Sample tray for temperature-sensitive reactions

Abstract

We provide sample trays for temperature-sensitive reactions. [Solution] The sample tray for temperature-sensitive reactions according to the present invention comprises a sample well, floating ribs, and a frame. Each sample well corresponds to two floating ribs. The sample wells are not connected to each other in the longitudinal direction of the frame, and each sample well is connected to the rectangular frame in the width direction of the rectangular frame by two floating ribs on both sides of the sample well.

Description

Technical Field

[0004] ,

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 750,119, filed with the United States Patent and Trademark Office on January 27, 2025. The entire contents of this application and those referred to below are hereby incorporated by reference in their entirety.

[0002] The present invention relates to a biological analysis apparatus, and more particularly to a sample tray for temperature-sensitive reactions that can include, but is not limited to, polymerase chain reaction (PCR), restriction enzyme digestion, or rapid diagnosis.

Background Art

[0003] Capillary electrophoresis (CE) separation is a microfluidic method of gel electrophoresis (a microchannel device that simplifies gel electrophoresis separation) and has the great advantage of a wide range of applications. CE technology is widely accepted in the biotechnology industry as a reliable, high-resolution, high-sensitivity separation and detection tool, particularly in nucleic acid-based tests. CE is also applied to DNA-related analyses such as, for example, the analysis of proteins, carbohydrates, and oligonucleotides, DNA sequencing, and the analysis of dsDNA fragments, and glycan profiling.

[0004] U.S. Patent Publication No. 8,778,155, jointly acquired by the assignees of the present invention, discloses a reusable cartridge-type portable electrophoresis system configured to use a pen-type bioseparation cartridge that has no moving parts, is easy to assemble and use, and has an integrated reagent (separation buffer) reservoir. The cartridge comprises a body defined with an opening that serves as a detection window for receiving an external optical detection element, and at least one capillary column supported within the body and having a first end extending beyond the first end of the body. A section along the capillary column is exposed through the detection window, and the detection window aligns the external optical element. The reservoir is attached to the second end of the body through which fluid flows to the second end of the capillary column. The reservoir is configured to be connected to a pneumatic pump or N2 tank that pressurizes the gel reservoir to remove and fill the capillary with a buffer / gel to serve as a separation support medium. The capillary column is capable of being injected with negatively charged samples and performing CE separation for analysis. Once the separation of the sample is complete, the used sample (negatively charged separated DNA / RNA fragments) is captured in the gel reservoir (large tank) located in an embedded electrode (anode) away from the output end of the capillary column. Then, other samples may be injected into the capillary column, and analysis is performed again using the same cartridge.

[0005] In relation to DNA / RNA analysis based on CE separation, polymerase chain reaction (PCR) is an experimental technique that rapidly generates (amplifies) millions to billions of copies of a specific region of DNA, eliminating the need to prepare large quantities of DNA / RNA samples beforehand and allowing for high-resolution, detailed study. PCR uses short synthetic DNA / RNA fragments called primers to select the genomic region to be amplified, and then multiple DNA / RNA synthesis cycles are performed before conventional gel electrophoresis or capillary electrophoresis (CE) separation, detection, and analysis. This amplified region allows for high-resolution results based on an initial small sample size (e.g., picoliters). The PCR process requires thermal circulation of the sample DNA region to prepare the sample for subsequent CE separation and analysis.

[0006] As described above, there has been a long-standing practice in which the PCR process is carried out independently using a thermal cycler that is not part of the CE separation and detection system / apparatus (e.g., an external one), and the prepared sample is then sent to the CE system for separation and analysis. U.S. Patent Publication No. 11,531,004, commonly acquired by the assignees of the present invention, discloses a CE apparatus having a temperature control mechanism such as a Peltier heating / cooling module connected (e.g., downward) to a workbench for supporting a sample vial, so as to be located below the capillary cartridge. The heating / cooling module is equipped with temperature control to facilitate PCR amplification and electrophoresis / detection in a single apparatus.

[0007] In conventional patent documents, for example, Patent Document 1 below discloses a portable electrophoresis system with an integrated thermal cycler, which comprises a thermal control module including a thermal platform thermally coupled to the thermoelectric module. The thermal platform comprises a base and at least one heating block supported to conduct heat to the base, the heating block being configured to receive a container containing at least one sample. The heating block comprises a body including a split longitudinal block having two longitudinal sides with defined valleys, the opposing walls of the sides each having a fan-shaped concave contour that conforms to the convex conical tubular contour of the bottom surface of the wells of the container tray, a heat sink, and a thermoelectric module connected to conduct heat between the base of the thermal platform and the heat sink, for heating / cooling the thermal platform based on the required heating / cooling temperature distribution.

