EP3965935A1 - Systems and methods for analyzing rna transcripts - Google Patents
Systems and methods for analyzing rna transcriptsInfo
- Publication number
- EP3965935A1 EP3965935A1 EP20822064.0A EP20822064A EP3965935A1 EP 3965935 A1 EP3965935 A1 EP 3965935A1 EP 20822064 A EP20822064 A EP 20822064A EP 3965935 A1 EP3965935 A1 EP 3965935A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- molecules
- rna transcript
- sample
- bound
- rna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1429—Signal processing
- G01N15/1433—Signal processing using image recognition
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N2015/144—Imaging characterised by its optical setup
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1486—Counting the particles
Definitions
- the subject matter disclosed herein relates to detecting target molecules, such as nucleic acid molecules and, more particularly, to systems for optical sensing of the target molecules.
- PCR Polymerase Chain Reaction
- An electronic sensor for detection of specific target nucleic acid molecules can include capture probes immobilized on a sensor surface between a set of paired electrodes.
- An example of a system and method for detecting target nucleic acid molecules is described in U.S. Patent No. 7,645,574, the entirety of which is herein incorporated by reference.
- amplified products or amplicons derived from targeted pathogen sequences are captured by the probes.
- Nano-gold clusters, functionalized with a complementary sequence, are used for localized hybridization to the amplicons.
- the nano-gold clusters serve as catalytic nucleation sites for metallization, which cascades into the development of a fully conductive film.
- the presence of the gold film shorts the gap between the electrodes and is measured by a drop in resistance, allowing the presence of the captured amplification products to be measured.
- sensors can be insensitive to small quantities of target molecules, resulting in false negative results or a failure to detect the target molecules.
- a system for analyzing molecules in a sample includes an imager, a flow cell, a magnet, and a processor.
- the flow cell includes a functionalized surface comprising a capture probe configured to bind molecules comprising a first RNA transcript.
- the magnet is positioned opposite the functionalized surface.
- the magnet is configured to direct the molecules comprising the first RNA transcript to the functionalized surface to bind to the capture probe.
- the light source configured to direct a light beam at the bound molecules comprising the first RNA transcript.
- the imager is configured to capture light from the bound molecules comprising the first RNA transcript.
- a processor configured to determine a quantity of the molecules in the sample comprising the first RNA transcript.
- the process is further configured to determine an expression level of the first RNA transcript in the sample based on the quantity of the molecules.
- a method for analyzing molecules in a sample uses an imager, a flow cell including a functionalized surface having a capture probe coupled to the functionalized surface, a magnet, and a light source.
- the method includes the following steps. Binding molecules comprising a first RNA transcript to magnetic particles. Directing the molecules comprising the first RNA transcript to the functionalized surface via the magnet. Binding the molecules comprising the first RNA transcript to the capture probe. Directing a light beam from the light source at the bound molecules comprising the first RNA transcript Capturing light from the bound molecules comprising the first RNA transcript. Determining a quantity of the molecules in the sample comprising the first RNA transcript. Determining an expression level of the first RNA transcript in the sample based on the quantity of the molecules.
- a method for analyzing molecules in a sample uses an imager, a magnet, a light source, and a flow cell that includes a functionalized surface having a plurality of capture probes. Each of the plurality of capture probes is configured to bind molecules in the sample comprising one of the plurality of RNA transcripts.
- the method includes the following steps. Binding molecules in the sample to a magnetic particle. Directing the molecules to the functionalized surface using the magnet. Binding each specific molecule of the molecules to one of the plurality of capture probes configured to bind the RNA transcript of the specific molecule. Directing a light beam from the light source at bound molecules bound on each of the plurality of capture probes. Capturing light from the bound molecules.
- FIG. 1 is a perspective view of a portable diagnostic assay system operative to accept one of a plurality of disposable cartridges configured to test fluid samples of collected blood/food/biological samples;
- FIG. 2 is an exploded perspective view of one of the disposable cartridges configured to test a blood/food/biological sample
- FIG. 3 is a top view of the one of the disposable cartridges illustrating a variety of assay chambers including a central assay chamber, one of which contains an assay chemical suitable to breakdown the fluid sample to detect a particular attribute of the tested fluid sample;
- FIG. 4 is a bottom view of the disposable cartridge shown in FIG. 3 illustrating a variety of channels operative to move at least a portion of the fluid sample from one chamber to another for the purpose of performing multiple operations on the fluid sample.
