 
         [0032]    Pump  1130 A is used to push fluid through the system. Pump  1130 B is used to pull fluid through the system. Valve  1125 F is a dual tubing 3 way pinch valve. Only air passes through the pumps. All liquids are deposited to waste. With valve  1125 F at P1, pump  1130 A can use an unrestricted air line to push fluid through the system. With valve  1125 F at P2, pump  1130 B uses a restricted air line to push fluid through the system and it operates at a lower flow rate. This lower flow rate can be set using the needle valves  1129 A and B (Needle valve parts available from Pneuaire Components, such as part number F-28 22-40-B80-K). Pump  1130 B operates in a similar manner but with the unrestricted and restricted air lines  1124  connected to the outlet  102  as opposed to the inlet  101 , such as shown in  FIG. 1 . 
         [0033]    The fluidic system control software system can be programmed in several states. First, the fluid system can operate for reagent reservoir loading through the activation of the “Reagent Loading” state. In this state, valve  1125 D and valve  1125 E are set to P1 and Valve  1123 D is set to on. Next, the system can be sent into “Begin” state in which input and control parameters (PCR temperatures, etc.) are entered into the system. The system then begins to verify the probes by setting Valve  1123 A to on, valve  1123 D to Off, and valves  1125 A, B, D, and E are set to P1. Pump  1130 B then switches “on” until a requisite amount of buffer/rinse is drawn from reservoir  1122 A. Then, valve  1123 A is set to off, valve  1123 D is set to on, and pump  1130 A is switched on. When fluid triggers sensor  1131 B, valves  1125 D and E switch to P2. When fluid triggers sensor  1131 A, pump  1130 A is switched “off” and valves  1125 D and E switch to P1. After this step, the laser  104  and CCD  105  are activated and the fluorescence emission spectrum is logged. 
         [0034]    The laser  104  and CCD  105 , such as shown in  FIG. 1 , are then deactivated, which activates the heater  109 ,  509 , such as shown in  FIGS. 1 and 5 . Once the detection temperature is reacted, the laser  104  and CCD  105  are reactivated and the fluorescence emission spectrum is logged again. The laser  104  and CCD  105  are again deactivated along with the heater  109 ,  509 . At this time, valves  1125 D and E switch to P2 and pump  1130 A is switched “on” until liquid deposited in waste chamber  1136 . Valves  1125 D and E then switch to P1. At this point, the device can proceed to the “Sample Loading” state unless the system has found that the probes are damaged. The user is then prompted to load a sample for the “Sample Loading” state, and the system will notify the user once loading is complete. Valves  1125 A and B are then switched to P2 and valve  1125 C is switched to P1. Pump  1130 B is switched “on” until sensor  1131 C is triggered. The reagents are then metered in the device during the “Reagent Metering” state by switching valve  1125 C to P2 and switching “on” pump  1130 B until sensor  1131 D is triggered. 
         [0035]    As depicted in  FIG. 12 , after the “Reagent Metering” state, the device enters the ‘Mixing’ state. Mixing occurs in the device through switching valve  1125 A to P1 and valve  1125 F to P2. When pump  1130 A is switch on, valve  1125 C switches between P1 and P2. As shown with arrows  1137 A, fluid then activates sensor  1131 C, and valves  1125 D and E are switched to P2. Fluid then activates sensor  1131 A, pump  1130  A switched off, and valves  1125 D and E are switched to P1. 
         [0036]    The PCR reaction then takes place within the device through the activation of the heater  109 ,  509  and temperature cycling during the “PCR Cycling” state. After the specified cycle number has been reached, the reaction chamber  513  is set to the detection temperature. This activates the laser  104  and CCD  105  and the fluorescence emission spectrum is logged. The laser  104  and CCD  105  are then deactivated and the reaction chamber  513  can be returned to temperature cycling. The above is repeated until end of PCR cycling. 
