JP4736056B2 - Integrated bioanalysis and sample preparation system - Google Patents

Description

Background of the Invention This application claims priority to US provisional patent application No. 60 / 514,381, filed on October 24, 2003. This provisional patent application is fully incorporated herein by reference. This application is based on US patent application Ser. No. 10 / 059,993 of the name “Multi-Channel Bio-Separation Cartride” filed on January 28, 2002; and the name “Optical Detection In A” filed on January 28, 2002. US Patent Application No. 10 / 060,052 for "Multi-Channel Bio-Separation System"; and US Patent Application No. 10 / 319,803 for "Optical Detection Alignment Coupling Apparatus" filed on December 13, 2002; and 2003 Continuation of the name “Optical Detection Alignment Cocepling Apparatus” filed on December 15; and the name “Multi-Conpillany Electrophoresis Cartridge Interface Mechanism” filed on April 12, 2004, and the assignee of the present invention. Bio Cal Technology Inc., which is incorporated herein by reference in its entirety.

All of the above applications, all other applications, documents and references are hereby fully incorporated by reference if disclosed herein.
The present invention relates to bioanalyses, and more particularly to bioanalytical systems that integrate sample preparation processes, and more particularly to multi-channel bioanalytical systems that integrate sample preparation processes.

2. Description of Related Art Bioanalysis, such as DNA analysis, is rapidly changing from pure scientific exploration for accuracy to routine procedures with proven high reliability. Medical researchers, pharmacologists, and forensic research teams all use DNA analysis to accomplish their work. However, due to the complexity of equipment for detecting and measuring DNA samples and the difficulty in preparing samples, existing DNA analysis procedures are often time consuming and expensive. Thus, the size of the device is reduced, the number of parts is reduced, the cost is reduced, the handling of the sample in operation is simplified, and generally a simplified, low-cost, sensitive detector is provided. A device is desirable.

  One type of DNA analyzer separates DNA molecules by relying on electrophoresis. Electrophoretic techniques can be used to isolate DNA fragments for genotyping applications such as human identity testing, expression analysis, pathogen detection, mutagen detection and pharmacological research. The term electrophoresis means that charged molecules move under the influence of an electric field. Electrophoresis can be used to separate molecules with equal charge / mass ratios but different masses. An example of such a molecule is a DNA fragment.

  There are a variety of commercially available instruments that use electrophoresis to analyze DNA samples. One such example is a capillary electrophoresis (CE) device. By applying electrophoresis in a fused silica capillary column carrying a buffer solution, the required sample size is significantly reduced, and the separation speed and resolution are many times higher than slab gel electrophoresis. Can also be improved. These DNA fragments are often detected in CE by illuminating the components separated from the fluorescently labeled sample through the walls of the capillary and detecting the fluorescence emission induced by the incident light. . The intensity of the radiation represents the concentration, amount and / or size of the sample components.

  Traditionally, CE devices are designed to work with products that are initially prepared by other devices and placed on the sample tray of the CE device. There may be several sample preparation procedures involving manual and / or automatic procedures. The detailed apparatus and system are designed to handle only sample preparation with steps such as extraction, purification, amplification, stabilization, etc. to produce samples suitable for separation by the CE apparatus. For example, a DNA sample can be prepared by a polymerase chain reaction (PCR) method to amplify a sufficient amount of sample from a small amount of DNA sample. The product of the PCR method may be left to the CE method to assess the completeness or status of the PCR method results. Transfer from individually prepared samples to the CE separation / analyzer requires significant manual intervention, which affects the overall throughput.

  In order to avoid interference between sample preparation and separation / analysis by the user, it is desirable to develop a fully integrated bioanalytical system such as a built-in sample preparation capillary.

SUMMARY OF THE INVENTION The present invention provides an integrated bioanalytical system with a built-in sample preparation capillary. In one aspect of the present invention, a bioanalyzer equipped with a built-in sample preparation device is provided. In one embodiment, the sample preparation method includes a biomolecular reaction method. In certain embodiments, the biomolecular reaction method is based on PCR. In a further embodiment of the invention, a Peltier unit in the sample preparation device provides a thermal cycle of the sample supported in the multiwell tray.

  In another aspect of the invention, the CE device is equipped with built-in sample preparation capability, which may comprise a biomolecular reaction method, for example a PCR-based sample preparation device.

  In another aspect of the invention, a biomolecular reaction system is provided with a built-in analytical device, such as a CE device, for evaluating the outcome of a reaction product that may be a modified biomolecule sample. In certain embodiments, the biomolecular reaction system prepares a sample based on PCR. In a further embodiment of the invention, a thermal cycling unit is provided in the sample preparation device. Samples prepared by PCR devices can be used for other analyzes of other systems.