- FIG. 5 is a diagram of an embodiment of a sensor system having a functionalized surface
- FIG. 6 is a flowchart illustrating an embodiment of a method of detecting a target molecule
- FIG. 7 is a diagram of the sensor system of FIG. 5 with target molecules bound to magnetic particles
- FIG. 8A is cross-sectional illustration of an embodiment of a magnetic particle
- FIG. 8B is a cross-sectional illustration of another embodiment of a magnetic particle
- FIG. 8C is an illustration of an embodiment of a magnetic particle bound with nanoparticles
- FIG. 8D is an illustration of another embodiment of a magnetic particle bound with nanoparticles
- FIG. 8E is an illustration of an embodiment of a target molecule bound with a magnetic particle and a nanoparticle
- FIG. 8F is an illustration of another embodiment of a target molecule bound with a magnetic particle and nanoparticles
- FIG. 8G is an illustration of yet another embodiment of a target molecule bound with a nanoparticle and magnetic particles
- FIG. 9 is a diagram of the sensor system of FIGS. 5 and 7 with the target molecules bound to the functionalized surface
- FIG. 10 is a diagram of the sensor system of FIGS. 5 and 7-8 with functionalized nanoparticles bound to the target molecules;
- FIG. 11 is a diagram of the sensor system of FIGS. 5, 7-8, and 10 with a light source directed at the functionalized nanoparticles;
- FIG. 12A is an embodiment of scattering signatures of 50 nm monodispersed nanoparticles under dark field microscopy
- FIG. 12B is an embodiment of scattering signatures of 100 nm monodispersed nanoparticles under dark field microscopy
- FIG. 13 is a comparison of scattering signatures of developed nanoparticles versus undeveloped nanoparticles under dark field microscopy
- FIG. 14 is an illustration of an embodiment of an optical sensor system
- FIG. 15A is an enlarged partial illustration of the optical sensor system of FIG. 14 with the magnet retracted;
- FIG. 15B is an enlarged partial illustration of the optical sensor system of FIG. 14 with the magnet extended;
- FIG. 16 is a side view illustration of an embodiment of an optical instrument incorporating the optical sensor system of FIG. 14;
- FIG. 17A is a diagram of another embodiment of a sensor system having a functionalized surface
- FIG. 17B is a diagram of the sensor system of FIG. 17A with target molecules bound to the functionalized surface;
- FIG. 17C is a diagram of the sensor system of FIGS. 17A-17B having nanoparticles bound to the target molecules;
- FIG. 18 depicts a method for analyzing a plurality of RNA transcripts in a sample
- FIG. 19 depicts a method for determining a risk level of a neurodegenerative disease by analyzing a plurality of RNA transcripts of a sample.
- FIG. 20 depicts another working example.
- a disposable cartridge is described for use in a portable/automated assay system such as that described in commonly-owned, U.S. Patent Application Ser. No. 15/157,584 filed May 18, 2016 entitled “Method and System for Sample Preparation” which is hereby included by reference in its entirety.
- the disposable cartridge may be used to detect any of a variety of diseases which may be found in either a blood, food or biological detecting hepatitis, autoimmune deficiency syndrome (AIDS/HIV), diabetes, leukemia, graves, lupus, multiple myeloma, etc., just naming a small fraction of the various blood borne diseases that the portable/automated assay system may be configured to detect
- Food diagnostic cartridges may be used to detect salmonella, e-coli, staphylococcus aureus or dysentery. Diagnostic cartridges may also be used to test samples from insects and specimen.
- blood diagnostic cartridges may be dedicated cartridges useful for animals to detect diseases such as malaria, encephalitis and the west Nile virus, to name but a few.
- a portable assay system 10 receives any one of a variety of disposable assay cartridges 20, each selectively configured for detecting a particular attribute of a fluid sample, each attribute potentially providing a marker for a blood, food or biological (animal borne) disease.
- the portable assay system 10 includes one or more linear and rotary actuators operative to move fluids into, and out of, various compartments or chambers of the disposable assay cartridge 20 for tiie purpose of identifying or detecting a fluid attribute. More specifically, the cartridge 20 includes a flow cell 21 extending horizontally therefrom.
- a rotary actuator (not shown) of the portable assay system 10 aligns one of a variety of ports 18P, disposed about a cylindrical rotor 18, with a syringe barrel 22B of a stationary cartridge body 22.
- the linear actuator 24 displaces a plunger shaft 26 so as to develop pressure i.e., positive or negative (vacuum) in the syringe barrel 22. That is, the plunger shaft 26 displaces an elastomer plunger 28 within the syringe 22 to move and or admix fluids contained in one or more of the chambers 30, 32.
- system 10 includes one or more processors 11 housed within a control board 12 of the body of the system 10 for receiving signals from the various components of the system 10.
- the disposable cartridge 20 provides an automated process for preparing the fluid sample for analysis and/or performing the fluid sample analysis.
- the sample preparation process allows for disruption of cells, sizing of DNA and RNA, and concentration/cleanup of the material for analysis. More specifically, the sample preparation process of the instant disclosure prepares fragments of DNA and RNA in a size range of between about 100 and 10,000 base pairs.
- the chambers can be used to deliver the reagents necessary for end-repair and kinase treatment. Enzymes may be stored dry and rehydrated in the disposable cartridge 20, or added to the disposable cartridge 20, just prior to use.
- a rotary actuator allows for a single plunger 26, 28 to draw and dispense fluid samples without the need for a complex system of valves to open and close at various times. This greatly reduces potential for leaks and failure of the device compared to conventional systems. Finally, it will also be appreciated that the system greatly diminishes the potential for human error.
- the cylindrical rotor 18 includes a central chamber 30 and a plurality of assay chambers 32, 34 surrounded, and separated by, one or more radial or circumferential walls.
- the central chamber 30 receives the fluid sample while the surrounding chambers 32, 34 contain a premeasured assay chemical or reagent for the purpose of detecting an attribute of the fluid sample.
- the chemical or reagents may be initially dry and rehydrated immediately prior to conducting a test
- Some of the chambers 32, 34 may be open to allow the introduction of an assay chemical while an assay procedure is underway or in-process.
- the chambers 30, 32, 34 are disposed in fluid communication, i.e., from one of the ports 18P to one of the chambers 30, 32, 34, by channels 40, 42 molded along a bottom panel 44, i.e., along underside surface of the rotor 18.
- a first port 18P corresponding to aperture 42, may be in fluid communication with the central chamber 30, via aperture 50.
- FIG. 5 illustrates an embodiment of a sensor system 70.
- the sensor system 70 includes an imager 72 configured to capture still images, video, or a combination thereof.
- the imager 72 can be configured to capture high resolution still images.
- the imager 72 includes a pixel array 74 and array circuitry 76.
- the pixel array 74 can include any suitable number of pixels.
- the pixel array 74 can be a high density array including at least six (6) megapixels.
- the camera can have a large field of view.