[0008] Furthermore, Patent Document 2 discloses a PCR temperature control module comprising a temperature control member and a heater. The temperature control member is formed from a mixture containing a resin and a substance having higher thermal conductivity than the resin. The heater controls the temperature of the temperature control member. The temperature control member is formed so that an eight-tube strip is inserted into it. The temperature control member is in contact with the bottom of the eight-tube strip into which it is inserted.

[0009] Furthermore, Patent Document 3 discloses a nucleic acid amplification reaction vessel and a nucleic acid amplification reaction apparatus. The nucleic acid amplification reaction vessel comprises a support and at least one transparent or translucent hose, with a sample inlet formed on the upper part of the support, the number of sample inlets corresponding to the number of hoses. The lower end of the sample inlet is connected to the upper part of the hose, which is used to inject an acid amplification reaction solution. A sealing port is formed at the bottom of the hose, and the support corresponds to the number of sample inlets and further includes a sealing cover for closing the sample inlets. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent Application Publication No. 20230330680A1 [Patent Document 2] Taiwan Patent Application Publication No. 201623938A Specification [Patent Document 3] Chinese Patent Application Publication No. 118580940A Specification [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] In PCR amplification, the precision and uniformity of temperature control are crucial. Inaccurate temperatures can lead to problems and failures in PCR amplification, such as decreased amplification efficiency, band disappearance or attenuation, the formation of nonspecific bands or primer dimers, and contaminated bands, sometimes making subsequent analysis impossible or requiring resampling.

[0012] This summary provides a brief explanation of concepts that will be further elaborated upon in the detailed description below. This summary and the background information provided are not intended to identify any significant or fundamental features of the claims. Furthermore, this summary is not intended to assist in determining the scope of the claims. [Means for solving the problem]

[0013] On the one hand, a sample tray for temperature-sensitive reactions comprises a frame and a number of floating ribs. Each sample well corresponds to two floating ribs. The number of sample wells are not connected to each other in the longitudinal direction of the frame, and each sample well is connected to the frame in the width direction by two of the floating ribs on either side of the sample well.

[0014] The sample tray according to the present invention features a novel free-floating rib design that allows each well to move freely in all directions, such as vertical, length, and width. This design allows each sample well to fit perfectly onto the corresponding heating column and maximizes the surface area for efficient heat transfer.

[0015] The sample tray is specially designed to minimize the cumulative tolerance between the heating column and the sample wells (tubes). This cumulative tolerance could create gaps between the heating column and the sample wells, potentially affecting the efficiency of the PCR. This could not only reduce the final PCR concentration but, in some cases, affect the test results, especially in multiplex PCR.

[0016] In one embodiment, each floating rib comprises a first rib section and a second rib section that are stepped and interconnected. The thickness of the first rib section is greater than the thickness of the second rib section.

[0017] In one embodiment, the first rib section gradually tapers towards the frame along the width direction.

[0018] In one embodiment, the second rib section gradually tapers towards the frame along the width direction.

[0019] In one embodiment, the two floating ribs allow for the displacement of each sample well in the vertical, longitudinal, and widthwise directions.

[0020] In one embodiment, the wall thickness of each sample well is in the range of 0.15 mm to 0.25 mm.

[0021] In one embodiment, the heating or cooling rate of the plurality of sample wells is 5°C / sec or higher.

[0022] In one embodiment, the sample tray is connected to the frame and further includes a support base for supporting the frame.

[0023] In one embodiment, the sample tray is integrally formed.

[0024] In one embodiment, the sample tray has a rectangular shape.

[0025] In one embodiment, the sample tray has a ring shape, the length direction is the circumferential direction of the sample tray, and the width direction is the radial direction of the sample tray.

[0026] From the descriptions in the following specification and drawings, at least the following matters will become clear.

Brief Description of the Drawings

[0027] [Figure 1] It is a schematic diagram showing a capillary electrophoresis (CE) system integrating a thermal cycling module / thermal cycler according to an embodiment of the present invention. [Figure 2A] It is a perspective view showing a capillary electrophoresis system including an integrated thermal cycling module / thermal cycler according to an embodiment of the present invention. [Figure 2B] It is a cross-sectional view taken along line A-A of FIG. 2A. [Figure 2C] It is a cross-sectional view showing a state where the lid of the thermal cycler according to an embodiment of the present invention is in contact with the sample tray. [Figure 2D] It is a cross-sectional view showing a state where the lid of the thermal cycler according to an embodiment of the present invention is in contact with the sample tray. [Figure 3A] It is a perspective view of the sample tray according to an embodiment of the present invention. [Figure 3B] It is a top view of the sample tray according to an embodiment of the present invention. [Figure 3C] This is a front view of a sample tray according to one embodiment of the present invention. [Figure 3D] This is a left side view of a sample tray according to one embodiment of the present invention. [Figure 3E] This is a perspective view of the bottom of a sample tray according to one embodiment of the present invention. [Figure 4A] This is a perspective view of a thermal cycling module / thermal cycler according to one embodiment of the present invention. [Figure 4B] This is a top view of a thermal cycling module / thermal cycler according to one embodiment of the present invention. [Figure 4C] This is a right side view of a thermal cycling module / thermal cycler according to one embodiment of the present invention. [Figure 5A] This shows the heating / cooling curves of the wells in a conventional 8-tube transceiver (PCR tube). [Figure 5B] The heating / cooling curves of the sample wells in the sample tray according to the present invention are shown. [Figure 6A] This graph shows the relationship between migration time and relative fluorescence units (RFU) in two wells of a conventional 8-tube PCR system. [Figure 6B] This graph shows the relationship between the migration time of two sample wells in a sample tray according to the present invention and the relative fluorescence units (RFU). [Figure 7] This is a top view showing a sample tray according to another embodiment of the present invention. [Modes for carrying out the invention]