Detailed Description of the Invention The present invention has been described with reference to various embodiments by reference to the following figures. While the invention has been described in terms of the best mode for accomplishing the objects of the invention, it will be appreciated by those skilled in the art that changes may be made without departing from the spirit or scope of the invention. Will be understood.

  The present invention provides an integrated bioanalytical system with sample preparation. In one aspect of the invention, a bioanalyzer is provided with a built-in sample preparation device. In one embodiment, the sample preparation method comprises a biomolecular reaction method. In certain embodiments, the biomolecular reaction method is based on PCR. In another aspect of the present invention, the CE device is equipped with built-in sample preparation capabilities, which can comprise a biomolecular reaction method, eg, a PCR-based sample preparation device. In another aspect of the present invention, a biomolecular reaction system is provided for evaluating the results of reaction products, such as CE devices, of built-in analytical devices, which can be modified biomolecule samples. In certain embodiments, the biomolecular reaction system prepares a sample based on PCR. In a further embodiment of the invention, a thermal cycle unit is provided in the sample preparation device. Samples prepared by PCR devices can be used for other analyzes in other systems.

  It is for purposes of illustrating the principles of the invention and is not intended to be limiting, and the invention has been described by reference to embodiments directed to CE analysis and PCR sample preparation. In an exemplary embodiment, the present invention provides a fully integrated system for PCR sample preparation in an automated multi-channel CE system for gene analysis.

  The assignee of the present invention, Biocal Technology, hic., Has developed a CE-based automation device (HDA-GT12 DNA Analyzer System). An exemplary embodiment of the automated device of the present invention is based on Biocal's CE device, which is a low-cost and sensitive optical detection technology to form a highly sensitive and accurate biological reagent detection (gene analysis) system. That is, it incorporates an integrated reagent cartridge and microfluidic electrophoresis principle for real-time fluorescence analysis. This system is designed for high throughput, simple use, portable, inexpensive, very robust and field operation / application.

  The cartridge is designed to be supported by the device and all essential cartridge elements are aligned in the device and coupled to the support element. The cartridge is maintained against a sample tray that can move relative to a capillary separation channel in the cartridge. The instrument has been improved with the provision of a PCR-based station that prepares the sample from the crude sample and loads it into a system for separation and analysis (all done in the instrument), so that once the automated instrument When a crude sample (eg, extracted DNA) is loaded into it, further interference by the user is minimized throughout the sample preparation (DNA amplification / PCR), separation and analysis methods.

  In the context of the present invention, crude samples (e.g., extracted DNA) may be in the form of intervening substances or samples taken in the field, which are pre-prepared before being loaded into a sample preparation station in an automated system. It can be left to the law. In the context of the present invention, a crude sample is a significant sample preparation method (e.g., PCR) in an automated device (which does not simply dilute the crude sample, but rather significantly different and / or modified forms of the sample and / or Or just put it into a state), or simply leave it to other methods that facilitate handling of the crude sample in subsequent methods. For example, crude samples are subject to biomolecular reaction methods in sample preparation devices. FIG. 6 is a block diagram illustrating the relationship between the various method steps described herein. With reference to the subsequent separation and / or analysis method, the product sample prepared by the sample preparation device is left to the subsequent separation and / or analysis method, which is left to separation and / or analysis. A crude sample is a sample that is input to a sample preparation device, which is a sample that is subject to processing by the sample preparation device. The crude sample may be a product before the primary treatment.

  For example, for a biological sample collected in the field, such as a subject's urine or blood, it is left to primary processing such as, but not limited to, extraction and purification to obtain a subject's representative DNA sample. Such extracted DNA fragments are loaded with the necessary chemicals (e.g., primers, polymerase, etc.) into the integrated system 200 of the present invention (below), more specifically, sample preparation devices for PCR amplification, etc. Is a “crude sample”. The output of the PCR method is a sample for subsequent separation and / or analysis.

The CE system overview system is designed and built for field robustness weighing less than 40lbs. This portable system also incorporates a built-in modular and integrated PCR thermocycler (with Peltier cooler device) for DNA amplification.