- the pixel array 74 is a light sensitive pixel array, such as an active array, passive array, planar Fourier capture array, angle sensitive array, photodiode array, a charge coupled device, a complementary metal-oxide semiconductor (CMOS), or a charge injection device.
- CMOS complementary metal-oxide semiconductor
- the sensor system 70 also includes a flow cell 78.
- the flow cell 78 can be formed of any suitable material, such as a polypropylene or polystyrene polymer or glass, among others. In an embodiment, the flow cell is formed by injection molding.
- the flow cell 78 includes a transparent or optically clear surface 80 and a transparent functionalized surface 82.
- the functionalized surface 82 includes a plurality of capture probes 84 in the form of a functionalized oxide surface allowing attachment and immobilization of capture probe molecules 84 on the surface 82.
- the capture probes 84 are designed to capture or bind target molecules 86 (FIG. 7) by interaction between complementary sequences.
- the target molecules 86 can be collected from a biological sample.
- the biological sample could be any suitable type of materials, such as blood, mucous, and skin, among others.
- the target molecules 86 can be protein ligands or DNA segments.
- An objective or lens 75 can optionally be positioned between the imager 72 and the flow cell 78.
- a magnet 88 can be positioned opposite the functionalized surface 82.
- the magnet 88 can be a single magnet or an array of magnets.
- the functionalized surface 82 includes an array of many different capture probes 84, and each capture probe 84 is configured to capture molecules having different RNA transcripts.
- the array of probes 84 can be deployed to capture, say, between 10-20 different RNA transcripts.
- multiple probes 84 may be configured to capture molecules having the same RNA transcripts, so that there is redundancy to allow for error checking of the results.
- an array of capture probes 84 can be designed to capture many different RNA transcripts to analyze the relative proportion of molecules in the sample that are expressing those RNA transcripts.
- one or more of the array of probes 84 can be configured to capture so-called housekeeping genes, which can serve as a baseline by which to normalize the number of other molecules found on other genes. For instance, if 400 molecules of the housekeeping gene are captured, then the number of a different captured molecule can be compared to 400 to give an absolute sense of how many of those molecules are captured as a percentage compared to the housekeeping genes, thus establishing an absolute scale.
- FIG. 6 illustrates an embodiment of a method 90 for detection of a target molecule.
- the method 90 can be employed by a sensor system, such as the sensor system 70.
- target molecules 86 are bound to magnetic particles 110, as illustrated in FIG. 7.
- the target molecules 86 are bound to the magnetic particles 110 before being introduced to the flow cell 78.
- the target molecules 86 and magnetic particles 110 are introduced to the flow cell 78 in an unbound state and the target molecules 86 bind to the magnetic particles 110 within the flow cell
- FIGS. 8A-8B illustrate two embodiments of magnetic particles 110.
- the magnetic particle 110A is a composite particle that has a magnetic core 112, formed of a magnetic material such as iron, and a nanoparticle coating 114.
- the coating 114 can be a gold coating.
- the coating 114 can be configured to act as a nucleation site for further nanoparticle development
- the magnetic particle 110A includes at least one binding site for a ligand A for binding to the target molecules 86.
- a chemical reactive group such as a thiol, amine, or aldehyde, can mediate or facilitate ligand binding.
- the magnetic particle 110B can have a magnetic body 116 formed of a magnetic material, such as iron.
- the magnetic particle 110B includes at least one binding site or ligand A for binding to a target molecule.
- the magnetic particle 11 OB further includes at least one binding site or ligand B for binding a magnetic nanoparticle.
- the magnetic particle can include any suitable combination of binding sites A, B.
- the magnetic particle can include both types of binding sites A, B or the magnetic particle can include only target molecule binding sites A.
- the magnetic particle 110 can include a magnetic body 112 and a plurality of nanoparticles 122 bound to the magnetic body 112.
- the magnetic particle 110 can be an alloy, such as a heterogeneous alloy, including a plurality of magnetic bodies 112 bound with a plurality of nanoparticles 122.
- the target molecule 86, magnetic particle 110, and nanoparticle 122 can be bound in a variety of arrangements. As illustrated in FIG. 8E, the magnetic particle 110 and nanoparticle 122 can each be bound directly to the target molecule 86. Alternatively and as illustrated in FIG. 8F, the magnetic particle 110 can be bound directly to the target molecule 86 and one or more nanoparticles 122 can be bound to the magnetic particle 110. Alternatively and as illustrated in FIG. 8G, a nanoparticle 122 can be bound directly to the target molecule 86 and one or more magnetic particles 110 can be bound to the nanoparticle 122.
- the bound magnetic particles 110 and target molecules 86 are directed or moved to the functionalized surface 82.
- the magnet 88 is coupled to an actuator (not shown) configured to move the magnet 88 toward (retracted) and away from (extended) the functionalized surface 82.
- the magnet 88 is moved away 118 from die functionalized surface 82, the magnetic particles 110, attracted to the magnet 88, move 120 toward die functionalized surface 82.
- the target molecules 86 are bound to the magnetic particles 110, the target molecules 86 are directed or drawn by the magnetic particles 110 toward the functionalized surface 82.
- the target molecules 86 are bound to the capture probes 84 of the functionalized surface 82 as illustrated in FIG. 9.
- die magnetic particles 110 remain bound to the bound target molecules 86.
- the target molecules 86 can be denatured to unbind the magnetic particles 110 from the target molecules 86 when the target molecules 86 reach the functionalized surface 82, following which the target molecules 86 can bind to the functionalized surface 82.
- functionalized nanoparticles 122 are introduced to the flow cell 78 and are bound to the target molecules 86, as illustrated in FIG. 10.
- the functionalized nanoparticles 122 are bound directly to the target molecules 86.
- the functionalized nanoparticles 122 are bound to the magnetic particles 110 bound to each target molecules 86.
