JP2022532170A - RNA transcript analysis system and method - Google Patents

Abstract

A system and method for analyzing molecules in a sample. The system includes an imager, a flow cell, a magnet, and a processor. The flow cell includes a functionalized surface with capture probes configured to bind molecules comprising the first RNA transcript. A magnet is placed opposite the functionalized surface. The magnet is configured to direct a molecule containing the first RNA transcript toward the functionalized surface to bind with the capture probe. A light source is configured to direct light to a binding molecule comprising the first RNA transcript. An imaging device is configured to capture light from the binding molecule containing the first RNA transcript. A processor is configured to determine the amount of molecules in the sample that include the first RNA transcript. The processor is further configured to determine the level of expression of the first RNA transcript within the sample based on the quantity of the molecule.

Description

Mutual reference to related applications This application claims priority over US Provisional Patent Application No. 62/669575, entitled "Alzheimer's Screening Techniques and Devices" filed May 10, 2018, 5/2019. US Patent Application No. 16/408970 filed on 10th May 2016 and US Provisional Patent Application 62 / entitled "Automatic Nucleometric Detection and Quantification Using Photon Detection" filed October 26, 2016. International application filed on October 26, 2017, claiming priority over Specification 413144, filed on April 25, 2019, which is a national phase transition under Article 371 of Specification PCT / US2017 / 058559. Claims the interests and priorities of US Patent Application No. 16/345175, the entire contents of which are incorporated herein by reference.

The subject matter disclosed herein relates to detecting a target molecule, such as a nucleic acid molecule, and more specifically to a photodetection system for the target molecule.

Various methods have been developed to analyze biologic samples and detect the presence of target molecules such as nucleic acid molecules. These methods can be used, for example, to detect pathogens in a sample.

Usually, the detection method is to extract and replicate nucleic acid molecules from a sample using a disruption technique such as the polymerase chain reaction (PCR). PCR is a technique that allows replication and amplification to a sufficient amount for analysis of trace amounts of DNA fragments. Therefore, PCR can be used for various applications such as DNA sequencing and detection of DNA fragments in samples.

An electronic sensor that detects a particular target nucleic acid molecule may include a capture probe immobilized on the sensor surface between a series of electrode pairs. An example of a system and method for detecting a target nucleic acid molecule is described in US Pat. No. 7,645,574, which is incorporated herein by reference in its entirety. After PCR, an amplicon from the amplified product or target pathogen sequence is captured by the probe. Nanogold clusters functionalized with complementary sequences are used for local hybridization to amplicon. Subsequently, using short-term treatment with gold developer reagents, the nanogold clusters act as catalytic nucleation sites for metallization, which leads to the growth of sufficiently conductive membranes. The presence of the gold film is measured by shorting the gap between the electrodes and reducing resistance, which confirms the presence of captured amplification products. However, such sensors may not react to small amounts of target molecules, resulting in false negative results or poor detection of target molecules.

In one embodiment, an analytical system for molecules in a sample is presented. The system includes an image pickup device, a flow cell, a magnet, and a processor. The flow cell comprises a functionalized surface comprising a capture probe configured to bind a molecule containing a first RNA transcript. The magnet is located on the opposite side of the functionalized surface. The magnet is configured to direct the molecule containing the first RNA transcript towards the functionalized surface and bind it to the capture probe. The light source is configured to direct the light beam towards the binding molecule containing the first RNA transcript. The imaging device is configured to capture light from the binding molecule containing the first RNA transcript. The processor is configured to determine the amount of molecule in the sample containing the first RNA transcript. The processor is further configured to determine the expression level of the first RNA transcript in the sample based on the amount of the molecule.

In another embodiment, a method for analyzing molecules in a sample is presented. The method uses an image pickup device, a flow cell including a functionalized surface having a capture probe coupled to the functionalized surface, a magnet, and a light source. The method comprises the following steps. Binding a molecule containing a first RNA transcript to a magnetic particle. Aiming the molecule containing the first RNA transcript through a magnet towards the functionalized surface. Binding a molecule containing a first RNA transcript to a capture probe. Directing light from a light source to a binding molecule containing a first RNA transcript. Incorporating light from a binding molecule containing a first RNA transcript. To determine the amount of molecules in a sample containing a first RNA transcript. To determine the expression level of the first RNA transcript in a sample based on the amount of molecule.

In another embodiment, a method for analyzing molecules in a sample is presented. The method uses an image pickup device, a magnet, a light source, and a flow cell including a functionalized surface having a plurality of capture probes. Each of the capture probes is configured to bind molecules within a sample containing one of the RNA transcripts. The method comprises the following steps. Bonding molecules in a sample to magnetic particles. Use a magnet to direct the molecule to the functional surface. Binding each particular molecule of a molecule to one of multiple capture probes configured to bind to the RNA transcript of the particular molecule. Directing a light beam from a light source to a bound molecule bound to each of multiple capture probes. Capturing light from bound molecules. To determine the amount of bound molecule bound to each of multiple capture probes based on the captured light. To determine multiple expression levels for multiple RNA transcripts based on the amount of bound molecule bound to each of the multiple capture probes configured to bind each of the multiple RNA transcripts.

The above embodiments are merely exemplary. Other embodiments are within the scope of the disclosed subject matter.

A detailed description of the invention may be provided by reference to certain embodiments, some of which are shown in the accompanying drawings, so that the features of the invention can be understood. However, it should be noted that the drawings only show specific embodiments of the invention and therefore should not be considered a limitation of that scope, as the scope of the disclosed subject matter embraces other embodiments as well. I want to be. The drawings are not necessarily proportional to actual size and are generally focused on showing the characteristics of certain embodiments of the invention. In the drawings, similar numbers are used to indicate similar parts throughout the various figures.

FIG. 1 is a perspective view of a portable diagnostic assay system that functions to accept one of a plurality of disposable cartridges configured to inspect a fluid sample of a collected blood / food / bioform sample. FIG. 2 is an exploded perspective view of one of the disposable cartridges configured to inspect blood / food / bioform samples. FIG. 3 is one of a disposable cartridge showing various assay chambers, including a central assay chamber, one of which contains assay chemicals suitable for destroying the fluid sample to detect specific attributes of the tested fluid sample. It is a top view. FIG. 4 is a bottom view of the disposable cartridge shown in FIG. 3, showing various channels that function to send at least a portion of the fluid sample from one chamber to another in order to perform multiple operations on the fluid sample. .. FIG. 5 is a diagram of an embodiment of a sensor system having a functional surface. FIG. 6 is a flowchart showing an embodiment of a method for detecting a target molecule. FIG. 7 is a diagram of the sensor system of FIG. 5 having target molecules bound to magnetic particles. FIG. 8A is a cross-sectional view of an embodiment of magnetic particles. FIG. 8B is a cross-sectional view of another embodiment of the magnetic particles. FIG. 8C is a diagram of an embodiment of magnetic particles bonded to nanoparticles. FIG. 8D is a diagram of another embodiment of magnetic particles bonded to nanoparticles. FIG. 8E is a diagram of an embodiment of a target molecule bound to magnetic particles and nanoparticles. FIG. 8F is a diagram of another embodiment of the target molecule bound to the magnetic particles and nanoparticles. FIG. 8G is a diagram of still another embodiment of the target molecule bound to nanoparticles and magnetic particles. FIG. 9 is a diagram of the sensor system of FIGS. 5 and 7 having a target molecule bound to a functionalized surface. FIG. 10 is a diagram of the sensor system of FIGS. 5 and 7-8 with functionalized nanoparticles bound to the target molecule. FIG. 11 is a diagram of the sensor system of FIGS. 5, 7-8, and 10 having a light source directed at the functionalized nanoparticles. FIG. 12A is an embodiment of signs of scattering of 50 nm monodisperse nanoparticles on darkfield microscopy. FIG. 12B is an embodiment of a sign of scattering of 100 nm monodisperse nanoparticles on darkfield microscopy. FIG. 13 is a comparison of signs of scattering of grown nanoparticles and undergrown nanoparticles on darkfield microscopy. FIG. 14 is a diagram of an embodiment of an optical sensor system. FIG. 15A is an enlarged partial view of the optical sensor system of FIG. 14 with the magnet contracted. FIG. 15B is an enlarged partial view of the optical sensor system of FIG. 14 with the magnet extended. FIG. 16 is a side view of an embodiment of an optical device incorporating the optical sensor system of FIG. 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 having a target molecule bound to a functionalized surface. FIG. 17C is a diagram of the sensor system of FIGS. 17A-17B with nanoparticles bound to the target molecule. FIG. 18 shows a method for analyzing a plurality of RNA transcripts in a sample. FIG. 19 represents a method of determining the risk level of neurodegenerative disease by analyzing multiple RNA transcripts of a sample. FIG. 20 represents another embodiment.