  FIG. 1 is a schematic diagram of a capillary electrophoresis (CE) system 200 according to an embodiment of the present invention. CE system 200 generally comprises a capillary separation column 22 (eg, 200-500 μm O.D.), which defines a separation channel 36 (eg, 25-200 μm I.D.). The capillary column 22 is made of fused silica, glass, polyamide, or other plastic / ceramic / glass material. The inner wall of the separation column 22 (ie, the wall of the separation channel 36) may be coated with a material that can build an electrostatic charge to facilitate electrophoresis and / or electrokinetic movement of sample components. Separation channel 36 is filled with a separation support medium, which can simply be a running buffer or a sieve gel matrix known in the art. For radioactively induced fluorescence detection, the gel matrix comprises a known fluorophore, such as ethidium bromide.

  One end of the capillary column 22 is submerged in the reservoir 28 of the running buffer / gel 34. The other end of the capillary column 22 is connected to a sample vial 26. It will be appreciated that the detection configuration shown in other embodiments may be equally performed in a system similar to the CE system 200. And the separation channel 36 can also be a straight capillary or a microchannel (with the section of the detection window closest to the gel reservoir at the exit end being the detection zone), which is currently the preferred embodiment of our invention. is there. The radiation detector 24 is located outside the transparent part of the capillary wall in the detection zone 30. The excitation fiber 16 extends from a radiation source 18 (eg, LED or laser) and is directed to a detection zone 30 outside the column wall. Electrodes 12 and 14, which are part of the cartridge assembly, are connected to buffer reservoir 26 and gel reservoir 28 to complete the electrophoresis path.

  In accordance with the present invention, system 200 involves a sample preparation device 250. The embodiment shown in FIG. 2 shows a suitable sample preparation device for DNA amplification by PCR. In particular, the sample preparation device 250 comprises a thermal cycler for PCR (comprising a heating / cooling element and controller that generates the necessary heating and cooling conditions). Biomolecule / DNA samples extracted from the field (soil, air and water) are loaded onto the microtiter plate 72 with the necessary chemicals and / or protocols and amplified (e.g., biomolecule reactions in the sample preparation device). Detection) during capillary electrophoresis becomes easier. After the PCR method is complete, the microtiter plate 72 is moved under the capillary column 140 and the cartridge 100 is moved perpendicular to the capillary column 140 to allow access to the wells of the microtiter plate 72. The amplified DNA sample (PCR product) is then automated and introduced by electrokinetic injection through the microchannel of the cartridge for molecular separation and fluorescence detection.

During CE separation and analysis overview operations, a crude sample taken from the field is prepared (eg, DNA is extracted, purified and amplified by PCR) into a sample suitable for CE. For example, a prepared biological sample (e.g., a DNA sample) in a sample vial 26 prepared by a sample preparation device 250 (i.e., a DNA amplification device in the illustrated embodiment) can be obtained from many methods (e.g., from a sample reservoir). Is introduced into the distal end of the capillary column 22 away from the detection zone 30. The sample binds to the fluorophore in the gel matrix supported in the capillary column 22.

  When a DC potential (e.g. 1-30 KV) is applied between electrodes 12 and 14, the sample moves along separation channel 36 (e.g. negatively charged DNA, as shown in FIG. The sieve gel with integrated dye matrix / fluorophore is moved towards the positive electrode) and separated into bands of sample components (DNA fragments). The degree and distance of separation moved along the separation channel 36 depends on a number of factors, for example, the moving separation of the sample components, the mass and size or length of the sample components, and the separation support medium. The driving force in the separation channel 36 for separating the sample can be electrostatic force, pressure, or electroosmotic flow (EOF) means.

  When the sample reaches the detection zone, excitation radiation is directed through the excitation fiber 16 in the detection zone. Each sample component emits fluorescence with an intensity proportional to the concentration of each sample component (proportional to the amount of fluorescent labeling substance). The detector 24 detects the intensity of fluorescence emitted at a wavelength different from the incident radiation. This detected radiation may be analyzed by known methods. For the automation system, the controller 32 controls the operation of the CE system 200.

Overview of PCR
The purpose of PCR is to prepare very many copies of the gene. This is essential in order to have sufficient starting template for DNA fragment analysis by CE. The following is a brief description of the PCR thermal cycling reaction.

1.Cycle reaction:
There are three main stages in PCR that are repeated over 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the sample vial with the reaction mixture in a very short time. As described below, a Peltier cooler / heater type mechanism may be integrated as part of the transport cartridge frame of the XY transport mechanism of a multi-channel capillary electrophoresis device for complete solutions for DNA amplification and analysis. DNA amplification by PCR is well known to those skilled in the art. Details of the protocol and chemicals (eg, primers, polymerase, etc.) are proportioned herein. Further reference may be made by any publication well documented in the art. According to one embodiment of PCR, the three major PCRs include the following three major steps:

1. Denaturation at 94 ° C:
During denaturation, the double strands melt and open into single stranded DNA, stopping all enzymatic reactions (eg: extension from the previous cycle).