         [0037]    As depicted in  FIG. 13 , during the “PCR Cycling” state, the feed line can be cleaned during the “Feed Line Cleaning” state by setting valves  1125 A, C, D, and E to P1, and valve  1125 B is set to P2. Valve  1123 A is then set to “on” and valve  1123 D is set to off. This activates pump  1130 B until requisite amount of detergent is drawn from reservoir  1122 C. Once this occurs, valve  1123 A is set to off, valve  1123 D is set to on, and pump  1130 A is switched on. As shown by arrows  1137 B, fluid is then pushed through system to a waste chamber  1136 . The above is repeated for detergent in reservoir  1122 B and buffer/rinse in reservoir  1122 A. 
         [0038]    During PCR cycling after detection has begun, real time data logging and analysis can be performed. The device presents the user with a graph of the logged fluorescence emission spectra. The device then performs a numerical analysis on the fluorescence emission spectra providing the user with numerical value (+/−) for the increase or decrease in fluorescence intensity for each target DNA sequence in both green and red. The device writes the chamber temperature and fluorescence spectra to spread sheet files. The user is alerted automatically of any positive targets. A control action can then be performed automatically by the device or manually by the user. 
         [0039]    Upon completion of PCR cycling, the device enters “Reaction Mixture Removal” state. During this state, valves  1125 D and E are set to P2 and valve  1123 D is set to on. Pump  1130 A then switches “on” until the reagent is sent to waste chamber  1136  or collected by the user via the tapping point. After this, the device enters “Chamber Cleaning” state as shown in  FIG. 14  where the device can self-clean the chamber via the steps for “Feed Line Cleaning” followed by switching valves  1125 D and E to P2, shown by arrows  1137 C. After this step, the device returns to “Probe Verification” state unless the user overrides the device and sends it to “End State” or the device automatically moves to “End State” due to lack of reagent. 
         [0040]    In operation, a sample is collected and prepared and is to be suspended in a liquid volume of 25 μl. The sample is loaded into the fluidics system, as shown in  FIGS. 11-14 , of the detection device using a standard pipette. The sample dosing point  1132 , such as shown in  FIG. 11 , consists of a manually removable cap allowing direct access to a tubing line. The cap is reattached once loading is complete. The 25 μl sample volume is mixed with a 75 μl volume containing the PCR reagents (primers, buffers, polymerase etc.) to give a total reaction volume of  1004  As shown in  FIG. 12 , mixing is achieved by alternating flow from the sample and reagent lines using a valve system  1123 ,  1125 ,  1129  and pumps  1130 . The 75 μl volume is drawn from a reservoir  1122  held at a temperature of 4 degrees Celsius to prevent degradation. The temperature of the reaction chamber  513 , as shown in  FIG. 5 , is controlled using a thermocouple  1131  while varying the voltage (and polarity) applied to the thermoelectric heater  509 , as shown in  FIG. 5 . A water block  520  attached to one side of the thermoelectric heater  509 , such as shown in  FIG. 5 , allows for a more stable operation of the heater. The water block  520  is connected to a radiator cooling system, shown in  FIGS. 11-14 , consisting of the radiator  1126 , the cooling system reservoir  1127 , and the cooling system pump  1128 . 
         [0041]    The 100 μl reaction volume is then deposited into the reaction chamber  513 ,  1113 . The inlet  101  and outlet  102  ports, such as shown in  FIG. 1 , of the chamber are then sealed by valves  1123 ,  1125 ,  1129 . The reaction volume is heated to 95 degrees Celsius to ensure cell lysis (where whole cells are introduced in the sample volume). Temperature is monitored by a thermocouple  1131  embedded in the reaction chamber  513 ,  1113  wall. 