[0028] The present invention will be described below through embodiments of the invention, but these embodiments are not intended to limit the invention as defined in the claims. Furthermore, not all combinations of features described in the embodiments are necessarily essential to the solution of the invention.

[0029] The embodiments will be described in more detail below with reference to the accompanying drawings. The accompanying drawings constitute part of this specification and illustrate specific embodiments. These embodiments are disclosed in sufficient detail to enable those skilled in the art to carry out the invention. However, the embodiments can be carried out in a variety of different forms and should not be construed as being limited to the embodiments described herein. Therefore, the following detailed description should not be construed as restrictive.

[0030] Biological analysis systems comprising detection systems disclosed in U.S. Patent Publications No. 8,778,155, 8,784,626, and 11,531,004, and U.S. Patent Application Publications No. 20150338347A1 and 20230330680A1 are referenced herein by reference, their entire contents incorporated herein by reference. These patents and patent applications are jointly assigned to BiOptic Inc., the inventor and assignee of the present invention. In more detail, these patents disclose simplified, low-cost, high-efficiency, high-sensitivity, and high-throughput bioseparation systems (e.g., capillary electrophoresis (CE) systems). The bioseparation system comprises a device configured to operate with a single-channel or multi-channel capillary cartridge, the device having a detection configuration including an optical element that irradiates an incident radiation onto a detection region along the separation channel and detects the exit radiation from the detection region, and the optical element does not need to be precisely aligned with the separation column to detect radiation emitted from the sample analyte (e.g., radiation-induced fluorescence emission). The device is configured to automatically perform bioseparation within the (multiple) separation channels of the bioseparation cartridge. The CE system has a simpler optical detection mechanism to reduce costs, and achieves ease of operation and high efficiency, sensitivity, and high-throughput rapid analysis. U.S. Patent Application Publication No. 20150338347A1 further discloses two-color fluorescence detection. U.S. Patent Application Publication No. US20230330680A1 further discloses a thermal control module for a bioseparation system. The present invention employs and modifies these systems, and the system (including the PCR thermal recycling module of the present invention) is improved based on the following disclosures.

[0031] Rather than limiting the present invention, the present invention will be described with reference to an embodiment of capillary electrophoresis using a single capillary separation column in order to explain its principles. While the present invention will be described in relation to radiation-induced fluorescence detection (e.g., a laser or excitation LED light source), the present invention is not limited thereto.

[0032] System Overview The miniaturization and automation of analytical instruments offer numerous advantages over conventional labor-intensive technologies (e.g., manual slab gel electrophoresis). These advantages include improved data accuracy and reproducibility, reduced analysis time, minimized sample consumption, and enhanced automation and integration of complex workflows.

[0033] In one embodiment, a fully automated bioseparation system, particularly a CE instrument incorporating a thermal recycling module / thermal cycler for sample preparation and PCR (hereinafter referred to as the PCR-CE instrument or system), provides direct endpoint PCR testing in high-speed amplification, high-speed separation, and fluorescence detection for clinical applications. Figure 1 shows a PCR-CE system / system 100 incorporating a thermal recycling module / thermal cycler according to one embodiment of the present invention. The PCR-CE system 100 schematically shown in Figure 1 incorporates a detection configuration. The PCR-CE system 100 typically includes a capillary separation column 10 (e.g., 200-500 μm OD) defining an internal separation channel 12 (e.g., 25-200 μm I.D.), which may be a capillary column 10 (only one separation channel / capillary column is shown for brevity). The capillary column 10 may be made of quartz glass, glass, polyimide, or other ceramic / glass material. The inner wall of the separation column 10 (for example, the inner wall which defines the separation channel 12) may be coated with a material capable of accumulating static electricity to promote the electrophoretic and / or electrokinetic transfer of the sample components. The separation channel 12 can be filled with a separation support medium, which may simply be a running buffer or sieving gel matrix (linear or nonlinear polymer composition) known in the art.