Throughout several figures, similar reference characters indicate similar parts. The examples set up herein illustrate some embodiments, but should not be construed in a limited way in any way.

Described in US Patent Application No. 15/157584 by the same applicant entitled "Sample Preparation Methods and Systems" filed May 18, 2016, all of which is incorporated herein by reference. Disposable cartridges, such as those for portable / automated assay systems, are described. The main uses of disposable cartridges include DNA testing, but only to name a few of the various blood infectious diseases that are configured for portable / automated assay systems to detect disposable cartridges, blood, food. , Or biologics to detect any of the various diseases that can be found in hepatitis, autoimmune deficiency syndrome (AIDS / HIV), diabetes, leukemia, Basedow's disease, lupus, multiple myeloma, etc. May be good. Food diagnostic cartridges may be used to detect Salmonella, Escherichia coli, Staphylococcus aureus, or Shigella. Diagnostic cartridges may also be used to inspect samples from insects and samples. For example, the blood diagnostic cartridge may be a dedicated cartridge useful for animals to detect diseases such as malaria, encephalitis, and West Nile virus, to name just a few.

More specifically, with reference to FIGS. 1 and 2, the portable assay system 10 has specific attributes of a fluid sample, each of which may provide a blood, food, or biologic (animal-mediated) disease marker. Accommodates any of a variety of disposable assay cartridges 20 selectively configured for detection. The portable assay system 10 includes one or more linear and rotary actuators that function to move the fluid in and out of the various compartments or chambers of the disposable assay cartridge 20 for identifying or detecting the attributes of the fluid. More specifically, the cartridge 20 includes a flow cell 21 extending horizontally from the cartridge 20. The rotary actuator (not shown) of the portable assay system 10 aligns one of the various ports 18P arranged around the cylindrical rotor 18 with the syringe barrel 22B of the fixed cartridge body 22. The linear actuator 24 moves the plunger shaft 26 to develop the pressure in the syringe barrel 22, ie, positive or negative (vacuum). That is, the plunger shaft 26 moves the elastomer plunger 28 in the syringe 22 to deliver and / or mix the fluid contained in one or more of the chambers 30, 32. Further, the system 10 includes one or more processors 11 housed in a control panel 12 of the system 10 main body to receive signals from various components of the system 10.

The disposable cartridge 20 provides an automated process for preparing and / or performing fluid sample analysis for analysis. The sample preparation process allows for cell destruction, DNA and RNA sizing, and enrichment / purification of analytical material. More specifically, the sample preparation step of the present disclosure prepares fragments of DNA and RNA within a size range of about 100-10000 base pairs. The chamber can be used to deliver the reagents required for end repair and kinase treatment. The enzyme may be dried and rehydrated in the disposable cartridge 20 and stored, or may be added to the disposable cartridge 20 immediately before use. The introduction of rotary actuators allows the single plungers 26, 28 to aspirate and administer fluid samples without the need for a complex system of valves that open and close multiple times. Compared to traditional systems, this significantly reduces the potential for leaks and equipment failure. It will also be understood that the system greatly reduces the possibility of human error.

In FIGS. 3 and 4, the cylindrical rotor 18 includes a central chamber 30 and a plurality of assay chambers 32, 34 surrounded and separated by one or more radiation or circumferential walls. In the described embodiment, the central chamber 30 houses the fluid sample, while the surrounding chambers 32, 34 contain pretreated assay chemicals or reagents for detecting the attributes of the fluid sample. Chemicals or reagents may first be dried and rehydrated just prior to testing. Some chambers 32, 34 may be open to allow assay chemicals to be introduced while the assay procedure is in progress or in step. Chambers 30, 32, 34 are fluid connected, ie, one of chambers 30, 32, 34 from one of the ports 18P by channels 40, 42 formed along the lower panel 44, i.e. along the back surface of the rotor 18. Placed between the two. For example, the first port 18P corresponding to the opening 42 may be in fluid connection with the central chamber 30 via the opening 50.

FIG. 5 shows an embodiment of the sensor system 70. The sensor system 70 includes an image pickup device 72 configured to capture still images, moving images, or a combination thereof. For example, the image pickup device 72 may be configured to capture a high resolution still image. In the illustrated embodiment, the image pickup device 72 includes a pixel array 74 and an array circuit 76. The pixel array 74 may include any number of suitable pixels. For example, the pixel array 74 can be a high density array containing at least 6 megapixels. In a further example, the camera may have a wide field of view. The pixel array 74 is a photosensitive pixel array such as an active array, a passive array, a flat Fourier acquisition array, an angle sensitive array, a photodiode array, a charge coupling device, a complementary metal oxide semiconductor (CMOS), or a charge injection device. ..

The sensor system 70 also includes a flow cell 78. The flow cell 78 can be formed from a suitable material, such as 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 transparent surface 80 and a transparent functionalized surface 82. The functionalized surface 82 includes a plurality of captured probe 84 in the form of a functionalized oxide surface that allows attachment and fixation of the captured probe molecule 84 on the surface 82. The capture probe 84 is designed to capture or bind the target molecule 86 (FIG. 7) by interaction between complementary sequences. The target molecule 86 can be taken from a biologic sample. The bioform sample can be a suitable type of material, such as blood, mucus, and skin, among others. For example, the target molecule 86 can be a protein ligand or a DNA segment.

The objective lens or the lens 75 can be arbitrarily arranged between the image pickup device 72 and the flow cell 78. The magnet 88 can be placed on the opposite side of the functionalized surface 82. The magnet 88 can be a single magnet or an array of magnets.

In one embodiment, the functionalized surface 82 comprises an array of many different capture probes 84, each capture probe 84 configured to capture molecules with different RNA transcripts. Thus, for example, an array of probes 84 can be arranged to capture 10-20 different RNA transcripts. In another example, multiple probes 84 may be configured to capture molecules with the same RNA transcript, thereby providing redundancy that allows error checking of the results. Thus, an array of capture probes 84 can be designed to capture many different RNA transcripts and analyze the relative proportions of molecules in the sample that express those RNA transcripts. In another example, one or more of the array of probes 84 can be configured to capture the so-called housekeeping gene, which can serve as a baseline to normalize the number of other molecules found in other genes. .. As an example, when 400 molecules of a housekeeping gene are captured, the number of different capture molecules is compared to 400, giving an absolute sense of how many of those molecules were captured as a percentage compared to the housekeeping gene. And thus an absolute scale is established.

FIG. 6 shows an embodiment of a method 90 for detecting a target molecule. The method 90 can be used by a sensor system such as the sensor system 70. In block 92, as shown in FIG. 7, the target molecule 86 binds to the magnetic particles 110. In an embodiment, the target molecule 86 binds to the magnetic particles 110 before being introduced into the flow cell 78. In another embodiment, the target molecule 86 and the magnetic particles 110 are introduced into the flow cell 78 in a non-bonded state, and the target molecule 86 binds to the magnetic particles 110 in the flow cell 78.

8A-8B show two embodiments of the magnetic particles 110. As shown in FIG. 8A, in one embodiment, the magnetic particles 110A are composite particles having a magnetic core 112 and a nanoparticle coating 114 formed of a magnetic material such as iron. For example, the coating 114 can be a gold coating. The coating 114 may be configured to act as a nucleation site for the growth of additional nanoparticles. The magnetic particle 110A contains at least one binding site of the ligand A for binding to the target molecule 86. Chemical reactive groups such as thiols, amines, or aldehydes mediate or promote ligand bonds.