         [0042]    Following this step a standard PCR thermocycling takes place. The number of cycles, denaturing, annealing and extension temperatures, final elongation and holding temperatures (if required) can be set by the user. The user can also input the number of cycles required before detection begins and detection temperature(s) (if required). When a pre-determined number of cycles have occurred, the sample is held at a detection temperature. As shown in  FIG. 1 , the oligonucleotide probes on the microarray are excited using a five milliwatt 532 nm laser  104  (532 nm DPSS Green Laser Module parts available from Lasermate, such as part number GMP-532-20F3-CP) spread into a line  137  by a plano concave lenses  138  (parts available from Thorlabs, such as part number LB1450-A). As shown in  FIGS. 1 and 8 , fluorescence is detected by focusing a double image of the microarray  108 ,  708  onto a 2048 pixel linear CCD  105 ,  805  (2048 Element Linear CCD array parts available from Ames Photonics Inc, such as part number Larry 2048) via two spherical lenses  106 ,  806  (parts available from Thorlabs, such as part number LB1450-A), with one half of the image filtered in red  821  and one half in green  822  (568 nm and 671 nm Band Pass Filters, available from Edmund Optics, such as part numbers NT43-127 and NT43-139).  FIG. 8  shows passage of light to the CCD  805  under excitation in the detection device of  FIGS. 5-7 . The emission intensity spectrum taken after each detection step is stored on the control computer. Analysis of the change in the ratio of green to red fluorescence is used to indicate whether a test sample is positive or negative for the target DNA (See  FIG. 2 ). 
         [0043]    After completion of the PCR and detection cycles, the reaction volume is removed from the reaction chamber  513 ,  1113 . It can be sent directly to a waste chamber  1136  or collected by the user via a tapping point as shown in  FIGS. 11-14 . The cleaning cycle begins while the PCR and detection steps are running. The sample loading line and mixing line are cleaned using a three step cleaning cycle. As shown in  FIG. 13 , the detergents and rinsing fluid are drawn from reservoirs  1122 . The spent fluid is deposited in the waste chamber  1136  via a line running parallel to the reaction chamber  513 ,  1113 , see arrows  1137 B. This reduces the amount of contamination entering the chamber between test runs. Upon removal of the reaction volume from the reaction chamber  513 ,  1113 , the same three step cleaning cycle is conducted in the chamber itself and deposited to the waste chamber  1136  (See  FIG. 14 , arrows  1137 C). The operation of the probes is then verified as described above. The operation of the oligonucleotide probes is checked after the cleaning cycle to ensure they are functioning correctly. The operator is alerted in case of a malfunction and an appropriate action is taken. The above described steps are then repeated. 
       EXAMPLE 
       [0044]    Four genes common to  E. coli  O157:H7 were selected for detection by the system. These genes are eaeA, hlyc, rfbE and stx1. To test the functionality of the reaction chamber  513 ,  1113  in amplifying these DNA sequences, 100 μl samples each containing target DNA and the relevant primers and buffers were prepared. Each sample was loaded into the reaction chamber  513 ,  1113  sequentially and underwent a 30 cycle PCR with temperature stages at 95° C., 50° C., 72° C. respectively. There was a 5 minute hold at 95° C. before cycling and a 10 minute hold at 72° C. after cycling after which the sample was cooled to 4° C. and removed from the reaction chamber  513 ,  1113 . The thermal response of the reaction chamber  513 ,  1113  is shown in  FIG. 9 . The overall reaction time was less than 80 minutes with maximum heating and cooling rates of 2.3° C./s and 3.1° C./s, respectively. The faster cooling rate was achieved by changing the polarity of the voltage supplied to the thermoelectric heater  509 . Gel electrophoresis was performed on each sample after PCR, an example of which is shown in  FIG. 10 , using a 1 kb ladder to confirm amplification had occurred. 
         [0045]    After studying the present disclosure, those skilled in the art will recognize that numerous modifications can be made to the specific implementations of the detection system described above. Therefore, the system is not to be limited to the specific embodiments illustrated and described above. The system, as originally presented and as it may be amended, encompasses variations, alternatives, modifications, improvements, equivalents, and substantial equivalents of the embodiments and teachings disclosed herein, including those that are presently unforeseen or unappreciated, and that, for example, may arise from applicants/patentees and others.

Technology Category: 7