- a plurality of functionalized nanoparticles 122 are bound to each target molecule 86. Any suitable method of hybridizing or binding the nanoparticles 122 to the target molecules 86 can be used, hi an embodiment, the functionalized nanoparticle 122 is a gold particle.
- the functionalized nanoparticle 122 is a catalytic nanoparticle, such as a gold catalyst reagent.
- the nanoparticles 122 are in the form of catalyst clusters.
- the nanoparticles 122 are bound to the target molecules 86 after the target molecules 86 are bound to the functionalized surface 82. In an alternative embodiment, the nanoparticles 122 can be bound to the target molecules 86 or magnetic particles 110 prior to binding of the target molecules 86 to the functionalized surface 82. [0059] Following binding of the nanoparticles 122 to the target molecules 86, an optional metallization step can be performed to metallize the nanoparticles 122 and develop or form enlarged nanoparticles or even a film. The developed nanoparticles can improve detection of the target molecules. In this metallization step, the nanoparticles 122 serve as nucleation sites for development of enlarged nanoparticles 124.
- a light source 126 directs a light beam 128 at the target molecules 86 and functionalized nanoparticles 122, 124 in the flow cell 78.
- the light beam 128 is aimed so that light is directed solely at the target molecules 86 and nanoparticles 122, 124 and no light 128 from the light source 126 is captured by the imager 72.
- the flow cell 78 is configured to prevent diffusion of the light beam 128 toward the imager 72.
- light 130 (FIG. 11) from the nanoparticles 122, 124, target molecules 86, magnetic particles 110, or a combination thereof, is captured by the imager 72.
- the light 130 can be reflected or emitted from the particles 86, 110, 122, 124, or a combination thereof.
- the light 130 captured by the imager 72 is analyzed to detect the number of target molecules 86 present.
- the captured light 130 can be analyzed using dark field microscopy.
- the spots of detected light are counted and quantified to determine the number of target molecules 86 present Counting and quantifying is accomplished using the one or more processors 11 (see FIG. 1).
- a single imager 72 and light beam 128 may be used as described above to sequentially examine each of the capture probes 84. Such sequential analysis may be achieved by providing, for example, for translation of the imager 72 and light beam 128 across the flow cell to allow each capture probe 84 to be examined one at a time.
- adjustable mirrors can be used to direct the light from the light beam 128 to one of the capture probes 84 and the imager 72 one at a time by adjusting the mirrors.
- an imager with a larger imaging capability can be fixed with the light beam 128 moving to each capture probe 84 one at a time.
- multiple imagers 72 can be used, with each one dedicated to one or several capture probes 84.
- FIGS. 12A-13 illustrate embodiments of light or scattering signatures or captured images of gold nanoparticles 122 captured under dark field microscopy.
- FIG. 12A is a scattering signature of 50 nm gold nanoparticles and
- FIG. 12B is a scattering signature of 100 nm gold nanoparticles.
- FIG. 13 is an image comparing the scattering signature 132 of undeveloped (20mn) nanoparticles 122 with the scattering signature 134 of developed (100mn) nanoparticles 124.
- a series of dark field images can be captured, hi this embodiment, a first image can be captured prior to development of the nanoparticles 122 and at least one additional image can be captured as the nanoparticles are developed. Alternatively, a first image can be captured prior to binding of the target molecules 86 and at least one additional image can be captured following binding of the nanoparticles 122.
- the captured images can be compared to removed background artifacts and improve analysis of the dark field images.
- a dye particle (not shown) is coupled to the target molecules 86 for detection of the target molecules 86.
- the light source 128 is tuned to the wavelength of the dye and regions covered by the dye will fluoresce. The fluoresce is detected by the imager 72.
- the functionalized surface is exposed to a radiation source (not shown).
- the regions of nanoparticles preferentially absorb the radiation, causing localized heating.
- the localized heating is captured and registered by the imager 72 to detect the presence of the target molecules 86. Based on the amount of heating registered, a count of the number of target molecules present is established.
- the system may be calibrated by allowing a known number of target molecules to be heated, and measuring the temperature, for example.
- FIG. 14 An example of an optical sensor system 140 is illustrated in FIG. 14. Similar to the optical sensor system 70 described above, the optical sensor system 140 includes an imager 142 and an objective or lens 144 coupled to the imager 142.
- the imager 142 is a high resolution imager having a wide angle or large field of view.
- the objective 144 is directed toward the flow cell 146.
- the interior of the flow cell 146 includes a functionalized surface for binding target molecules.
- One or more feeder lines 147 can be coupled to the flow cell 146 to facilitate the introduction of various particles to the flow cell 146.
- a light source 148 is directed at the flow cell 146.
- the light source 148 can be any suitable light source.
- the light source 148 can provide light at a predetermined frequency.
- the light source 148 can be a white light.
- the light source 148 is directed or aimed solely at the flow cell 146. Li the illustrated embodiment, the light source 148 is directed orthogonally to the axis X on which the objective is positioned.
- the flow cell 146 is configured to channel the light from the light source 148 toward the particles within the flow cell 146, rattier than toward the imager 142 and to prevent light diffusion from the light source 148 to the imager 142.
- a magnet 150 is positioned opposite the flow cell 146 from the objective 144.
- An actuator 152 such as a solenoid, is coupled to the magnet 150 and is configured to move the magnet. As illustrated in FIGS. 15A-15B, in an embodiment, the actuator 152 is configured to retract or move the magnet 150 toward (FIG. 15A) the flow cell 146 and to extend or move the magnet 150 away (FIG. 15B) from the flow cell 146.
- FIG. 16 illustrates an embodiment of an analysis system 160 including an optical system, such as the optical sensor systems 70, 140.
- the analysis system 160 includes a base 162 and a head 164.