As shown in FIG. 8B, in another embodiment, the magnetic particles 110B may have a magnetic material 116 formed of a magnetic material such as iron. The magnetic particle 110B contains at least one binding site or ligand A for binding to the target molecule. In the illustrated embodiment, the magnetic particles 110B further include at least one binding site or ligand B for binding to the magnetic nanoparticles. It should be understood that the magnetic particles may contain suitable combinations of binding sites A, B. For example, the magnetic particles may contain both types of binding sites A and B, or the magnetic particles may contain only the target molecular binding site A.

As shown in FIG. 8C, rather than the nanoparticles coating on the magnetic core, the magnetic particles 110 may include a magnetic material 112 and a plurality of nanoparticles 122 bonded to the magnetic material 112. Alternatively, as shown in FIG. 8D, the magnetic particles 110 can be an alloy, such as a non-uniform alloy, comprising a plurality of magnetic bodies 112 bonded to the plurality of nanoparticles 122.

As shown in FIGS. 8E-8G, the target molecule 86, the magnetic particles 110, and the nanoparticles 122 can be bonded in various arrangements. As shown in FIG. 8E, the magnetic particles 110 and the nanoparticles 122 can be directly bound to the target molecule 86, respectively. Alternatively, as shown in FIG. 8F, the magnetic particles 110 can be directly attached to the target molecule 86, and one or more nanoparticles 122 can be attached to the magnetic particles 110. Alternatively, as shown in FIG. 8G, the nanoparticles 122 can be directly attached to the target molecule 86, and one or more magnetic particles 110 can be attached to the nanoparticles 122.

Returning to FIG. 6, in the block 94, the bound magnetic particles 110 and the target molecule 86 are directed and transferred to the functionalized surface 82. Referring to FIG. 7, the magnet 88 is coupled to an actuator (not shown) configured to move the magnet 88 closer to (shrink) (shrink) or away from (extend) the functional surface 82. .. 118 where the magnet 88 is separated from the functional surface 82 and 120 where the magnetic particles 110 attracted to the magnet 88 approach the functional surface 82. When the target molecule 86 binds to the magnetic particles 110, the magnetic particles 110 direct and attract the target molecule 86 to the functionalized surface 82.

Returning to FIG. 6, in block 96, the target molecule 86 binds to the capture probe 84 of the functionalized surface 82, as shown in FIG. In the illustrated embodiment, the magnetic particles 110 remain bound to the bound target molecule 86. Alternatively, when the target molecule 86 reaches the functional surface 82, the target molecule 86 may be denatured to unwind the magnetic particles 110 from the target molecule 86, and then the target molecule 86 may bind to the functional surface 82.

Returning to FIG. 6, in block 98, as shown in FIG. 10, the functionalized nanoparticles 122 are introduced into the flow cell 78 and bound to the target molecule 86. In embodiments, the functionalized nanoparticles 122 bind directly to the target molecule 86. Alternatively, the functionalized nanoparticles 122 bind to the magnetic particles 110 bound to their respective target molecules 86. In an embodiment, the plurality of functionalized nanoparticles 122 bind to their respective target molecules 86. Any suitable method of hybridizing or binding the nanoparticles 122 to the target molecule 86 can be used. In an embodiment, the functionalized nanoparticles 122 are gold particles. In another embodiment, the functionalized nanoparticles 122 are catalytic nanoparticles such as gold catalytic reagents. In embodiments, the nanoparticles 122 are in the form of catalytic clusters.

In the illustrated embodiment, the nanoparticles 122 bind to the target molecule 86 after the target molecule 86 binds to the functionalized surface 82. In another embodiment, the nanoparticles 122 may bind to the target molecule 86 or the magnetic particles 110 before the target molecule 86 binds to the functionalized surface 82.

After the nanoparticles 122 are bound to the target molecule 86, any metallization step can be performed to metallize the nanoparticles 122 to grow or form expanded nanoparticles or even a film. The grown nanoparticles can improve the detection of target molecules. In this metallization step, the nanoparticles 122 serve as nucleation sites for the growth of the enlarged nanoparticles 124.

Returning to FIG. 6, in the block 100, in the flow cell 78, the light source 126 directs the light beam 128 toward the target molecule 86 and the functionalized nanoparticles 122, 124. The light is directed exclusively at the target molecule 86 and the nanoparticles 122, 124, and the light beam 128 is directed so that the light 128 from the light source 126 is not captured by the image pickup device 72. In an embodiment, the flow cell 78 is configured to prevent the light beam 128 from diffusing into the image pickup device 72.

Referring to FIG. 6, in the block 102, the light 130 (FIG. 11) from the nanoparticles 122, 124, the target molecule 86, the magnetic particles 110, or a combination thereof is taken in by the image pickup apparatus 72. In embodiments, the light 130 can be reflected or emitted from the particles 86, 110, 122, 124, or a combination thereof. At block 104, the light 130 captured by the image pickup device 72 is analyzed to detect the number of target molecules 86 present. For example, the captured light 130 can be analyzed using darkfield microscopy. In this embodiment, the detected spots of light are calculated and quantified to determine the number of target molecules 86 present. Calculations and quantifications are accomplished using one or more processors 11 (see FIG. 1).

In one embodiment, as described above, a single image pickup device 72 and a ray 128 may be used, and the respective capture probes 84 are sequentially examined. Such a sequential analysis may be realized, for example, by providing a translation of the image pickup apparatus 72 and a ray 128 across the flow cell so that each capture probe 84 can be examined one at a time. In another embodiment, an adjustable mirror can be used, which adjusts the light to direct the light from the light beam 128 to one of the capture probes 84 and one at a time to the image pickup device 72. In a further embodiment, an image pickup device with higher imaging capability can be immobilized on a ray 128 moving one at a time to each capture probe 84. In other embodiments, a plurality of imaging devices 72, each specialized for one or more capture probes 84, can be used.

FIGS. 12A-13 show embodiments of light or scattering of gold nanoparticles 122 captured by darkfield microscopy, or captured images. FIG. 12A is a sign of scattering of 50 nm gold nanoparticles, and FIG. 12B is a sign of scattering of 100 nm gold nanoparticles. FIG. 13 is an image comparing the scatter sign 132 of the undergrown (20 nm) nanoparticles 122 with the scatter sign 134 of the grown (100 nm) nanoparticles 124. In embodiments, a series of darkfield images can be captured. In this embodiment, the first image can be captured before the nanoparticles 122 grow, and at least one additional image can be captured when the nanoparticles grow. Alternatively, the first image can be captured before the binding of the target molecule 86 and at least one additional image can be captured after the binding of the nanoparticles 122. The captured image can be compared with the removed background artifacts, which can improve the analysis of the darkfield image.

In another embodiment, dye particles (not shown) bind to the target molecule 86 for detection of the target molecule 86. In this embodiment, the light source 128 is tuned to the wavelength of the dye and the dye-covered region fluoresces. Fluorescence is detected by the image pickup device 72.

In another embodiment, the functionalized surface is exposed to a radiation source (not shown) after binding of the target molecule 86 to the nanoparticles 122 to detect the presence of the target molecule 86. When exposed to a radiation source, the nanoparticle region preferentially absorbs radiation and causes local heating. The local heating is captured and recorded in the image pickup apparatus 72, and the presence of the target molecule 86 is detected. Establish a calculation of the number of target molecules present based on the amount of heating recorded. For example, the system may be calibrated by heating a known number of target molecules and measuring their temperature.

An example of the optical sensor system 140 is shown in FIG. Similar to the optical sensor system 70 described above, the optical sensor system 140 includes an image pickup device 142 and an objective lens or lens 144 coupled to the image pickup device 142. In an embodiment, the image pickup device 142 is a high resolution image pickup device having a wide angle or a wide field of view. The objective lens 144 is directed at the flow cell 146. As mentioned earlier, the interior of the flow cell 146 contains a functionalized surface for binding the target molecule. One or more shunts 147 can be coupled to the flow cell 146 to facilitate the introduction of various particles into the flow cell 146.