2. Annealing at 54 ° C:
Primers move around by Brownian motion. An ionic bond is steadily formed and broken between the single stranded primer and the single stranded template. More stable binding lasts only slightly longer (primer that engages correctly) and continues with a portion of double-stranded DNA (template and primer) that has little, allowing the polymerase to bind and begin to copy the template. Once only a few bases are incorporated, the ionic bond is very strong between the template and the primer and is no longer broken.

3. Elongation at 72 ° C:
This is an ideal effective temperature for a polymerase. The primer has a stronger attractive force on the template than a force that breaks this attractive force when only a few bases are incorporated. Primers in positions that do not match exactly will loosen again (due to the higher temperature) and will not cause fragment extension. The base (complementary to the template) binds to the primer on the 3 'side (the polymerase adds dNTP from 5' to 3 ', reads the template from 3' to 5 ', and the base complements the template. ). For example, the advancement step may be modified, but for the time being, provides PCR or DNA amplification without departing from the scope and spirit of the present invention.

CE system based on multiple capillary cartridges According to the present invention, PCR amplification is integrated into a microfluidic electrophoresis system for nucleic acid detection. Crude samples of DNA or RNA extracted from the field are purified and then loaded into sample vials (e.g. 96-well microtiter plates) in automated microfluidic electrophoresis systems for PCR amplification (using standard PCR plate preparation steps) ). Primer pairs for specific genetic markers will be used for PCR analysis. Furthermore, according to the present invention, the completed PCR product in the sample vial is automatically introduced into the multichannel gel cartridge by electrokinetic sample injection for high resolution separation and fluorescence detection.

  FIG. 2 shows an overall perspective view of a CE system 200 (eg, a DNA analyzer). CE system 200 incorporates an interface mechanism 300 in accordance with an embodiment of the present invention. The interface mechanism 300 supports the multi-channel cartridge 100 according to an embodiment of the present invention, which facilitates handling of the multi-channel separation column and allows optical coupling of the detection zone to the detection optics of the CE system 200 . For details of the interface mechanism 300, reference is made to US patent application Ser. No. 10 / 823,382, which is incorporated by reference herein in its entirety. The fully automated DNA analysis system 200 has a base 74 (supporting a modular X-Z mechanism 80 with a sample tray support frame 81). XZ mechanism 80 is supported by interface mechanism 300 with PCR sample preparation device 250 (discussed in detail below with reference to FIGS. 4 and 5) (supporting 96-well microtiter plate 72 and buffer plate 70). The multi-capillary cartridge 100 is moved. Specifically, the mechanism 80 includes an X mechanism 82 that moves the support frame 81 along the X direction with respect to the cartridge 100, and a Z mechanism 83 that moves the cartridge relative to the support frame 81 in the Z-axis direction. . The PCR sample preparation device 250 is controlled by the PCR thermoelectric controller 68.

PCR Sample Preparation Device Referring to FIGS. 4 and 5, the PCR sample preparation device includes a heating unit, such as a Peltier thermoelectric unit 251, which is the size of a complementary well 255 that is sized at the top to receive the bottom of well 73. A thermal interface structure 252 having The Peltier thermoelectric unit 251 may be controlled by the controller 68 to heat or cool the well 73 and leave the contents in the well 73 to the thermal cycle required by the PCR method. At the bottom of the Peltier thermoelectric unit 251, a sink is provided to remove heat from the Peltier thermoelectric unit 251 to remove excess heat during the unit's cooling cycle and / or during the heating cycle. . The PCR sample preparation device 250 is supported on the transport frame 81 of the transport mechanism 80.

The control system 200 of the automation system 200 is responsible for facilitating the handling of the multi-channel separation column and easily allows optical coupling of the detection zone to the detection optics of the CE system 200. Operation of the CE system 200 with the interface mechanism 300 is controlled by a controller 32 that interfaces with an external user control interface (eg, PC 918). The PCR thermoelectric controller 68 may operably couple the controller 32 and / or PC918 so that the remaining control of the PCR sample preparation device and system 200 is tuned to achieve the functions described herein. May be good.