- the imager 142 and objective 144 are positioned in the base 162.
- the flow cell 146 is positioned on the top surface of the base 162, aligned with the objective 144.
- the magnet 150 is positioned in the head 164 and is configured to extend to and engage with the flow cell 146.
- the imager 142 and objective 144 are not aligned along an axis, as illustrated in FIG. 14. Rather, the imager 142 is aligned along an axis Y extending longitudinally through the base 162 between the side surfaces 166, 167 of the base 162.
- the objective 144 is positioned orthogonal to the axis Y and extends upward through the base 162.
- a mirror 168 is positioned below the objective 144 and angled toward the imager 142 to create an optical path 170 between the objective 144 and the imager 142.
- FIGS. 17A-17C illustrate an alternative embodiment of a sensor system 180.
- the sensor system 180 includes a prism type substrate 182 having a Kretshmann configuration.
- the substrate 182 has a surface 184 coated with a metal film 186 suitable for surface plasmon resonance or Raman scattering.
- the metal film can be gold, silver, copper, titanium, or chromium.
- the film 186 is functionalized with a bio-specific coating to include capture probes 84.
- a light source 188 directs a light beam 190 through the prism substrate 182 toward the film 186 and a detector 192 captures light 194 from the film 186, such as light reflected or emitted by the film 186.
- a baseline measurement of the captured light is taken.
- the baseline measurement of the captured light is used to calibrate the absorbance angle (FIG. 17A).
- the baseline measurement can be sued to identify contaminants or debris on die sensor prior to binding of the target molecules 86 or prior to development of increased nanoparticle size, as discussed below.
- a sample containing target molecules 86 is introduced to the sensor system 180 and the target molecules 86 bind to the capture probes 84 (FIG. 17B).
- the target molecules 86 can be directed to the surface film 186 via magnetic particles as described above.
- Functionalized nanoparticles 122 are introduced to the system 180 and allowed to bind to the target molecules 86 (FIG. 17C).
- a plating bath can optionally be used to increase the size of the bound nanoparticles 122.
- the beam 190 is directed toward the film 186 and the light 194 from the film 186, such as reflected or emitted, is captured. Any difference in reflectivity or intensity between the baseline measurement and the final measurement is observed in order to detect the presence of the target molecules 86.
- a quantitative count of target molecules 86 may be established by comparison with calibrated tests of the reflectivity or intensity for known number of target molecules.
- the baseline measurement can be used to subtract particles identified as debris from the final measurement
- FIG. 18 depicts a method 1800 for analyzing a plurality of RNA transcripts in a sample.
- transcript array technology has involved identification of genes which are up regulated or down regulated in various diseased states. Additional uses for transcript arrays have included the analyses of members of signaling pathways, and the identification of targets for various drugs. Transcript arrays can be beneficial in monitoring the level of either disease states or effect of therapies.
- RNA profiling is a process useful to monitor disease state or treatment efficacy by monitoring the expression levels of key RNA transcripts in a sample.
- Key RNA transcripts can be identified using techniques such as an Afiymetrix array to screen all transcripts in a cell. By profiling multiple patients at different stages of disease, a set of key indicator transcripts can be identified and used on an array, such as that of the current invention which targets the key indicators.
- some housekeeping genes which do not vary due to the disease are also monitored to establish a baseline with which to compare expression of the key indicators. Statistical methods are used to determine which indicators are needed to reliably monitor disease progression.
- RNA probes which can monitor expression of thousands of genes at a time, such as the Afiymetrix array.
- Afiymetrix makes quartz chips for analysis of DNA Microarrays called GeneChip arrays.
- Affymetrix's GeneChip arrays assist researchers in quickly scanning for the presence of particular genes in a biological sample.
- Asymetrix is focused on oligonucleotide microarrays. These microarrays are used to determine which genes exist in a sample by detecting specific pieces of mRNA. A single chip can be used to analyze thousands of genes in one assay.
- these systems are expensive to run and their use can be limited in monitoring disease states and treatment efficacy.
- RNA for analysis in the present techniques can be derived from a variety of samples, including but not limited to blood, plasma, leukocytes, other blood fractions, sputum, saliva, urine, stool, vaginal swabs, and tissue samples.
- cartridges have the capability for automated sample disruption and isolation of RNA.
- automation of all sample processing improves reliability of RNA isolation without degradation.
- automation of sample processing enables testing to be run outside of traditional laboratories.
- the method and system described with respect to FIG. 18 provides a system and methods for determining or monitoring the progression of disease states or the efficacy of therapeutic regimens in a subject, preferably a human patient.
- the technique relates to methods for monitoring disease states or therapies by monitoring changes in mRNA expression levels.
- the current technique utilizes a simple, easy to use system which can monitor expression of a number of gene transcripts for rapid diagnosis, to enable better treatment.
- RNA profiling to monitor disease state or effectiveness of treatment.
- the identification of changes in gene expression caused either by the actions of disease states or by therapeutic regimens, such as drug regimens, for disease states is a problem of great commercial and human importance. Most of the decisions that need to be made to run efficient clinical trials and to properly manage the health of patients rely on assays that monitor changes in cells in the body.
- RNA can be difficult to handle and is susceptible to degradation by
- the closed system automates disruption of the sample, cleaning and concentrating of the RNA.
- the sample is treated with a guanidine hydrochloride solution, which lyses the cells and disrupts enzymes, thereby stabilizing the RNA
- a guanidine hydrochloride solution which lyses the cells and disrupts enzymes, thereby stabilizing the RNA
- the system further provides a multiplexed array to monitor the expression levels of multiple RNA transcripts.
- the sensor array has multiple sites specific for individual transcripts. Capture probes specific to a sequence of nucleotides in a transcript are printed at a site.