The light source 148 is directed at the flow cell 146. The light source 148 can be a suitable light source. For example, the light source 148 can provide light of a predetermined frequency. For example, the light source 148 can be white light. The light source 148 is directed exclusively at the flow cell 146. In the illustrated embodiment, the light source 148 is oriented at right angles to the X-axis where the objective lens is located. The flow cell 146 is configured to direct the light from the light source 148 toward the particles in the flow cell 146 instead of the image pickup device 142, and prevent the light diffusion from the light source 148 to the image pickup device 142.

The magnet 150 is arranged from the objective lens 144 on the opposite side of the flow cell 146. An actuator 152 such as a solenoid is configured to be coupled to a magnet 150 to move the magnet. As shown in FIGS. 15A-15B, in embodiments, the actuator 152 is configured to retract or move the magnet 150 closer to or closer to the flow cell 146 (FIG. 15A) and to extend or detach the magnet 150 from the flow cell 146 (FIG. 15B). Magnet.

FIG. 16 shows an embodiment of an analysis system 160 including an optical system such as an optical sensor system 70, 140. In this embodiment, the analysis system 160 includes a base 162 and a head 164. The image pickup device 142 and the objective lens 144 are arranged in the base 162. The flow cell 146 is arranged on the upper surface of the base 162 so as to be aligned with the objective lens 144. The magnet 150 is arranged in the head 164 and is configured to extend to the flow cell 146 and mesh with the flow cell 146.

In the illustrated embodiment, the image pickup device 142 and the objective lens 144 are not along the axis as shown in FIG. 14 in order to minimize the footprint of the analysis system 160. On the contrary, the image pickup apparatus 142 is along the Y-axis extending longitudinally through the base 162 between the sides 166, 167 of the base 162. The objective lens 144 is arranged at right angles to the Y axis and extends upward through the base 162. The mirror 168 is arranged under the objective lens 144 and tilts with respect to the image pickup device 142 to form an optical path 170 between the objective lens 144 and the image pickup device 142.

17A-17C show another embodiment of the sensor system 180. In this embodiment, the sensor system 180 includes a prismatic substrate 182 with a Kletchman arrangement. The substrate 182 has a surface 184 coated with a metal film 186 suitable for surface plasmon resonance or Raman scattering. For example, the metal film can be gold, silver, copper, titanium, or chromium. Membrane 186 is functionalized with a biospecific coating and contains a capture probe 84. The light source 188 directs the light beam 190 toward the film 186 through the prism substrate 182, and the detector 192 captures the light 194 from the film 186, such as the light reflected or emitted by the film 186.

In the operation, the baseline measurement of the captured light is performed. In embodiments, baseline measurements of captured light are used to calibrate the absorbance angle (FIG. 17A). In addition, as described below, baseline measurements can be used to identify contaminants or debris on the sensor prior to binding of the target molecule 86 or growth of increased nanoparticle size. After baseline measurement, the sample containing the target molecule 86 is introduced into the sensor system 180 and the target molecule 86 binds to the capture probe 84 (FIG. 17B). In the embodiment, as mentioned above, the target molecule 86 can be directed at the face film 186 via the magnetic particles. The functionalized nanoparticles 122 are introduced into the system 180 and bound to the target molecule 86 (FIG. 17C). A plating bath can optionally be used to increase the size of the bound nanoparticles 122. In order to detect the presence of the target molecule 86, the light beam 190 is directed at the film 186 to capture the light 194 from the reflected or emitted film 186. To detect the presence of the target molecule 86, observe the difference in reflectance or intensity between the baseline measurement and the final measurement. A quantitative calculation of the target molecule 86 may be established by comparison with a calibration test of reflectance or intensity for a known number of target molecules. In embodiments, baseline measurements can be used to subtract particles identified as debris from the final measurement.

FIG. 18 represents a method of analyzing a plurality of RNA transcripts in a sample, 1800.

As a background, several techniques have made it possible to measure the expression levels of many intracellular transcripts at any given time (eg, Science et al., 1995, Complementary monitoring of genome expression patterns with a). ｃｏｍｐｌｅｍｅｎｔａｒｙ ＤＮＡ ｍｉｃｒｏ－ａｒｒａｙ， Ｓｃｉｅｎｃｅ ２７０：４６７－４７０； Ｌｏｃｋｈａｒｔ ｅｔ ａｌ．， １９９６， Ｅｘｐｒｅｓｓｉｏｎ ｍｏｎｉｔｏｒｉｎｇ ｂｙ ｈｙｂｒｉｄｉｚａｔｉｏｎ ｔｏ ｈｉｇｈ－ｄｅｎｓｉｔｙ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ａｒｒａｙｓ， Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ １４：１６７５－１６８０； Ｂｌａｎｃｈａｒｄ ｅｔ ａｌ．， １９９６， Ｓｅｑｕｅｎｃｅ ｔｏ ａｒｒａｙ： Proving the genome's secrets, Nature Biotechnology 14, 1649; 1996, see US Pat. No. 5,569,588, entitled "Pharmaceutical Screening Method," patented October 29, 1996 by Ashby et al.).

The application of transcript array techniques has involved the identification of genes that are up-regulated or down-regulated in various pathological conditions. The additional use of transcript arrays has included analysis of elements of signaling pathways and identification of targets for various agents. Transcript arrays can be useful in measuring the stage of a medical condition or therapeutic effect.

RNA profiling is a step that helps measure the pathology or therapeutic effect by measuring the expression level of key RNA transcripts in a sample. Techniques such as the Affymetrix Array can be used to identify key RNA transcripts and screen all intracellular transcripts. By profiling multiple patients with different stages, a series of key indicator transcripts can be identified and used in arrays such as the present invention targeting the key indicators. In addition, some housekeeping genes that do not change with disease are also measured to establish a baseline for comparison with the expression of key indicators. Statistical methods are used to determine which indicators are needed to reliably measure disease progression.

Many of these techniques involve large arrays of RNA probes that can measure the expression of thousands of genes at once, such as the Affymetrix array. Affymetrix manufactures quartz chips for the analysis of DNA microarrays, called gene chip arrays. Affymetrix gene chip arrays help researchers quickly determine the presence of specific genes in biological samples. Within this area, Affymetrix focuses on oligonucleotide microarrays. These microarrays are used to determine what genes are present in a sample by detecting a specific portion of mRNA. A single chip can be used to analyze thousands of genes in a single assay. However, implementing these systems is costly and their use may be limited to measuring medical conditions and therapeutic effects.

RNA for analysis with current technology is obtained 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. be able to. In various embodiments, the cartridge has the ability of automated sample destruction and RNA isolation. Advantageously, automation of all sample processing improves the certainty of RNA isolation without degradation. In addition, automation of sample processing allows for inspection outside the old-fashioned laboratory.

As an illustration, the methods and systems described with respect to FIG. 18 provide systems and methods for determining or measuring the progression of a condition or the effectiveness of a treatment in a subject, preferably a human patient. In particular, the technique relates to a method of measuring a pathological condition or therapeutic effect by measuring changes in mRNA expression levels. Current techniques utilize a simple and easy-to-use system that can measure the expression of many gene transcripts for rapid diagnosis to enable better treatment. The present disclosure provides systems and methods for RNA profiling to measure the efficacy of a medical condition or treatment. Identification of changes in gene expression caused by treatments such as pathogenic effects or dosing regimens for the pathology is a crucial issue for commerce and humans. Much of the decisions needed to conduct efficient clinical trials and properly manage patient health rely on assays to measure changes in cells in the body.

The system provides an automated and closed system for isolating RNA from patient samples. RNA is difficult to handle and is susceptible to degradation by RNase. The closed system automates sample destruction, RNA washing and enrichment. During isolation, the sample is treated with a guanidine hydrochloride solution that lyses cells and destroys the enzyme, thereby stabilizing RNA. By automating the process within the closed cartridge, the risk of introducing RNase into the sample during handling is eliminated.

The system further provides multiple arrays for measuring expression levels of multiple RNA transcripts. As described below, the sensor array has multiple sites specific for each transcript. A capture probe specific for the nucleotide sequence in the transcript is printed on the site. Multiple arrays enable quantification of transcript expression, which is a key indicator, and measurement of housekeeping genes to establish baseline expression. The system then compares the expression of the key indicator gene with the expression of the housekeeping gene to determine its expression relative to the baseline. Algorithms are used to analyze changes in key gene expression to determine pathology.