[0034] One end of the capillary column 10 is connected to a reservoir 14 of running buffer. The other end of the capillary column 10 is connected to another reservoir 16 which alternately contains the sample (injected into the separation channel 12) and the running buffer (after the sample is injected and then separated). A power supply 18 supplies high voltage to the reservoirs 14 and 16 via electrodes 20 and 22. The capillary column is supported by a cartridge C disclosed in U.S. Patent Publication No. 8,778,155, which is incorporated herein by reference and described in more detail below.

[0035] The mechanisms of electrophoresis and radiation-induced fluorescence are outside the scope of this invention in themselves. For completeness, the operation of the PCR-CE system 100 will be briefly described. In the operation, a prepared biological sample labeled with at least one known fluorescent dye is introduced to the far end of the capillary column away from the detection region by some method not part of this invention (e.g., electrokinetic injection from a sample reservoir or injection by physical pressure using a syringe pump). When a DC potential (e.g., 1-30 kV) is applied to the electrodes 20 and 22 from the power supply 18, the sample moves along the separation channel 12 in direction 24 due to the applied potential (e.g., negatively charged sample moves toward the positive electrode 22, as shown in Figure 1), and is separated into bands of sample components. The separation range and migration distance along the separation channel 12 depend on several factors, such as the mobility of the sample components, the mass and size or length of the sample components, and the separation support medium. The driving force for separating the sample in the separation channel 12 may be electrophoresis, pressure, or electroosmotic flow (EOF).

[0036] When the sample reaches the detection region 32, excitation light is irradiated in the direction 35 of the detection region 32 by the excitation fiber 34. The sample components fluoresce with an intensity proportional to the concentration of each sample component (proportional to the amount of fluorescently labeled substance). The detector 42 detects the intensity of the fluorescence irradiated in the direction 37 via the emission fiber 36 at one or more wavelengths different from the incident radiation. The detected irradiation light may be analyzed by a multicolor (e.g., two-color) detection scheme (see U.S. Patent Application Publication No. 20150338347A1, which is incorporated herein by reference).

[0037] In the automated system, the controller 26 (e.g., a laptop or desktop computer, or a computing unit built into the device) has a processor and controls the operation of various components of the PCR-CE system 100, and controls the capillary electrophoresis separation, data acquisition, and other functions described below. The controller 26 may provide a user interface and program experimental / test settings and parameters. The controller includes necessary application software routines, which may include data reduction applications. The controller 26 may be an integrated part of the system 100 (e.g., part of a system board including application routines coded in ASICs) or it may be an independent unit connected to / coupled to the PCR-CE system 100. Figure 1 shows an embodiment of the PCR-CE system 100 with a separate controller 26.

[0038] In the embodiment shown in Figure 1, the controller is in the form of a desktop or laptop computer, externally connected to the housing of the PCR-CE system 100, and connected to the PCR-CE system 100 via a system board such as a USB interface. The external controller 26 may include a mass storage device, a display, a keyboard, etc. Alternatively, some of these user interface elements may be configured to be integrated into the PCR-CE apparatus (for example, the display and keyboard are integrated into the front housing). Or, without departing from the spirit of the invention, the system board may be incorporated as part of the external controller 26. Specific implementations of such control disclosed herein are within the scope of knowledge familiar to those skilled in the art.

[0039] Figure 2A is a perspective view showing a capillary electrophoresis system in which a thermal cycler / thermal recycling module according to one embodiment of the present invention is integrated. Figure 2B is a cross-sectional view along line AA in Figure 2A. The cartridge C accesses the sample wells 62 of the sample tray 6, which is supported vertically within the capillary column 10 and vertically supported from one end by the first heating column 5, and the reagents in the reagent wells 45 of the reagent tray 4, which is transported by a transport mechanism (not shown, e.g., a transport mechanism that transports various trays in two axis directions XZ (horizontal and vertical) or XY(r)-Z (horizontal and vertical) by a stepping motor). Further details can be found in the patent publications referenced herein. Incidentally, the sample wells 62 of the sample tray 6 corresponding to the reservoir 16 are shown in the schematic diagram of Figure 1. The reagent tray 4 includes, for example, reagent wells 45 for containing buffers accessed by the cartridge C. In the illustrated embodiment, the reagent tray 4 is provided with two small additional reagent wells 45 for additional reagents required for a particular CE protocol, such as alignment markers (AM) and size markers (SM).

[0040] For example, using the PCR-CE system 100, the entire SARS-CoV-2 detection assay can be completed in 90 minutes through high-speed, low-volume (1-20 μl) thermal circulation and integrated high-speed electrophoretic separation and detection. The detection limit for multiplex amplification of genomic DNA in this system is low, at 10-20 copies. The preferred disposable gel cartridge method is a simple yet very robust design method, suitable for mass production using easy-to-operate capillary gel electrophoresis (CGE) equipment, significantly reduces background noise, improves the signal-to-noise ratio, has a very low run cost per sample, and provides high detection sensitivity for biomolecules (e.g., pathogens).