- the multiplexed array allows for quantitation of expression of key indicator transcripts and monitoring of housekeeping genes in order to establish baseline expression. The system will then compare expression of the key indicator genes against the housekeeping genes to determine expression relative to the baseline. Variance of key gene expression will be analyzed using an algorithm to determine disease state.
- the current invention Rather than monitoring all transcripts in a cell, which requires thousands of sensors, the current invention provides an array to monitor between one and hundreds of key transcripts. Additional sensors for one to tens of housekeeping genes would also be included. Optimally the number of key transcripts to be monitored would be between three and twenty. Keeping the number of sites low will minimize the cost of the array but allows for targeted testing of key indicators of a disease.
- the current invention has critical functions needed to capture and quantitate RNA expression levels rapidly.
- the current invention provides a magnetic approach to concentrate and move RNAs to the sensors to allow for rapid testing without loss of sensitivity.
- the current system provides a method to quantitate the RNA transcripts at each sensor.
- the method 1800 may be performed using a sensor (eg., sensor system 70 of FIG. 5 or any other example set forth herein) comprising an imager (e.g., imager 72 of FIG. 5 or any other example set forth herein), a flow cell (e.g., flow cell 78 of FIG. 5 or any other example set forth herein) comprising a functionalized surface (e.g., functionalized surface 78 of FIG. 5 or any other example set forth herein) having a plurality of capture probes (e.g., probe molecules 84 of FIG.
- a sensor eg., sensor system 70 of FIG. 5 or any other example set forth herein
- an imager e.g., imager 72 of FIG. 5 or any other example set forth herein
- a flow cell e.g., flow cell 78 of FIG. 5 or any other example set forth herein
- a functionalized surface e.g., functionalized surface 78 of FIG. 5 or any other example set forth herein
- capture probes e
- the method starts at block 1801 and includes the following steps:
- Step 1810 Binding molecules comprising a first RNA transcript to magnetic particles.
- Step 1820 Directing the molecules comprising the first RNA transcript to the functionalized surface via the magnet
- Step 1830 Binding the molecules comprising the first RNA transcript to the capture probe.
- Step 1840 Directing a light beam from the light source at the bound molecules comprising the first RNA transcript
- Step 1850 Capturing light from the bound molecules comprising the first RNA transcript.
- Step 1860 Determining a quantity of the molecules in the sample comprising the first RNA transcript
- Step 1870 Determining an expression level of the first RNA transcript in the sample based on the quantity of the molecules.
- the method 1800 further includes calculating a disease state or a treatment efficacy based on the plurality of expression levels of the plurality of RNA transcripts.
- the plurality of capture probes corresponds to between 10 and 20 RNA transcripts.
- the method further includes monitoring a housekeeping gene for determining a baseline for comparison of the plurality of expression levels of the plurality of RNA transcripts.
- the method further includes receiving and processing the sample without external exposure.
- the method further includes binding a plurality of magnetic particles to the molecules and interacting the magnet with the magnetic particles to direct the molecules to the functionalized surface.
- the method further includes comprising preventing diffusion of the light beam toward the imager.
- the method further includes binding the molecule to a nanoparticle when the molecule is bound to the functionalized surface, wherein the nanoparticle reflects the light beam toward the imager.
- FIG. 19 depicts a method for determining a risk level of a neurodegenerative disease by monitoring expression levels of a plurality of RNA transcripts of a sample.
- Problem 1 is the accuracy of diagnosis.
- the number of conditions that can cause cognitive deficits that may look like Alzheimer’s disease is pages long. Some of these conditions, such as vitamin B deficiency, can be cured, while others may not be curable but might be managed with appropriate intervention. The possibility of cure or potential management require an accurate diagnosis.
- Step 1 it would be possible to detect disease before significant brain damage has occurred.
- Step 2 would call for early effective treatment to halt or slow disease progression so that affected persons could live the rest of their lives free of disease symptoms. Free of the shakes and motor losses of Parkinson’s disease or free of the memory and cognitive defects of Alzheimer’s disease.
- Step 1 This“silent period” for many age-related neurodegenerative diseases creates a window of opportunity for a two-step process.
- Step 1 it would be possible to detect disease before significant brain damage has occurred.
- Step 2 would call for early effective treatment to halt or slow disease progression so that affected persons could live the rest of their lives free of disease symptoms. Free of the shakes and motor losses of Parkinson’s disease or free of the memory and cognitive defects of Alzheimer’s disease.
- This application addresses Step 1, accurate diagnosis and early diagnosis.
- a variety of methods have been used to show that early diagnosis of Alzheimer’s disease is real.
- Positron Emission Tomography (PET) scanning of the brain in living people has shown Alzheimer pathology in some people as young as the teens or 20s.
- Microscopic pathological examination of more than 3,000 brains of people who died between the ages of 10 and 100 has shown the start of Alzheimer’s in people in their 20’s.
- sophisticated cognitive testing has been able to establish indications of Alzheimer’s 15 years before the disease became clinically detectable (REF).
- the application presented describes methods for obtaining a blood sample, for inserting a sample into an apparatus capable of determining levels of expression of multiple RNA species which have been determined to be useful in multivariate analyses to diagnose and predict Alzheimer’s disease.
- the method described can provide a diagnosis or probability of a future diagnosis in the field within about 30 minutes fin a cost we estimate to be less than $100.
- the system presented is capable of application to screening of a mass population of people without symptoms but who may be at risk of a future diagnosis of Alzheimer’s disease.