Rather than measuring all intracellular transcripts that require thousands of sensors, the invention provides an array that measures key transcripts between one and hundreds. It also includes additional sensors for one to dozens of housekeeping genes. Optimally, the number of key transcripts measured is 3-20. Reducing the number of sites minimizes the cost of the array, but allows targeted testing of key disease indicators.

The present invention has important functions required for rapidly capturing RNA and quantifying RNA expression levels. In particular, the present invention provides a magnetic approach that concentrates RNA and moves it to the sensor, enabling rapid testing without loss of sensitivity. In addition, the system provides a method for quantifying RNA transcripts at each sensor.

Returning to FIG. 18, a description of how these features are performed is provided. As an example, method 1800 includes an image pickup device (eg, the image pickup device 72 of FIG. 5 or another example shown herein), a functionalized surface (eg, a functionalized surface 78 of FIG. 5 or another example shown herein). A flow cell with a functionalized surface having a plurality of capture probes bound to (eg, probe molecule 84 of FIG. 5 or other example shown herein) (eg, flow cell 78 of FIG. 5 or other example shown herein). , A magnet (eg, magnet 88 of FIG. 5 or other example shown herein), and a light source (eg, light source 126 of FIG. 6 or other example shown herein) and a sensor (eg, the sensor system of FIG. 5). 70 or other examples shown here) may be used. In the embodiment of FIG. 18, the method begins with block 1801 and comprises the following steps:

Step 1810-Binding a molecule containing a first RNA transcript to a magnetic particle.

Step 1820-To direct the molecule containing the first RNA transcript to the functionalized surface via a magnet.

Step 1830-Binding the molecule containing the first RNA transcript to the capture probe.

Step 1840-Directing a ray from a light source to a bound molecule containing a first RNA transcript.

Step 1850-Incorporating light from a binding molecule containing the first RNA transcript.

Step 1860-Determining the amount of molecules in the sample containing the first RNA transcript.

Step 1870-Determining the expression level of the first RNA transcript in the sample based on the amount of molecules.

In one embodiment, Method 1800 further comprises calculating the pathology or therapeutic effect based on multiple expression levels of multiple RNA transcripts. In other embodiments, the plurality of capture probes corresponds to 10-20 RNA transcripts. In a further embodiment, the method further comprises measuring a housekeeping gene to determine a baseline for comparing multiple expression levels of multiple RNA transcripts.

In one example, the method further comprises accommodating the sample and processing it without external exposure. In another example, the method further comprises binding a plurality of magnetic particles to a molecule and allowing a magnet to interact with the magnetic particles to direct the molecule to a functionalized surface. In another example, the method further comprises preventing the diffusion of light rays into the image pickup device. In a further example, the method further comprises binding the molecule to the nanoparticles when the molecule binds to the functionalized surface, which reflects the light beam to the imager.

FIG. 19 represents a method of determining the risk level of neurodegenerative disease by measuring the expression level of multiple RNA transcripts of a sample.

As a background, in the diagnosis of Alzheimer's disease addressed by the technique proposed in this patent application, there are two challenges: 1) accuracy of diagnosis of symptomatic patients, and 2) early detection of disease in asymptomatic patients. There is.

Problem 1 is the accuracy of diagnosis. The number of diseases that can cause cognitive impairment that may appear to be Alzheimer's disease spans long pages. While some of these disorders, such as vitamin B deficiency, can be cured, others may not be cured even if managed with appropriate intervention. Accurate diagnosis is required for curative or promising management. Unfortunately, studies have established that the diagnosis of Alzheimer's disease is inaccurate. When diagnosed by a general practitioner, there is a 50% chance that the diagnosis is correct (Connolly, A., Gaehl, E., Martin, H., Morris, J., Planandare, N., 2011. Under. diagnosis of diagnosis in prevalence (variations in the observed prevalence and comparisons to the exposed prevalence. Aging Men. 98). When diagnosed by one of the 30 federal government-recognized and financially funded Alzheimer's centers in the United States, the accuracy is about 75% (Beach, TG, Monsell, SE, Phillips). , LE, Kukull, W., 2012. Accuracy of the clinical diagnosis of Alzheimer disease at National Instrumente on The system proposed here will increase the accuracy of diagnosis by more than 90%.

Problem 2 is early detection of illness. We now understand that Alzheimer's disease, Parkinson's disease, and many other age-related neurodegenerative diseases begin decades before reaching the clinically detectable stage of brain damage. There is. In Parkinson's disease, 80% of the neurons in the substantia nigra, the most susceptible area of the disease, are lost before the symptoms that lead to the diagnosis of the disease appear. Examination of Alzheimer's disease lesions in more than 3000 brains of people who died between the ages of 10 and 100 showed early Alzheimer's lesions in 20% of the brains of people who died in their late 20s and early 30s. (Braak, H., Braak, E., 1997. Frequency of lesions of Alzheimer-relatated leases in diffuse categories. Neurobiol. 35, Age1. Twenty percent of people will not be clinically diagnosed with Alzheimer's disease by 50 years.

This "silent period" for many age-related neurodegenerative diseases creates a great opportunity for a two-step process. Step 1 makes it possible to detect the disease before serious brain damage occurs. Step 2 allows the sick person to spend the rest of his life without symptoms of the illness, for example, with Parkinson's disease without tremors and impaired motor function, or with Alzheimer's disease without memory and cognitive deficits. Demands early and effective treatment to stop or slow the progression of the disease. This application addresses step 1, that is, accurate and early diagnosis.

Various methods have been used to show that early diagnosis of Alzheimer's disease is feasible. Positron emission tomography (PET) scans of the brains of survivors showed Alzheimer's lesions in some young people, such as teens or twenties. Microscopic pathological examination of more than 3000 brains of people who died between the ages of 10 and 100 showed the onset of Alzheimer's disease in people in their twenties. And advanced cognitive tests were able to establish signs of Alzheimer's disease 15 years before the disease became clinically detectable (REF) (Kawas, CH, Corrada, M.M., Brookmeyer). , R., Morrison, A., Resnick, SM, Zonderman, AB, Arnberg, D., 2003. Visual memory predicts Alzheimer's disease .). These studies are the basis for establishing that Alzheimer's disease exists in the brain for decades before it is damaged to the point where it presents with overt memory and cognitive problems. However, the cost of the study or the fact that the study relies on post-mortem samples makes the study very expensive for the detection of the disease or the potential for future illness in the general population.

There are some problems with the current state of the technology. Several techniques have been used to establish the presence of changes in the brain that precede the clinical diagnosis of Alzheimer's disease for years or even decades. Some of these techniques require sophisticated equipment and costly expertise. PET imaging is a prime example of this, with PET scans costing on the order of $ 5,000. This is too high to apply to mass screening, which is necessary for the detection of early Alzheimer's disease.

Another technique currently in use is the analysis of proteins in the cerebrospinal fluid, which requires an invasive procedure for spinal puncture performed by a physician. The cost of this procedure can also be on the order of thousands of dollars.

Many studies have reported that extensive cognitive testing can reveal early and presymptomatic signs of Alzheimer's disease. Most of these studies require valuable time and considerable cost on both sides of the patient and the examiner.

In addition to recognizing these costs and other shortcomings, the need to improve diagnostic accuracy and presymptomatic disease detection led to the search for Alzheimer's disease and the explanation of blood tests for Alzheimer's disease. Some of these are towards the detection of already obvious diseases. Some also include the ability to detect presymptomatic illnesses. At present, everything requires sending samples to a central laboratory for relatively expensive and time-consuming procedures. The system proposed here can test for Alzheimer's disease or other neurodegenerative diseases quickly and at minimal cost in the field.

We present here how to achieve detection of a disease that is low cost, minimally invasive, and therefore practical for use in large numbers of people, or a diagnosis of potential disease in the future. Detection of early-stage disease, perhaps in populations over the age of 30, requires a minimally invasive, low-cost method that does not require specialist personnel. We present here how to achieve these goals. It is a way to provide a more accurate diagnosis of people with symptoms of Alzheimer's disease and to determine a person's risk for future diagnosis of Alzheimer's disease. The present application is a method of obtaining a blood sample and a method of introducing the sample into a device capable of determining the expression level of multiple RNA species that have been confirmed to be useful for multivariate analysis for diagnosing and predicting Alzheimer's disease. Will be described. The method described can provide diagnostic or future diagnostic potential in the field within about 30 minutes at a cost estimated at less than $ 100. The system presented can be applied to screen a large population of people who are asymptomatic but may be at risk for future diagnosis of Alzheimer's disease.