[0041] Gel Cartridge C The reusable single-channel pen-type gel cartridge C allows for easy plug-and-play use with a robust injection-molded body that integrates a gel reservoir design incorporating a microfluidic glass capillary column 10 (e.g., 20-100 μm ID) with an effective separation length of 11 cm. The reduced capillary length allows for a reduction in operating voltage (1-15 KV), eliminating the need for expensive cooling systems such as recirculation chillers. The design includes upper and lower electrodes (anode and cathode), an exposed detection area, and an embedded RFID chip / label that includes the gel cartridge type ID and tracks the number of runs performed per cartridge. Each cartridge contains a linear gel matrix, and analysis on samples can be performed in as little as 2 minutes per run, with 100-300 samples consumed directly from the PCR sample well immediately after polymerase chain reaction. The pen-type cartridge design with a small-bore capillary facilitates sample injection from the PCR sample well, which is directly accessible from the integrated thermal recycling module. This is an open system without a lid / door on the sample well 62 of the sample tray 5 supported by the first heating column 5. The amplified PCR product is then injected directly into the capillary, which is filled with a gel containing a fluorescent dye (gel matrix) for electrophoretic analysis. The reusable / disposable gel cartridge is a simple yet highly robust design method, suitable for mass production with easy-to-operate CGE instruments, significantly reduces background noise, improves the signal-to-noise ratio, has a very low run cost per sample, and provides high detection sensitivity for biomolecules.

[0042] The pen-type gel cartridge integrates an upper buffer / gel reservoir, which is directly connected to a modular pneumatic pump (external to the device). The pneumatic pump (or N2 gas tank) provides the air pressure necessary to fill the capillary (microfluidic channel) with the separation gel / buffer (dynamic coating of the capillary). Based on the viscosity of the separation gel / buffer, the upper buffer / gel reservoir forces the glass capillary to a pressure of up to 60 PSI. Each cartridge reservoir includes a built-in electrode (anode) that automatically connects to an HV power supply for electrophoresis when installed in the device. The anode is embedded in the gel reservoir to capture DNA fragments separated from the large cell of the gel and prevents reinjection into the capillary column during the removal phase between runs. A test sample (coated with mineral oil) is introduced into the separation capillary (microfluidic channel) so as to be electrokinetically injected directly from a PCR sample tray. Using a high-voltage power supply, an electric field of 0-20KV is applied to the capillary to perform electrokinetic injection and separation of biomolecules. An excitation LED with broadband light energy (FWHM=50nm) and a field of view of 100° is connected at a flat input end (polished or cleaved end) to a thick core excitation fiber (100-1000μm). A line filter (bandpass line filter with FWHM=2-50nm) is used in front of the LED, and then the light is coupled to the 300μm core by a 500μm ball-end excitation fiber to reduce background noise. The fluorescence emission signal generated from the separated analyte is collected in the detection region of the capillary using a ball-end fiber (thick core fiber with a 500μm ball) and transmitted by a built-in emission filter (bandpass filter) to the detector module for monochromatic or bichromatic detection (using a PMT, SiPMT, or CCD).

[0043] Thermal Cycling Module / Thermal Cycler The PCR-CE system 100 has an integrated thermal recycling module / thermal cycler. In PCR amplification of DNA / RNA samples, the thermal cycling module is in the form of a thermal cycler and is capable of heating and cooling the sample and / or performing heating and cooling cycles of the sample. It should be noted that in some embodiments, the thermal cycling module can be used as a standalone thermal cycler that is not combined with the CE separation system.