  In accordance with an embodiment of the present invention, referring also to FIG. 3, a block diagram of the controller 32 for the CE system 200 is shown. The controller 32 is a digital corresponding detector signal from the detector 24 (eg, PMT) (from the LEDScan PCBA interface 914 for transporting and receiving the system from each part of the CE system 200 as directed by the CPU 910). The A / D (LED processor PCBA) interface 912, which includes a processor as part of an A / D board (LED processor PCBA) 912 with a CPU 910 to convert to signals, is connected to various actuators in the interface mechanism 300. Connected (by using interface mechanism 300) at least high voltage source 76, compressed air 78 (hidden from the interface mechanism 300 point of view in FIG. 2), motor control (XZ sample / buffer tray) 80 And interlocking devices (cartridges and transport doors) 61 and 62 (in FIG. Over scan mechanism 300 not shown in) to control and linking. The A / D or LED processor PCBA912 also includes a high voltage source 76, a sample injection and electrophoresis function of the CE system 200, a circuit 914 (LEDScan board) and a CE system for adjusting the excitation radiation source (eg LED) 921 Controls 200 detector modules 24. See pending US patent application Ser. No. 10 / 060,052, incorporated by reference herein for details of adjustment of the excitation radiation source.

  The A / D (LED processor PCBA) 912 may be further coupled to an external personal computer 918, which in turn uses additional control functions for data processing and CE system 200, for example, using BioCal's BioCalculator software. To control various features and functions of an automated multi-channel CE system 200 (eg, an integrated PCR sample preparation device).

  The PCR sample preparation device 250 is controlled by the controller 68 in order to perform the sequence of the thermal cycle reaction described above. Thermoelectric unit 251 (supported by X-Z transport mechanism 80) is designed for 96 well microtiter plate 72. The thermoelectric unit 251 is controlled directly by the thermal cycle controller module 68 (A / D or microprocessor board 912, and as shown in FIG. 3, the control firmware is connected to the user interface (BioCalculator in PC918 via RS232 cable). The thermoelectric controller 68 controlled by) is integrated into the controller 32 in an alternative embodiment.

  The components of controller 32, except for PC918, are mounted on electrical board 64 (FIG. 2) and on cooling fan 63, CE system 200 and electrically coupled to PC918 via a serial port (not shown). Or they may be part of a separate controller module external to the CE system 200. CPU 210 and / or PC 218 are programmed to achieve various control functions and features for CE system 200. In one embodiment, the PC 218 may be designed to provide a user control interface for the CE system 200 (e.g., user initiated initiation sequence of the interface mechanism 300). Those skilled in the art will be within the scope of executing the program code of the functions and features described herein. In an alternative embodiment, the controller 32 or components thereof may be incorporated as part of the PC 918.

Capillary cartridge The multi-channel capillary cartridge 200 includes 12 detection zones (shown schematically as 30 in FIG. 1), which are defined by capillaries maintained in the cartridge body. The cartridge 100 includes a 12 channel fused silicon capillary array that is used for sample separation and detection as part of a disposable and / or portable, replaceable cartridge assembly 100. The cartridge 100 shown in FIG. 2 includes a maximum of 12 capillaries 140 (length 12-18 cm). The cartridge 100 is integrated with an upper, outlet buffer reservoir 130 common to all capillaries 140, which is connected by an interface mechanism 300 to a modular compressed gas source 78 (eg, inert, compatible or non-reactive ( for example, nitrogen, etc. CO ₂₎ is connected directly to the replaceable compressed gas cartridge or pressure pump). Appropriate pressure tubes such as tubing, pressure bubbles and solenoid controls are provided. (Details of such tubes are proportionate because they are well known to those skilled in the art of designing tubes that provide the functionality, features and operation of the system 200 described herein.) The pressure source 78 fills all 12 capillaries with the sieve gel contained in the reservoir 130 and supplies the gas pressure necessary to purge the gel from the previous run during the refill process from the capillaries. Depending on the viscosity of the gel, a pressure of up to 40 PSI may be applied to the capillary 140 via the gel filling reservoir 130. The cartridge gel reservoir 130 includes a built-in common cathode 134 for all 12 capillaries, which are automatically installed inside the system 200 by the interface mechanism 300 to a high voltage supply 76 (FIG. 2) for electrophoresis. Will be linked. A fan or Peltier cooler (not shown) for the structure adjacent to the cartridge 100 may be provided to provide temperature control of the cartridge. In addition or alternatively, the cartridge has vent holes (inlet and outlet) for air circulation (temperature-controlled air introduced into the cartridge from the device side). Depending on the heat generated during CE separation, the cartridge has no incidental cooling features and may simply be exposed to ambient temperature. The power supply 66 (FIG. 2) provides DC power to the CE system 200.

  For further details of the cartridge, see pending application 10 / 059,993, which is incorporated by reference herein.