Returning to FIG. 19, the method 1900 is an image pickup device (eg, the image pickup device 72 of FIG. 5 or another example shown herein), a functionalized surface (eg, a functionalized surface 78 of FIG. 5 or other examples shown herein). A flow cell with a functionalized surface having a plurality of capture probes (eg, probe molecule 84 in FIG. 5 or other examples shown herein) attached to an example) (eg, flow cell 78 in FIG. 5 or other shown herein). Example), a sensor with a magnet (eg, magnet 88 of FIG. 5 or other example shown herein), and a light source (eg, light source 126 of FIG. 6 or other example shown herein) (eg, FIG. 5). It may be performed using the sensor system 70 or other examples shown herein).

In the embodiment of FIG. 19, the method begins at block 1901, including the following steps.

Step 1910-Binding the molecules in the sample to the magnetic particles.

Step 1920-Molecules are directed to the functional surface using magnets.

Step 1930-Binding each particular molecule of a molecule to one of a plurality of capture probes configured to bind to the RNA transcript of the particular molecule.

Step 1940-Directing a ray from a light source to a bound molecule bound to each of multiple capture probes.

Step 1950-Capturing light from bound molecules.

Step 1960-Determining the amount of bound molecule bound to each of the multiple capture probes based on the captured light.

Step 1970-Determining multiple expression levels for multiple RNA transcripts based on the amount of bound molecule bound to each of the multiple capture probes configured to bind each of the multiple RNA transcripts. ..

Step 1980-Calculating the risk of neurodegenerative disease based on multiple expression levels of multiple RNA transcripts.

In one embodiment, Method 1900 further comprises calculating the risk of diagnosis of future neurodegenerative diseases. In another embodiment, Method 1900 further comprises calculating the risk of diagnosis of current neurodegenerative diseases.

In a further embodiment, calculating the risk of step 1980 of the method comprises using an equation of the form F = a ₀ + a ₁ X ₁ + a ₂ X ₂ + ... + a _p X _p + e, where F is proportional to risk, p is the number of multiple RNA transcripts, X _n for n = 1 to p is the expression level of each of the multiple RNA transcripts, and an for _n = 1 to p is multiple. It is a discrimination coefficient for each RNA transcript, and e is an error term.

In a specific embodiment, the plurality of RNA transcripts comprises eight DNA transcripts, HSP27, HSP90, GAPDH, FTH, FTL, COX1, COX2, and TFR for n = 1-8, and the plurality of RNA transcripts. The discriminant coefficient an for _n = 1 to 8 is -4.25936, 3.671572, 2.685682, -5.295300, 1.973631, 2.506241, 0.495803, and -1.392785. include.

In one example, Method 1900 further comprises binding a plurality of magnetic particles to a molecule and interacting a magnet with the magnetic particles to direct the molecule to a functionalized surface. In another example, method 1900 further comprises preventing the diffusion of light rays into the image pickup device. In a further example, method 1900 further comprises binding the molecule to the nanoparticles when the molecule binds to the functionalized surface, the nanoparticles reflecting the light beam to the image pickup device.

Further implementation details are described in Detailed Examples of the Technique.

In summary, our basic laboratory studies have quantified the expression of multiple nucleotide RNA species in the sample and then combined these data to estimate disease diagnosis or to diagnose future diseases. Established the possibility of producing numbers that provide possibilities. In the latter case, the endpoint is a biomarker of the disease for which a sufficient amount is present to distinguish it from the disease-free state but is not yet at a level indicating a clinically diagnosable disease. In one example, the systems and methods presented herein are used to determine the amount of 10-20 RNA species in each sample. The RNA species investigated will be selected based on previous and ongoing studies. Then multiply the expression level of each of these genes by a number. The numbers are based on previous and ongoing studies and will vary with each RNA species. The multiplier for the same RNA species is the same for all samples. The resulting product of 10 to 20 multiplications is then combined by an algorithm to form a single number indicating the possibility of diagnosing Alzheimer's disease or the possibility of diagnosing Alzheimer's disease in the future.

Details on how to get a blood sample. Blood is drawn from a person for the purpose of determining the risk of diagnosis or future diagnosis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and frontotemporal dementia. Blood is usually taken from the median cubital vein, but may be taken from any vein or placed in a syringe. Alternatively, blood may be obtained by collecting blood with the finger pad. When collecting from the finger, discard the first drop, then squeeze the finger and collect blood in Whatman P Card. The blood is dried overnight at room temperature and then the sample is stored at −20 ° C. Using the Qiagen kit of your choice, we used QiAmp and exoRNeasy to isolate RNA from filter paper. Work on dry ice when transporting samples. For example, a blood sample of 2.5 ml or less may be obtained from a person using a syringe and a needle or a similar device from any vein. Alternatively, a small amount of blood (2 to 3 drops) may be obtained by collecting blood with a finger pad using a disinfecting needle, or by another device that achieves blood collection with a finger pad or a blood source other than the finger. In the case of "blood sampling from the finger pad", blood is collected on filter paper or an equivalent. The sample is given an identification alphanumerical name that tracks the sample through all further processing. RNA is then extracted from the sample using one of several methods that may use trizol, PAXgene tubes, phenol / chloroform and the like. As an example, the procedure for collecting blood in a PAXgene tube is as follows.

Blood collection procedure using PAXgene blood RNA tube

Make sure the PAXgene blood RNA tube is at 18 ° C to 25 ° C before use and is properly labeled with the patient ID or code.

If the PAXgene blood RNA tube is the only tube used for blood collection, blood must be collected in a "disposal tube" before blood is collected in the PAXgene tube. Otherwise, the PAXgene tube should be the last tube to be collected in the phlebotomy procedure.

Blood is drawn into a PAXgene tube according to your facility's recommended procedure for standard venipuncture.

Keep the PAXgene blood RNA tube vertical under the arm of the blood donor during blood sampling.

Allow at least 10 seconds for a complete blood sampling. Before removing the tube from the holder, make sure that blood has stopped flowing into the tube.

After blood sampling, gently invert and mix the PAXgene tube 8-10 times.

Store the PAXgene tube in an upright position.

The tube can also be placed at room temperature for 2 hours and then at −20 ° C., or upright on a wire mesh stand immediately after blood collection.

Storage and transportation procedures

To freeze the PAXgene tube, stand the tube upright on a wire mesh stand. Do not freeze the tube upright on a styrofoam tray as it will cause the tube to crack.

For long-term storage at -80 ° C, the tubes must be stored at -20 ° C for at least 24 hours before being placed in the -80 ° C freezer.

In order to transport the tube on dry ice, the tube should be frozen at −20 ° C. for at least 24 hours before being placed on dry ice.

Note: Frozen PAXgene blood RNA tubes are vulnerable to impact. Frozen tubes should be treated in the same way as glass tubes to reduce the risk of breakage in transit. It is recommended to wrap the tube with a bubble wrap or other type of treatment for protection. For research purposes, when thawing a PaxGene tube at room temperature, the tube must be miscible at least 6 turns to completely mix the contents. Blood can then be drawn from the PaxGene tube and introduced directly into the cartridge supplied by INT.

An example of a method for isolating RNA from blood collected in an EDTA tube is as follows. RNA is extracted from leukocytes by the manufacturer's procedure using an mRNA isolation kit for blood / bone marrow (Roche). In short, red blood cells are selectively lysed and collected by centrifugation. Then, leukocytes are lysed, and all nucleic acids are recovered by non-specific adsorption to magnetic beads and magnetic separation. After a series of washes and elution of nucleic acids, the mRNA is captured by biotin-labeled oligo (dT) and streptavidin-coated magnetic particles. After removal of other nucleic acids (DNA, rRNA, and tRNA) by washing, mRNA samples are collected and stored at -80 ° C. The quality and quantity of RNA is confirmed by a 260/280 ratio and gel electrophoresis. Messenger RNA is amplified and radioactively labeled with 32P CTP. The labeled amplified RNA is hybridized and quantified to a custom cDNA array.