[0044] Figures 3A to 3E are perspective views, top view, front view, left side view, and bottom perspective view of the sample tray 6 according to one embodiment of the present invention, respectively. As shown in Figures 3A to 3E, the sample tray 6 is manufactured from a plastic material, which may be any polymer that can be injection molded to have good physical strength, uniformity, and smoothness, such as polyurethane, polypropylene (PP), PE, PTE, PTFE, urethane, etc. The integrally formed sample tray 6 comprises a frame 61, a plurality of sample wells 62, a support base 63, and a plurality of floating ribs 64. Each sample well 62 corresponds to two floating ribs 64 and is configured to hold, for example, 2 to 100 μl of fluid sample. In the illustrated embodiment, the number of sample wells 62 is 8, but other numbers may be used. The frame 61 is preferably oblong or rectangular and may be athletics field shaped, ring-shaped, or polygonal. In the case of a ring-shaped frame 61, the length direction is defined as the circumferential direction of the ring, and the width direction is defined as the radial direction of the ring. The plurality of sample wells 62 are independent of each other (unconnected) in the length direction L of the rectangular frame 61, and each sample well 62 is connected to the rectangular frame 61 in the width direction W of the rectangular frame 61 by two floating ribs 64 on both sides of the sample well 62. In a preferred embodiment, each floating rib 64 comprises a stepped first rib section 64A and a second rib section 64B that are connected to each other. The first rib section 64A is connected to the sample well 62 and the second rib section 64B, and the second rib section 64B is connected to the first rib section 64A and the frame 61. The thickness H1 of the first rib section 64A is greater than the thickness H2 of the second rib section 64B. For example, the thickness H1 is approximately 1.9 mm, and the thickness H2 is approximately 0.4 mm. Figures 2C and 2D are cross-sectional views showing a thermal recycling module / thermal cycler according to one embodiment of the present invention.Referring to Figures 2C and 2D, in the illustrated embodiment, the openable upper cover 8 of the thermal cycler is contactable with the first rib section 64A to bias a constant pressure over the sample well 62. This ensures good contact between the sample well 62 and the first heating column 5 to further improve the heating state. The first rib section 64A gradually tapers toward the frame 61 along the width direction W (opposite direction of the sample well 62), and the second rib section 64B gradually tapers toward the frame 61 along the width direction W (opposite direction of the sample well 62). In the illustrated embodiment, the support base 63 comprises four support legs, each positioned close to the lower corners of the frame 61. The support base 63 allows the sample tray 6 to be placed directly on a table without the use of a tube holder. Figure 7 is a top view of the sample tray 6 according to another embodiment of the present invention. The sample tray 6 shown in Figure 6 is similar to the sample tray 6 shown in Figures 3A to 3E, but differs in the following respects. It differs from the sample tray 6 shown in Figures 3A to 3E in that the frame 61 is circular and ring-shaped. In the illustrated embodiment, the length direction is defined as the circumferential direction of the ring-shaped frame 61, and the width direction is defined as the radial direction of the circular ring frame 61. The details of the sample tray 6 shown in Figure 6 are the same as those shown in Figures 3A to 3E, so their explanation will not be repeated here.

[0045] Figures 4A to 4C are perspective views, top views, and right side views, respectively, of a thermal recycling module / thermal cycler according to an embodiment of the present invention. Referring to Figures 4A to 4C and Figures 2A and 2B, the thermal cycler comprises a thermal platform P, a thermoelectric module E located below the thermal platform P, and a heat sink (not shown) located below the thermoelectric module E. As is well known in the art, the thermoelectric module E can be operated based on the Peltier effect, which creates a temperature difference by transferring heat between two electrical contacts when a voltage is applied across the contacts. Therefore, the thermoelectric module E adjusts / controls the temperature of the thermal platform P based on the required heating / cooling temperature distribution and dissipates the heat through the heat sink.

[0046] The thermal platform P has a base B that supports a plurality of first heating columns 5 and a plurality of second heating columns 7 to conduct heat. The number of first heating columns 5 corresponds to the number of sample wells 62, and by receiving the corresponding sample wells 62 and supporting them to conduct heat, the thermal platform P heats or cools the fluid sample in the sample wells 62. In the embodiment shown in Figure 2A, a single thermoelectric module E adjusts / controls the temperature of the first heating columns 5 and / or the second heating columns 7 based on the required heating / cooling temperature distribution. According to another embodiment shown in Figure 4A, the thermoelectric module E has a first thermoelectric module E1 and a second thermoelectric module E2 that can be controlled independently. The temperatures of the plurality of first heating columns 5 and the plurality of second heating columns 7 are controlled separately by the first thermoelectric module E1 and the second thermoelectric module E2. The base B, the plurality of first heating columns 5, and the plurality of second heating columns 7 are typically made of metal (e.g., aluminum) or an alloy. The base B, the plurality of first heating columns 5, and the plurality of second heating columns 7 may be independent structures connected to each other to conduct heat, or they may be integrally molded. In the illustrated embodiment, the eight first heating columns 5 are connected to each other in the longitudinal direction L. In other embodiments, the eight first heating columns 5 are connected to each other in the longitudinal direction.

[0047] As shown in Figure 2A, the plurality of first heating columns 5 and second heating columns 7 may be equipped with one or more first temperature sensors (e.g., thermocouples, not shown) for temperature feedback to control the temperature distribution of the thermoelectric module E. Alternatively, as shown in Figure 4A, the plurality of first heating columns 5 may be equipped with one or more first temperature sensors (e.g., thermocouples, not shown) for temperature feedback to control the temperature distribution of the first thermoelectric module E1. The plurality of second heating columns 7 may also be equipped with one or more second temperature sensors (e.g., thermocouples, not shown) for temperature feedback to control the temperature distribution of the second thermoelectric module E2. The contour of the outer surface 621 of each sample well 62 gradually tapers, and each first heating column 5 is equipped with a cone-shaped hole 51 that matches the contour of the outer surface 621 of the sample well 62. Furthermore, the unconnected sample wells 62 and floating ribs 64 in the longitudinal direction L allow each sample well 62 to be displaced to a limited extent in the vertical, longitudinal, and width directions. This ensures that each first heating column 5 is in close contact with the outer surface 621 of the corresponding sample well 62, improving the heat transfer efficiency between them.