Detection System US patent application 10 / 060,052, incorporated by reference herein, relates in more detail to a time division / multiple detection scheme that can be applied in CE system 200.

Interface mechanism
The structure and operation of the CE system 200 interface mechanism is referred to US patent application Ser. No. 10 / 823,382, incorporated herein by reference. The cartridge interface allows a quick and reliable interface to be connected to the disposable gel-containing capillary cartridge 100. These interface connections include gas compression connections, high voltage connections, and precision optical connections. The interface also positions the cartridge element in relation to the support element in the CE system 200, e.g., the position of the capillary chip in relation to the external sample or buffer reservoir found on the 96 well titer plate, etc. Provides accurate and repeatable cartridge positioning. In addition, the interface provides separate electrical, optical and pneumatic connections for each separation channel, thus independent of the channel from both electrical and optical crosstalk and the remaining device from high voltage. Insulate against

Sample handling tray transport mechanism 80 (with 96 well plate (8x12)) 72 and 70 are used to introduce amplified DNA samples (or analytes) into each capillary 140 during CE system operation . The XZ transport mechanism 80 guides the array of sample transport wells 73 of the microtiter plate 72 below the array of capillary chips 140, and the chips are immersed in the wells. By applying a voltage, motorized injection moves a known amount of DNA sample to the beginning of the separation column 140. After injection, the DNA sample from sample tray 72 may be replaced with the running buffer from tray 70. Instead, after injection, transport mechanism 80 moves to a position below the cartridge to replace 12 rows of wells 73 in titer plate 72 containing buffer solution with 12 wells containing DNA samples. Can lead to.

  By applying a high voltage over the entire length of the capillary 140, separation of the DNA sample into DNA fragments is achieved. As the fragment approaches the end of the capillary 140 and enters the detection zone, excitation light energy (eg, derived from 12 LEDs delivered by an optical fiber) is detected in the detection zone and the moving DNA fragment is irradiated. The detection scheme may be time-resolved as described in pending US patent application Ser. No. 10 / 060,052.

  To prepare for the next run with a different sample, the old gel from the previous run is purged by applying pressure to the reservoir and the capillary is refilled with fresh gel. Trays 70 and / or 72 carry cleaning solutions, waste collections, and samples. The purged gel is collected by one of trays 70 and 72 by placing the capillary tip in line with one waste collection well in the tray. The capillary tip may be washed with water or with a washing solution by immersing the capillary tip in such a solution in a suitable tray well. When the capillary is refilled and ready for the next run, the capillary tip is immersed in the sample by repositioning the tray 72. The sequence of the above method may be programmed as one of the automation functions of the controller 32. The interface mechanism 300 provides an interface to the support element cartridge in the CE system 200, eg, high voltage, gas pressure, LED radiation source, and detection optics as described above. Compared to conventional methods (using separate PCR machines and electrophoresis (slab or gel capillary electrophoresis) equipment), the new portable automation system offers the combined functionality of sample preparation and analysis (eg PCR and electrophoresis) Are designed to be integrated as a single device. The portable microfluidic electrophoresis-based device of the present invention comprising an integrated PCR device with a multi-channel and multi-color fluorescence detection system is adapted to be applied for genotype analysis faster and at a lower cost It is a reliable device.

  The system of the present invention may be a benchtop system designed for high-throughput biofactor material (DNA) detection / analysis as shown in FIG. The 12 channel separation / detection cartridge can analyze multiple samples simultaneously. A 12-channel separation / detection cartridge combined with an integrated PCR plate can complete 96-well sample plate amplification and electrophoresis in a very short time with predictable results. The system is simple, reliable and fast. The entire method from PCR to DNA separation is completed in about 2 hours. Analysis times for 12 PCR samples are targeted within a few minutes. The cost of the proposed device will be 10 times less than current multi-channel high-end capillary electrophoresis equipment. On average, the cost / analysis of chemicals is estimated to be significantly lower than the cost of prior art CE systems.

  The automated device of the present invention is a sample handling tray with direct detection of nucleic acid molecule amplification, electrophoresis / separation, an integrated PCR device for fluorescence detection, and improved detection of any bound dye for immunoassays. Including mechanisms. A fully automated and portable system is controlled by a common computer. Data analysis on detection results is automated to provide spot amplification (PCR), peak identification and semi-quantitative analysis. With this new, inexpensive and high-throughput analyzer, the DNA laboratory will have the ability to perform high-throughput and rapid bioagent identification in a single unit with less cost and very accurately. The detection capability of this system is that the capability of the system can be designed to detect only certain potential bioagents and pathogens.