An example of a method for isolating RNA from blood collected in a PAXgene tube is as follows. Approximately 2.5 mL of fresh whole blood is collected in a PAXgene blood RNA tube (BD diagnostics / Qiagen) and mixed in 4 turns. All RNA is extracted from leukocytes using the PAXgene blood RNA kit (Qiagen) or the PAXgene blood miRNA kit (Qiagen) as directed by the manufacturer. All RNA is stored at -80 ° C until later use. RNA integrity is determined by analysis using an Agilent 2100 bioanalyzer. The suitability of the sample for analysis is based on RNA Integrity Number, and it is considered that rare samples with RINs less than 2 cannot be used.

An example of a method for converting RNA into cDNA is as follows. Reverse transcriptase 1.0 μg of all RNA is performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNA was diluted 1: 5 and used with TaqMan® Gene Expression Master Mix® and TaqMan® Gene Expression Associates®, iCyler iQ®. It is used as a template in the qRT-PCR reaction performed in Biorad) or an equivalent device. Quantitative RT-PCR amplification is performed 3 times under thermal cycle conditions of 10 minutes at 95 ° C., followed by 40 cycles of denaturation at 95 ° C. for 15 seconds and annealing and elongation at 60 ° C. for 1 minute. Beta-glucuronidase (GUSB) is used as an endogenous control as it is known to be the same between samples. After normalization, the data are shown as ratios using the 2 (-Delta Delta C (T)) method (Livek and Schmittgen, 2001).

In one embodiment, if blood is obtained from a vein, 2.5 ml is collected in a PaxGene tube or equivalent, followed by introduction of blood into the cartridge of the system for internal preparation, according to a typical procedure.

In one embodiment, the following RNA species may be used to calculate the risk of neurodegenerative disease. RNA species for study are selected based on known mechanisms of neurodegenerative diseases and experimental results in preliminary studies. Some examples are shown in Table 1.

The weight assigned to each independent variable is corrected for the interaction between all variables. The weight is called the discrimination coefficient. The instrument accurately quantifies the amount of RNA in a sample and detects the presence and amount of each of a number of specific RNA or DNA species. In the examples given, eight specific RNA species have been identified, but the identities, specific species, and numbers thereof may differ. Information about the amount of each selected RNA or DNA species in the sample may be transmitted to an external device such as a mobile phone or other portable computing device. Alternatively, the value may be transmitted to an arithmetic circuit in the same device. The amount detected by the sensor element for each species is multiplied in the arithmetic unit by a weight specific to the RNA or DNA species. The weight may be referred to as a standardized discrimination coefficient (abbreviation: COX1, prostaglandin endoperoxide synthase 1; COX2, prostaglandin endoperoxide synthase 2; FTH, ferritin, heavy chain polypeptide; FTL, ferritin, light chain poly). Peptides; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSP27, heat shock 27 kDa protein 1; HSP90, heat shock protein 90 kDa, class B1; TFR, transferrin receptor).

In this example, this producting method yields eight values that represent the product of the amount of nucleotide species in the sample, multiplied by the weight. The weights may be the result of prior research or the result of introducing into the system a method of determining weights based on the known identities of the samples submitted to the system. In the examples given, these eight products of the amount of nucleotides weighted for each nucleotide are combined by an algorithm that yields a single number that represents the characteristics of the sample introduced into the system. And this number represents the possibility of detecting current or future diagnostic potential for Alzheimer's disease or other neurodegenerative diseases such as Parkinson's disease and amyotrophic lateral sclerosis. The resulting numbers may represent the possibility of diagnosing a neurodegenerative disease or clinically diagnosing the disease at some point in the future. This information can be shown on the display of the device itself, a device such as a local or remote computer, and the data may be transmitted wirelessly to the secure host computer side. In the latter case, these data can be combined with data from multiple locations to assess potential relationships between illness, geography, socioeconomic status, and so on.

The purpose of discriminant analysis is to obtain a model that predicts a single qualitative variable from one or more independent variables. Discriminant analysis derives an expression as a combination of independent variables that best discriminates between groups within a dependent variable. This combination is known as a discriminant function. The weight assigned to each independent variable is corrected for the interaction between all variables. The weight is called the discrimination coefficient.

The discriminant is F = a ₀ + a ₁ X ₁ + a ₂ X ₂ + ... + a _p X _p + e.

Here, F consists of a linear combination of dependent variables, X ₁ , X ₂ , ... X _p is an independent variable of p, e is an error term, and a ₀ , a ₁ , a ₂ , ...・_Ap is a discrimination coefficient.

As described in Table 2, the normalization discrimination coefficient is calculated for each of the eight transcripts.

By comparison, examples of quantifying the amount of specific RNA species in a sample using cDNA or nucleotide arrays are: The cDNA clones represented in the array highlighted cDNA clones that test the hypothesis that stress, inflammation, and cell cycle-related transcripts are affected in leukocytes in the case of Alzheimer's disease. The 172 cDNAs selected for this purpose were printed on a nylon membrane using a 96-pin replicator (Narujununk) with four of each cDNA arranged. The array was probed with labeled amplified RNA generated from RNA extracted from leukocyte samples and exposed to a stored phosphor screen. The hybridization intensity of each spot is quantified by laser densitometry scanning (Phosphor Imager, molecular dynamics). As a control, the amount of cDNA deposited at each spot in the array is quantified by stripping the membrane and reprobing the membrane with T7 promoter-specific oligonucleotides present in all vectors. The relevant aspects of this example may be combined with the other examples shown here.

By comparison, examples of quantifying the amount of specific RNA species in a sample by quantitative reverse transcriptase-polymerase chain reaction (qPCR) are as follows. qRT-PCR may be performed using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). In short, 3.0 μg of all RNA is reverse transcribed for each sample using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). 2 μL of the 1: 5 diluted cDNA is mixed with TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems) and TaqMan Gene Expression Assay in a 10 μL reaction setting with a CAS-1200 liquid treatment system. The qRT-PCR amplification may be performed with an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). Common thermal cycle conditions are 40 cycles of denaturation at 95 ° C. for 15 seconds and annealing and elongation at 60 ° C. for 1 minute after 10 minutes at 95 ° C. Amplification efficiency is close to 100% for all assays by analysis of different dilutions of cDNA. Beta-glucuronidase (GUSB) is used as an endogenous control as expression is known to be invariant throughout all samples. After data normalization, the data may be shown as a ratio using the 2 (-Delta C (T)) method (Livek and Schmittgen, 2001). The relevant aspects of this example may be combined with the other examples shown here.

An example of the analysis result is shown in Table 3.

The table above shows the results of quantitative PCR analysis of COX1 in 8 non-dementia cases (ND) and 8 Alzheimer's cases (AD) (upper left corner). Each number below the "Sample" column is a non-specific number given to each case tracked throughout all analyses. The "Situation" column indicates whether the case is ND or AD. The column named COX1-GUSB has a mathematical relationship with the qPCR signal-derived invariant signal to beta-glucuronidase (GUSB), an enzyme used as an endogenous control because its expression is invariant throughout all samples. It is the result of qPCR of the signal intensity derived from qPCR of COX1. The next column, named "std Dev", is the standard deviation of each measurement in the above column, based on the fact that each measurement is performed three times. The column named "Ratio COX1 / GUSB" is the ratio of the formula for the ratio of COX1 and GUSB expression to each case. The column "COX1 for calibrator" is arbitrarily set to 1 for the purpose of comparison with the value of the AD case (similar to the column named "magnification change").

The columns for AD (Alzheimer's disease) cases are named "COX1 for calibrators" and "magnification change", which represent the relationship between the mean values for ND (non-dementia) data and AD (Alzheimer's disease) data. The same is true except for the columns. Appendix I contains all correspondence tables for each RNA species in this demonstration. Then, these data are multiplied by the determined weights for each RNA species.