[0048] Referring to Taiwan Patent Application Publication No. 201623938A and China Patent Application Publication No. 118580940A, the sample wells of a conventional sample tray (8-tube strip / PCR tube) are connected in series along the length. In practice, the wells of the 8-tube strip may not be able to make close contact with the heating block, and due to the cumulative tolerance between the wells and the heating block, the contact fails, resulting in a decrease in PCR amplification efficiency. After PCR amplification, one or more of the 8 samples could not be subjected to subsequent CE separation and analysis. To solve the above problem, the sample tray according to the present invention and the conventional 8-tube strip were used separately, and the same PCR protocol was performed using the same sample. The experimental results showed that performing PCR on the sample sample using the sample tray according to the present invention resulted in a higher yield, and the problem of low heating efficiency due to cumulative tolerance was overcome.

[0049] In the illustrated embodiment, the wall thickness of each sample well 62 of the sample tray 6 is in the range of 0.15 mm to 0.25 mm, preferably 0.2 mm. In contrast, the wall thickness of the sample wells in the sample tray according to the prior art is 0.5 mm. The performance of the combination of the sample wells and the first heating column configuration was verified. Experimental results showed that using the sample wells and the first heating column configuration according to the present invention resulted in a maximum heating rate and maximum cooling rate of 5 to 5.5°C / sec for the sample wells, which is far superior to the 1.8 to 3°C / sec of the prior art. In addition to the improved thermal conductivity, the thin wall thickness allows the sample wells 62 to flexibly fit into the conical holes of the corresponding first heating column 5, thereby improving thermal efficiency. As shown in Figure 2A, the sample wells 62 of the sample tray 6 are inserted into the conical holes of the first heating column 5, and the support base 63 is joined to the upper surface of the base B.

[0050] Heating / Cooling Performance Verification In this test, a data logger and two K-type thermocouples were used to verify the heating rate, cooling rate, and uniformity of the combination of the sample tray 6 and the first heating column 5 according to the present invention, and the combination of the conventional 8-tube strip (PCR tube) and heating block. Figure 5A shows the heating / cooling curve of the well of the conventional 8-tube strip, and Figure 5B shows the heating / cooling curve of the sample well according to the present invention. As shown in Figures 5A and 5B, the well of the conventional 8-tube strip had a maximum heating rate of 3°C / s and a maximum cooling rate of 1.8°C / s. In contrast, the sample well according to the present invention had a maximum heating rate of 5°C / s and a maximum cooling rate of 5.5°C / s. In addition, the sample well with the floating rib design was shown to have better temperature uniformity compared to the conventional 8-tube strip.

[0051] PCR test this inspection An ALDH2 (aldehyde dehydrogenase) gene testing kit has been introduced. ALDH2 is located on chromosome 12 and is responsible for the oxidation reaction of acetaldehyde. The single nucleotide polymorphism (SNP) in the ALDH2 gene is a substitution from G to A, resulting in the formation of the Glu504Lys allele of ALDH2, thus resulting in three different genotypes: G / G homozygous (wild type, WT), G / A heterozygous (heterozygous), and A / A homozygous (mutant, MT). The G to A mutation alters the protein structure, reduces acetaldehyde metabolism, and also reduces alcohol tolerance. The incidence of the ALDH2 gene SNP in different East Asian countries ranges from 35% to 57%. This is the reason why Asians are more prone to flushing reactions when they consume alcohol.

[0052] Multiplex polymerase chain reaction (multiplex PCR) was performed on the same sample using the combination of the sample tray 6 and the first heating column 5 according to the present invention, and the combination of the conventional 8-tube strip and heating block. Figure 6A is a graph showing the relationship between the migration time and relative fluorescence units (RFU) of two tubes (wells) of the conventional 8-tube strip, and Figure 6B is a graph showing the relationship between the migration time and relative fluorescence units (RFU) of two sample wells 62 of the sample tray 6 according to the present invention. In this test, the ratio of the mutant (MT) peak to the wild-type (WT) peak must be less than 1:5. As shown in Figure 6A, when a multiplex PCR reaction was performed on the sample in two tubes (wells) of the conventional 8-tube strip, the MT / WT ratio of one tube exceeded the above ratio. As shown in Figure 6A, after the multiplex PCR reaction, the MT / WT ratio of both sample wells 62 was less than 1:5. The results described above indicate that the combination of the first heating column 5 and the sample tray 6 exhibits excellent thermal uniformity and improves heating efficiency by eliminating the cumulative tolerance.