  In one embodiment of the invention, the crude sample is introduced into a 96-well titer plate in a thermal cycler (hot and cold thermal cycle process) that is subject to various temperatures before, during or after the sample is injected into the gel cartridge. Is done. Thermal cyclers have several purposes or functions during sample preparation prior to biomolecule separation and analysis. One function is for straight PCR amplification for straight PCR amplification of biomolecules and the other is for changing the state / condition of the sample prior to electrophoresis through a gel capillary cartridge. The stage for various temperature cycles of the sample / biomolecule (sample preparation stage). The 12 channel cartridge can also function as a microdispenser. For example, one skilled in the art can apply electrokinetics at the initial position of the sample tray (position A on a 96-well plate) to inject negatively charged DNA into the capillary or micro-channel of the cartridge, and It will be understood that the system can return the HV feed electrode to position B of the 96-well plate to mix the DNA sample (or biomolecule) from well A and the sample from well B (biomolecule reaction). Let's go. The cartridge can function as a sample dispenser (liquid dispenser). The heat block can be used to heat or cool any biomolecule and electrophorese it for analysis by a CE cartridge. All biomolecules (ie DNA, RNA, proteins, antibodies and antigens) may be analyzed by pre-integrated sample preparation and electrophoresis mechanisms in a very flexible manner. For example, one skilled in the art would heat a sample up to 90 ° C., then lower it to 80 ° C. or 70 ° C. and quickly evaluate it on the CE cartridge / device for the effect of DNA and probe binding before and after temperature cycling be able to. Or, similar to the microarray concept of hybridization, one skilled in the art can manipulate the sample temperature to wash away point mutations and perform electrophoresis to quickly quantify it or simply sample The amount of can be checked quantitatively. This approach / method will enable one skilled in the art to check the location of detected peaks quickly for quantitative performance, which is useful in natural mutation detection. The increase in detection sensitivity is due to the fact that the current fluorescence detection mechanism (i.e. LED excitation to laser excitation) optical detection system reduces the number of thermal cycle (PCR) steps by heat block during electrophoresis. Can be achieved by improving to a higher value. This thermoelectric module also allows one skilled in the art to store PCR-sampled samples for the purpose of storage or preservation at lower temperature sets. By the method of the present invention, the PCR step can be simplified to detect fragments (mutation analysis) or single-stranded analysis or protein analysis that are detected correctly or incorrectly due to temperature changes. . This system of thermal cycle shutoff / method for sample preparation can also be used as a PCR step / method for samples injected inside capillary tubes. A person skilled in the art simply injects the sample electrodynamically from the sample well, then let the oil go to the other wells in it, and immerse the capillary tip into the tip of the oil to be injected inside the micro-channel. The sample temperature cycle can be started (internal capillary PCR) and electrophoresed. This system of multichannel capillary electrophoresis combined with sample plate thermal cycling characteristics (sample plate heat block) for sample preparation is similar to microarray or real-time PCR for a complete and one-step solution for biomolecular analysis Would provide flexibility.

  While the invention has been shown and described in detail by reference to the preferred embodiments, various changes in form and detail will occur to those skilled in the art without departing from the spirit and scope of the invention. You will understand that it is good.

  The sample preparation station may be designed for methods that are performed to produce samples in a form suitable for CE analysis rather than PCR. The automation system 200 may be designed to perform other types of analysis that are different from or in addition to CE separation and analysis. For example, protein or bioagent detection combined with a microfluidic electrophoresis system may also be used. Protein extraction from the culture is used for immunoassays. Amplification signals through the interaction of antibodies and antigens bound to fluorescent dyes can be automatically applied to multi-channel cartridges for high resolution detection within minutes.

  The interface mechanism may be designed to accept other structural design capillary cartridges. One skilled in the art will appreciate that a device incorporating the essence of the present invention may be used for other biomolecular analyzes that are not DNA analyses. For example, by replacing separation gels or buffers, the device can be modified to analyze biomolecules such as proteins, carbohydrates, and lipids.

  For purposes of illustration and not limitation, the detection scheme of the present invention has been described in the context of capillary electrophoresis and radiation-induced fluorescence detection. The present invention is also applicable to the detection of analytes separated on the basis of other biological separation phenomena that are not electrophoresis, and other radiation emissions that are not fluorescent radiation, such as other types of radioactive radiation, for example It will be appreciated that it is applicable for the detection of absorbance based on phosphorescence, luminescence and chemiluminescence, and detection.

  Furthermore, while the separation channel in the described embodiment is defined by a cylindrical column or tube, the concept of the present invention applies to channels defined by etching in a substrate, such as microchannels (eg, square, rectangular or essentially It can be seen that the present invention is equally applicable to separation channels (microfluidic devices or biochips) defined by a semicircular cross-section.