FIG. 20 represents another embodiment for determining the risk of future diagnosis of Alzheimer's disease by being homozygous for APOE4 ++. In this example, we are concerned about the risk of future diagnosis of AD in people who are currently cognitive. Having two copies of the APOE4 gene (one from each parent-APOE4 ++) poses a significant risk for future diagnosis of Alzheimer's disease. The bar graph below shows our blood test scores for people who are all cognitive. Those who have two copies of APOE4 (APOE4 ++ ND) and are at high risk of future diagnosis of Alzheimer's disease have a score of those who do not have the APOE4 gene variant and are not at high risk of future diagnosis of Alzheimer's disease. It is clearly distinguished from the score of.

In summary, the discriminant numbers produced by our analysis may represent a diagnosis of neurodegenerative disease in symptomatic patients, and in the case of cognitively healthy controls, a clinical diagnosis of the disease at some point in the future. Can represent the possibility of. This information can be shown on the display of the device itself, a device such as a local or remote computer, and the data may be transmitted wirelessly to the secure host computer side. In the latter case, these data can be combined with data from multiple locations to assess potential relationships between illness, geography, socioeconomic status, and so on.

Promising advantages of the above method include improved sensitivity of target molecule detection and improved detection of small amounts of target molecule. Further, the above method includes improving the detection rate of the target molecule. The above method allows detection of a target molecule, for example, without the initial replication of the target molecule, such as in a PCR step.

Although the present invention has been specifically shown and described with reference to specific preferred embodiments, those skilled in the art will be able to detail various modifications without departing from the spirit and scope of the invention which may be supported by the specification and drawings. Will be understood. Further, as the preferred embodiment is described with reference to certain elements, it will be appreciated that the preferred embodiment can be implemented with less than or more elements.

The patentable scope of the present invention is defined by the claims and may include other embodiments conceived by those skilled in the art. Such other embodiments are claimed if they have structural elements that are not different from the literal words of the claim, or if they contain structural elements that are not substantially different from the literal words of the claim and are equivalent. It is intended to be within the scope of the terms.

As long as the claims describe the expression "at least one of" for multiple elements, this description is intended to mean at least one of the listed elements and is limited to at least one of each element. do not do. For example, "at least one of element A, element B, and element C" is intended to indicate element A only, element B only, element C only, or any combination thereof. "At least one of element A, element B, and element C" is not intended to be limited to at least one of element A, at least one of element B, and at least one of element C.

A Target molecule binding site B Nanoparticle binding site X Axis Y Axis 10 Portable assay system 18 Rotor 18P Port 20 Disposable assay cartridge 21 Flow cell 22 Cartridge body 22 B Syringe barrel 24 Linear actuator 26 Plunger shaft 28 Elastomer plunger 30 Central chamber 32 Assay chamber 34 Assay Chamber 40 Channel 42 Channel 44 Bottom Panel 50 Opening 70 Sensor System 72 Imaging Device 74 Pixel Array 75 Lens / Objective Lens 76 Array Circuit 78 Flow Cell 80 Surface 82 Functionalized Surface 84 Capture Probe 86 Target Molecule 88 Magnet 90 Method 92-104 Step of Method 110 Magnetic Particle 110A Magnetic Particle 110B Magnetic Particle 112 Magnetic Core 114 Nano Particle Coating 116 Magnetic Body 118 Movement 120 Movement 122 Nano Particle 124 Enlarged Nano Particle 126 Light Source 128 Light 130 Light 132 Signs of Scattering (Image)
134 Signs of scattering (image)
140 Optical sensor system 142 Imaging device 144 Objective lens / lens 146 Flow cell 147 Minute wiring 148 Light source 150 Magnet 152 Actuator 160 Analysis system 162 Base 164 Head 166 Side 167 Side 168 Mirror 170 Optical path 180 Sensor system 182 Prism substrate 184 Surface 186 Film 188 Light source 190 Ray 192 Detector 194 Light

Claims (17)

An analysis system for molecules in a sample
Imaging device and
A flow cell with a functionalized surface with a capture probe configured to bind a molecule containing a first RNA transcript, and
A magnet located on the opposite side of the functionalized surface and configured to direct the molecule containing the first RNA transcript towards the functionalized surface and bind to the capture probe.
A light source configured to direct a light beam toward the binding molecule containing the first RNA transcript and the imaging device to capture light from the binding molecule containing the first RNA transcript.
With a processor configured to determine the expression level of the first RNA transcript in the sample based on the amount of the molecule in the sample containing the first RNA transcript and the amount of the molecule. , A system. The system according to claim 1, wherein the system is further configured to calculate a pathological condition or therapeutic effect based on the expression level of the first RNA transcript. The functionalized surface comprises a second capture probe configured to bind a molecule containing the second RNA transcript, the light source directing other light to the binding molecule containing the second RNA transcript. The imaging device is configured to direct and capture light from the binding molecule containing the second RNA transcript, and the processor is configured to capture the molecule in the sample containing the second RNA transcript. The system of claim 1, configured to determine the amount of. The system of claim 1, wherein the system comprises a cartridge for accommodating the sample and the sample is processed within the system without being exposed to the outside. The first aspect of claim 1, wherein the plurality of magnetic particles are configured to bind to the molecule in the sample, and the magnet interacts with the magnetic particle to direct the molecule to the functionalized surface. system. Each of the binding molecules, including the first RNA transcript, is configured to bind to the nanoparticles when bound to the functionalized surface, and the nanoparticles are configured to reflect the rays to the imaging device. The system according to claim 1. The system according to claim 5, wherein the nanoparticles are gold nanoparticles. The system of claim 5, wherein the nanoparticles are configured to act as nucleation sites for the growth of further expanded nanoparticles. The system according to claim 1, further comprising a lens arranged between the image pickup apparatus and the flow cell. A method for analyzing molecules in a sample using an image pickup apparatus, a flow cell having the functionalized surface having a capture probe coupled to the functionalized surface, a magnet, and a light source.
Binding a molecule containing a first RNA transcript to a magnetic particle,
Directing the molecule containing the first RNA transcript towards the functionalized surface via the magnet.
Binding the molecule, including the first RNA transcript, to the capture probe.
Directing light from the light source to the binding molecule containing the first RNA transcript,
Incorporating light from the binding molecule, including the first RNA transcript,
Determining the amount of the molecule in the sample containing the first RNA transcript, and determining the expression level of the first RNA transcript in the sample based on the amount of the molecule. Including, method. 10. The method of claim 10, further comprising calculating the pathology or therapeutic effect based on the expression level of the first RNA transcript. The method comprises a second capture probe configured such that the functionalized surface binds a molecule containing a second RNA transcript.
Directing another ray at the binding molecule containing the second RNA transcript,
The tenth aspect of the present invention further comprises taking in light from the binding molecule containing the second RNA transcript and determining the amount of the molecule in the sample containing the second RNA transcript. The method described. 10. The method of claim 10, further comprising accommodating the sample and processing it without exposing it to the outside. 10. The method of claim 10, further comprising binding a plurality of magnetic particles to the molecule in the sample and directing the molecule to the functionalized surface using the magnet. 10. The method of claim 10, further comprising interfering with the diffusion of the light beam onto the image pickup apparatus. It further comprises binding each of the binding molecules, including the first RNA transcript, to the nanoparticles when bound to the functionalized surface, the nanoparticles being configured to reflect the rays to the imaging device. The method according to claim 10. A method for analyzing molecules in a sample using an image pickup device, a magnet, a light source, and a flow cell having a functionalized surface having a plurality of capture probes, wherein each of the plurality of capture probes is a plurality of RNA transcriptions. The method is configured to bind molecules in the sample containing one of the objects.
Bonding the molecules in the sample to the magnetic particles,
Using the magnet to direct the molecule toward the functionalized surface,
Binding each particular molecule of the molecule to one of the plurality of capture probes configured to bind to the RNA transcript of the particular molecule.
Directing the light beam from the light source to the bound molecule bound to each of the plurality of capture probes.
Taking in light from the bound molecule,
The amount of the bound molecule bound to each of the plurality of capture probes is determined based on the captured light, and each of the plurality of capture probes configured to bind each of the plurality of RNA transcripts. A method comprising determining a plurality of expression levels corresponding to said plurality of RNA transcripts based on said amount of said bound molecule bound to.

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