- the method 1900 may be performed using a sensor (e.g., sensor system 70 of FIG. 5 or any other example set forth herein) comprising an imager (e.g., imager 72 of FIG. 5 or any other example set forth herein), a flow cell (e.g., flow cell 78 of FIG. 5 or any other example set forth herein) comprising a functionalized surface (e.g, functionalized surface 78 of FIG. 5 or any other example set forth herein) having a plurality of capture probes (e.g, probe molecules 84 of FIG. 5 or any other example set forth herein) coupled to the functionalized surface, a magnet (e.g., magnet 88 of FIG. 5 or any other example set forth herein), and a light source (e.g., light source 126 of FIG. 6 or any other example set forth herein).
- a sensor e.g., sensor system 70 of FIG. 5 or any other example set forth herein
- an imager e.g., imager 72 of FIG. 5 or any other
- the method starts at block 1901 and includes the following steps:
- Step 1910 Binding molecules in the sample to a magnetic particle.
- Step 1920 Directing the molecules to the functionalized surface using the magnet
- Step 1930 Binding each specific molecule of the molecules to one of the plurality of capture probes configured to bind the RNA transcript of the specific molecule.
- Step 1940 Directing a light beam from the light source at bound molecules bound on each of the plurality of capture probes.
- Step 1950 Capturing light from the bound molecules.
- Step 1960 Determining a quantity of the bound molecules bound on each of the plurality of capture probes based on the captured light.
- Step 1970 Determining a plurality of expression levels corresponding to the plurality of RNA transcripts based on the quantity of the bound molecules bound on each of the plurality of capture probes configured to bind each of the plurality of RNA transcript.
- Step 1980 Calculating a risk of the neurodegenerative disease based on the plurality of expression levels of the plurality of RNA transcripts.
- the method 1900 further includes calculating the risk of a future diagnosis of the neurodegenerative disease. In another embodiment, the method 1900 further includes calculating the risk of a present diagnosis of the neurodegenerative disease.
- the method 1900 further includes binding a plurality of magnetic particles to the molecules and interacting the magnet with the magnetic particles to direct the molecules to the functionalized surface.
- the method 1900 further includes preventing diffusion of the light beam toward the imager, hi a further example, the method 1900 further includes binding the molecule to a nanoparticle when the molecule is bound to the functionalized surface, wherein the nanoparticle reflects the light beam toward the imager.
- the number will be based on prior and ongoing research and will be different for each RNA species.
- the multiplier numbers will be the same for the same RNA species in all samples.
- the resulting 10-20 multiplication products will then be combined into a single number by an algorithm that will indicate the probability of an Alzheimer’s disease diagnosis OR the probability of a future diagnosis of Alzheimer’s disease.
- Blood is drawn from a person for the purpose of determining a diagnosis of or risk of a future diagnosis of a neurodegenerative disease such as Alzheimer’s disease, Parkinson’s disease, Frontotemporal dementia, etc.
- Blood may be drawn from any vein, usually the median cubital vein, and drawn into a syringe. Alternately, blood may be obtained from a finger stick. If drawn from a finger, discard the first drop, then squeeze finger and collect blood onto a Whatman P Card. Let blood dry overnight at room temperature, then store sample at -20 C. Isolate RNA from die filter paper using QIAGEN kit of your choice - we have used QiAmp and exoRNeasy.
- protocol for blood drawn into a PAXgene tube is as follows.
- PAXgene Blood RNA Tube is the only tube to be drawn, blood should be drawn into a“Discard Tube” prior to drawing blood into the PAXgene tube. Otherwise, the PAXgene tube should be the last tube drawn in the phlebotomy procedure.
- Tubes can either be left at room temperature for 2 hours then placed at - 20o C or they can be placed at -20o C upright in a wire rack immediately after the blood draw.
- RNA is extracted from leukocytes using the mKNA Isolation Kit for Blood/Bone Marrow (Roche) per manufacturer's protocol.
- erythrocytes are selectively lysed and collected by centrifugation.
- the leukocytes are then lysed and the total nucleic acids is collected by nonspecific adsorption to magnetic beads and magnetic separation.
- mKNA is captured by biotin-labeled oligo(dT) and streptavidin-coated magnetic particles.
- RNA samples are collected and stored at -80 °C. RNA quality and abundance are confirmed by 260/280 ratios and by gel electrophoresis.
- Messenger RNA is amplified and radioactively labeled with 32P CTP. The labeled amplified RNA is hybridized to custom cDNA arrays and quantified.
- RNA integrity is determined by analysis with an Agilent 2100 Bioanalyzer. The suitability of samples for analysis is based on RNA Integrity Number, with the rare sample with a RIN a number less than 2 considered not usable.
- RNA to cDNA An example method for converting RNA to cDNA is as follows: 1.0 pg of total RNA is reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNA is diluted 1:5 and used as template in qRT-PCR reactions using TaqMan® Gene Expression Master Mix (Applied Biosystems) and TaqMan® Gene Expression Assays (Applied Biosystems) and run on an iCycler iQ TM (Bio-Rad) or equivalent equipment. The quantitative RT-PCR amplifications are run in triplicate at thermal cycling conditions of 10 minutes at 95°C, 40 cycles of denaturation at 95°C for 15 seconds, and annealing and extension at 60°C for 1 minute.
- Beta glucuronidase (GUSB) is used as an endogenous control since it has been found to be the same from sample to sample. Following normalization, data are then presented as a ratio using the 2(-Delta Delta C(T)) method (Livak and Schmittgen, 2001).
- RNA species may be used for calculating risk of neurodegenerative disease.
- RNA species for study are selected on the basis of known mechanisms of neurodegenerative diseases and on the basis of laboratory findings in preliminary studies. Some examples are shown in the Table 1.
- the weights assigned to each independent variable are corrected for the interrelationships among all the variables.
- the weights are referred to as discriminant coefficients.