  The transport mechanism may be designed to move the tray horizontally, and a further transport mechanism may be provided to allow the tray to access the tray vertically.

  The output of the sample from the sample preparation device may be an input that does not need to be left to separation to the analytical device.

  Accordingly, the disclosed invention is to be considered merely by way of explanation and limitation, as specified only in the appended claims.

For a full understanding of the nature and advantages of the present invention, and the preferred method of use, reference should be made to the following detailed description in conjunction with the drawings. In the following drawings, reference numbers refer to similar parts throughout the drawings.
1 is a cross-sectional view of a capillary electrophoresis system comprising a sample preparation device according to an embodiment of the present invention. 1 is a cross-sectional view of a capillary electrophoresis system comprising a sample preparation device according to an embodiment of the present invention. 1 is a block diagram of a control system of a capillary electrophoresis system according to an embodiment of the present invention. FIG. FIG. 3 is a perspective view of a sample preparation device. FIG. 5 is a cross-sectional view of the sample preparation device taken along line 5-5 of FIG. It is a block diagram explaining the relationship between various sample processing steps.

Claims (15)

Biological analysis system:
A sample preparation device that accepts a crude sample that is processed into a suitable form of sample for later analysis;
An integrated transport mechanism that positions the sample preparation device to be accessible by the sample analysis device; and a sample analysis device that receives a sample from the sample preparation device that is subject to analysis;
Comprising
Here, the sample preparation device and the sample analysis device are operably coupled in an integrated automated system and the crude sample is loaded into the sample preparation device with additional user intervention. A biological analysis system in which the output of the sample from the sample preparation device is an input to the sample analysis device.   The biological analysis system of claim 1, wherein the sample analysis device comprises a separation capability for separating a sample into components.   The bioanalytical system of claim 2, wherein the sample analysis device further comprises a detection capability responsible for analyzing separated components.   4. The bioanalytical system of claim 3, wherein the sample analysis device comprises capillary electrophoresis that provides separation and detection capabilities.   The bioanalytical system of claim 1, wherein the sample preparation device is constructed and configured to submit a crude sample to a biomolecular reaction.   6. The bioanalysis of claim 5, wherein the sample preparation device is constructed and configured to provide thermal cycling of the crude sample to output the crude sample that is amplified from the crude sample. system.   The bioanalytical system of claim 1, wherein the sample preparation device comprises a thermoelectric unit that is a controller that circulates the temperature of the crude sample such that a biomolecular reaction occurs.   The bioanalytical system of claim 7, wherein the sample preparation device is constructed and configured such that DNA amplification occurs by PCR in a biomolecular reaction.   The bioanalytical system of claim 8, wherein the analytical device comprises a capillary electrophoresis system.   The bioanalytical system of claim 9, wherein the capillary electrophoresis system comprises multichannel separation and analysis.   The bioanalytical system of claim 10, wherein the crude sample comprises DNA extracted from a field sample. The crude sample is loaded into the sample well in the sample preparation device Rutotomoni, the integrated transport mechanism to position the sample wells so as to be accessible by the capillary electrophoresis system, organism according to claim 9 Analysis system.   The bioanalytical system of claim 1, wherein the sample preparation device comprises an apparatus for initiating a thermal cycle. PCR system:
A thermal cycler performing PCR, which receives the crude DNA sample amplified by PCR and put into a form suitable for subsequent analysis;
A capillary electrophoresis system that accepts the DNA sample from the thermal cycler that is subjected to capillary electrophoresis and analyzes the results of the PCR; and
An integrated transport mechanism that positions the thermal cycler to be accessible by the capillary electrophoresis system;
Comprising
By wherein said capillary electrophoresis system and a thermal cycler is operatively coupled to at within the integrated automation system Rukoto, without user further intervention, DNA samples output from the thermal cycler capillary electrophoresis inputs and ing PCR system to system. A method for analyzing a crude sample comprising:
Providing a sample preparation device that accepts a crude sample to be processed into a sample in a form suitable for subsequent analysis;
Providing a sample analysis device, wherein the sample preparation device accepts a sample to be analyzed from the sample preparation device; and positioning the sample preparation device to be accessible by the sample analysis device; Providing an integrated transport mechanism,
Comprising
Here, the sample preparation device and the sample analysis device are operatively coupled within an integrated automated system so that the sample output from the sample preparation device can be sample analysis device without further user intervention. Method for analyzing crude samples as input to

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