[0053] As shown in Figures 2A, 2B, and 4A-4C, in the illustrated embodiments, the second heating column 7 is configured to pre-treat the sample. Each second heating column 7 receives a sample tube 71 having a conical bottom and has a conical cavity for heating or cooling the sample in the sample tube 71. In the illustrated embodiments, the sample tube functions to inactivate enzymes such as protease K. The pre-treated sample is then transferred to the sample well 62 of the sample tray 6 for PCR amplification.

[0054] In other alternative embodiments, the plurality of first heating columns 5 and the plurality of second heating columns 7 are used to perform PCR thermal circulation, and a transport mechanism is used to enable automated analysis of up to 16 PCR products.

[0055] Incidentally, the thermal recycling module / thermal cycler according to the present invention is an open system that does not require a lid. The sample in the sample well 62 of the sample tray 6 is covered with a thin layer of mineral oil to prevent evaporation and heat loss. When mineral oil is used, the sample well 62 does not require a separate cover or lid. Such an open system allows CE to be performed with fewer PCR cycles, thus reducing the overall operating time.

[0056] Incidentally, the aforementioned thermal cycling module, equipped with the sample tray 6, can be used for other temperature-sensitive reactions besides polymerase chain reaction (PCR), such as restriction enzyme digestion and rapid diagnostic tests, but the present invention is not limited to these.

[0057] As described above, specific embodiments of the present invention are described herein for illustrative purposes, but various modifications are possible that do not depart from the scope of the invention. Therefore, the present invention is limited only by the appended claims.

[0058] Although the present invention has been described above using embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be obvious to those skilled in the art that various modifications or improvements can be made to the above embodiments. Furthermore, it will be obvious to those skilled in the art that such modifications or improvements can be made. Moreover, it will be clear from the claims that such modified or improved forms may also be included within the technical scope of the present invention. [Explanation of symbols]

[0059] B Bass C Cartridge E Thermoelectric Module E1 Thermoelectric Module No. 1 E2 Second Thermoelectric Module L (Length direction) P Heating Platform W (width direction) 4 Reagent trays 5. First heating column 6 Sample Trays 7. Second heating column 8. Top cover 10 capillary columns 12 Separation Channels 14 Reservoirs 16 Reservoirs 18 Power supply 20 electrodes 24 directions 32 Detection area 34 Excitation Fibers 35 directions 36 Light-emitting fibers 37 directions 42 detectors 45 reagent wells 61 frames 62 sample wells 63 Support stand 64 Floating Ribs 64A First Rib Section 64B Second Rib Section 100 PCR-CE Systems

Claims

1. Multiple sample wells, Frame and, A plurality of floating ribs, each of the plurality of sample wells comprising a plurality of floating ribs corresponding to two of the plurality of floating ribs, A sample tray for temperature-sensitive reactions, characterized in that the plurality of sample wells are not connected to each other in the longitudinal direction of the frame, and each of the plurality of sample wells is connected to the frame in the width direction of the frame by two of the plurality of floating ribs on either side of the sample well.

2. Each of the plurality of floating ribs is stepped and comprises a first rib section and a second rib section connected to each other, the first rib section being connected to the corresponding sample well and the second rib section, the second rib section being connected to the first rib section and the frame, and the thickness of the first rib section being greater than the thickness of the second rib section, as described in claim 1, for a temperature-sensitive reaction sample tray.

3. The sample tray for a temperature-sensitive reaction according to claim 2, characterized in that the first rib section gradually tapers toward the frame along the width direction.

4. The sample tray for a temperature-sensitive reaction according to claim 2, characterized in that the second rib section gradually tapers toward the frame along the width direction.

5. The sample tray for a temperature-sensitive reaction according to claim 2, characterized in that the two stepped floating ribs allow for the displacement of the corresponding sample wells in the vertical, longitudinal, and width directions.

6. The sample tray for temperature-sensitive reactions according to claim 1, characterized in that the wall thickness of each of the plurality of sample wells is in the range of 0.15 mm to 0.25 mm.

7. The sample tray for temperature-sensitive reactions according to claim 1, characterized in that the heating rate or cooling rate of the plurality of sample wells is 5°C / sec or higher.

8. The sample tray for a temperature-sensitive reaction according to claim 1, further comprising a support base for supporting the frame, which is connected to the frame.

9. The sample tray for a temperature-sensitive reaction according to claim 1 is characterized in that the sample tray is integrally molded.

10. The sample tray for a temperature-sensitive reaction according to claim 1 is characterized in that the sample tray is rectangular in shape.

11. The sample tray for a temperature-sensitive reaction according to claim 1, characterized in that the frame is ring-shaped, the length direction is the circumferential direction of the frame, and the width direction is the radial direction of the frame.

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