- the present apparatus quantifies amount of RNA in a sample accurately detect the presence and amount of each of any number of specific RNA or DNA species, hi the example provided, 8 specific RNA species are specified, but the identity, specific species and their number may be different.
- This information about amount of each selected RNA or DNA specie in the sample may be transmitted to an external device such as a cell phone or other mobile computing device. Or, the value may be transmitted to a computing circuit within the same device.
- the amount detected by the sensor elements for each specie is multiplied in the computing device by a weight that is unique to that
- RNA or DNA specie The weight may be referred to as the standardized discriminant coefficient.
- this multiplication results in 8 values representing the product of amount of nucleotide specie in the sample multiplied by a weight
- the weights may be a product of prior investigation or they may be a result of entering into the system a method for determining weights on the basis of known identity of samples submitted to the system.
- these 8 products of amount of nucleotide multiplied by the weight for each nucleotide are combined by any algorithm that results in a single number that represents a characterization of the sample entered into the system. This number then represents the probability for the detection of a currently diagnosed or probable future diagnosis of Alzheimer’s disease or some other neurodegenerative disease, such as Parkinson's disease or amyotrophic lateral stenosis.
- the resulting number may represent either a diagnosis of a neurodegenerative disease or a probability of a clinical diagnosis of that disease at some time in the future.
- This information can be presented to any device such as a display in the equipment itself, a local or remote computer or it may wirelessly report data to a secure central site. In the latter case these data can be combined with data from multiple sites to evaluate potential relationships among disease, geography, socio-economic status, etc.
- discriminant analysis The purpose of discriminant analysis is to obtain a model to predict a single qualitative variable from one or more independent variable(s). Discriminant analysis derives an equation as a combination of the independent variables that will discriminate best between the groups in the dependent variable. This combination is known as the discriminant function. The weights assigned to each independent variable are corrected for the interrelationships among all the variables. The weights are referred to as discriminant coefficients.
- F is formed by the linear combination of the dependent variable, Xi, X 2 ,...
- X p are the p independent variables
- e is the error term
- ao, a 1 , a ,..., a p are the discriminant coefficients.
- RNA species in a sample with cDNA or nucleotide arrays is as follows:
- the cDNA clones represented in arrays emphasized those that would test the hypothesis that transcripts related to stress, inflammation, and cell cycle would be affected in leukocytes from AD cases.
- 172 cDNAs selected for this purpose were printed on nylon membranes using a 96-pin replicator (Nalge Nunc) with each cDNA spotted in quadruplicate. Arrays are probed with labeled amplified RNA generated from extracted RNA from leukocyte samples and then exposed to a storage phosphor screen.
- Hybridization intensity of each spot is quantified by laser densitometric scanning (Phosphorlmager, Molecular Dynamics).
- the amount of cDNA deposited on each spot in the array is quantified by stripping and reprobing the membrane with an oligonucleotide specific for the T7 promoter present in all vectors. Any relevant aspects of this example may be combined with other examples set forth herein.
- qPCR quantitative reverse transcriptase polymerase chain reaction
- the qRT-PCR amplifications may be on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). Universal thermal cycling conditions are: 10 minutes at 95 °C, 40 cycles of denaturation at 95 °C for 15 seconds, and annealing and extension at 60 °C for 1 minute. Amplification efficiencies is close to 100% for all assays according to analyses of different dilutions of the cDNA. Beta glucuronidase (GUSB) is used as an endogenous control since its expression is found to be invariant across all samples. Following data normalization, data may be then presented as a ratio using the 2(-Delta C(T)) method (Livak and Schmittgen, 2001). Any relevant aspects of this example may be combined with other examples set forth herein.
- the table above shows the results of a quantitative PCR analysis of COX1 (upper left comer) of 8 non-demented (ND) and 8 Alzheimer cases (AD).
- Each number under the column“Sample” is the de-identified number given to each case that follows that case through all analyses.
- the column“Status” indicates whether cases were ND or AD.
- the column labelled COXl-GUSB is the qPCR result of the strength of signal from qPCR of COX1 for that case in mathematical relation to the invariant signal from the qPCR signal for the enzyme beta glucuronidase (GUSB) which is used as an endogenous control since its expression is invariant across all samples.
- GUSB beta glucuronidase
- the next column labelled“std Dev” is the standard deviation of each measure in the preceding column, and is based on the fact that each measure is made in triplicate.
- the column labelled “Ratio COX1/GUSB” is a ratio mathematical expression of the ratio between expression of COX1 and GUSB for each case.
- the column“COX1 relative to calibrator” is arbitrarily set at 1 (as is the column labelled“fold change”) for purposes of comparisons with the values from AD cases.
- FIG. 20 depicts another working example, for determining risk of a future diagnosis of Alzheimer’s disease by virtue of being APOE4-H- homozygotes.
- this example we are concerned with the risk of a future diagnosis of AD in persons who are presently cognitively intact. Having two copies of the APOE4 gene (one from each parent - APOE4++) constitutes significant risk for a future diagnosis of Alzheimer’s disease.
- the below bar graph showing our blood test scores for people all of whom are cognitively intact.
- the discriminant number resulting from our analysis resulting may represent either a diagnosis of a neurodegenerative disease in a symptomatic person or in the case of a normal control person who is cognitively intact it can represent the probability of a clinical diagnosis of that disease at some time in the future.
- This information can be presented to any device such as a display in the equipment itself, a local or remote computer or it may wirelessly report data to a secure central site. In the latter case these data can be combined with data from multiple sites to evaluate potential relationships among disease, geography, socio-economic status, etc.
- Possible advantages of the above described method include improved sensitivity of target molecule detection and improved detection of small quantities of target molecules.
- the above described method includes increased speed in detection of target molecules.
- the above described method can permit detection of target molecules without initial replication of the target molecules, such as in a PCR process.
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