Sample tray for temperature-sentitive reactions

Abstract

A sample tray for temperature-sensitive reactions includes wells and a frame. The wells are disconnected one another in a length direction of the frame, and each well is connected to the frame in a width direction of the frame through two floating ribs on both sides of the well.

Description

[Technical Field]

[0001] This invention relates to a bioanalysis, and more specifically to a sample tray for temperature-sensitive reactions, such as, but not limited to, polymerase chain reaction (PCR), restriction enzyme digestion, or rapid diagnostics. [Previous Technology]

[0002] Capillary electrophoresis (CE) separation is a microfluidic method or a simplified microchannel device for gel electrophoresis separation, with its greatest advantage being its wide range of applications. As a reliable, high-resolution, and highly sensitive separation and detection tool, CE technology has been widely accepted by the biotechnology industry, especially for nucleic acid-based assays. CE has been applied to the analysis of proteins, carbohydrates, and DNA, such as oligonucleotide analysis, DNA sequencing, dsDNA fragment analysis, and glycan analysis.

[0003] U.S. Patent Publication No. 8,778,155, jointly assigned to the present invention patentee, discloses a reusable, cartridge-based portable electrophoresis system comprising a pen-shaped bio-separation cartridge that is easy to assemble and use, has no moving elements, and integrates a reagent (separation buffer) reservoir. The cartridge includes a body defining an opening as a detection window for receiving external detection optics, and at least one capillary column supported therein, having a first end extending beyond a first end of the body, wherein a portion of the detection window is exposed along the capillary column, the external optics are aligned with the capillary column through the detection window, and a gel reservoir is connected to a second end of the body, allowing fluid flow communication with the second end of the capillary column. The gel reservoir is configured to be connected to a pneumatic pump or nitrogen tank to pressurize the gel reservoir and purge and fill the capillary with buffer / gel as a separation support medium. Negatively charged samples can be loaded into the capillary column and CE separation performed for analysis. When sample separation is complete, the used sample (the negatively charged separated DNA / RNA fragments) is captured in a gel reservoir (large tank) located away from the capillary column outlet, near the embedded electrode (anode). Another sample can then be loaded into the capillary column and analyzed in a subsequent run using the same cartridge.

[0004] Polymerase chain reaction (PCR) is a laboratory technique associated with CE-based DNA / RNA analysis for the rapid generation (amplifying) of millions to billions of specific DNA fragments, which can then be studied; more detail at high resolution can be obtained without requiring a large initial sample size. PCR involves using short synthetic DNA / RNA fragments called primers to select the genomic fragment to be amplified, and then performing multiple DNA / RNA synthesiss to amplify that fragment before separation and detection analysis by conventional gel electrophoresis or capillary electrophoresis (CE), thus providing high-resolution results based on an initial low sample intake (e.g., a few picoliters). The PCR process requires thermal cycling of the sample DNA fragments to prepare the sample for subsequent CE separation analysis.

[0005] To date, the conventional practice has been to perform a separate PCR process using a thermal cycler, which is not part of the CE separation and detection system / instrument, and then transfer the prepared sample to the CE separation and analysis system. U.S. Patent No. 11,531,004, commonly assigned to the present invention patent holder, discloses a CE instrument that includes a temperature control mechanism, such as a Peltier heating / cooling module, connected (e.g., connected below) to a stage supporting sample vials to be positioned below a capillary cartridge. The heating / cooling module provides temperature control to facilitate PCR amplification and electrophoresis / detection within the same instrument.

[0006] US Patent Publication No. US20230330680A1 discloses a portable electrophoresis system integrating a thermal cycler, wherein the thermal control module of the thermal cycler includes: a thermal platform thermally coupled to a thermoelectric module, comprising: a base, and at least one heating block thermally supported on the base, the heating block being configured to receive a sample tray containing at least one sample, wherein the heating block includes a body comprising a split longitudinal block having two longitudinal sides defining a valley, wherein the opposing walls of the two longitudinal sides each have a fan-shaped concave profile conforming to the convex conical tube shape profile of the bottom surface of the sample well of the sample tray; a heat sink; and a thermoelectric module thermally coupled between the base of the thermal platform and the heat sink, heating / cooling the thermal platform according to a desired heating / cooling temperature distribution.

[0007] Republic of China Patent Publication No. TW201623938A discloses a temperature control module for PCR thermal cycling, which includes a temperature adjustment component and a heater. The temperature adjustment component is made of resin and a substance with a higher thermal conductivity than resin. The heater is used to control the temperature of the temperature adjustment component. One of the 8-tube reaction tubes is inserted into the temperature adjustment component, which contacts the bottom of the 8-tube reaction tube.

[0008] China Mainland Patent Publication No. CN118580940A discloses a nucleic acid amplification reaction container and a nucleic acid amplification reaction apparatus. The nucleic acid amplification reaction container includes a support and at least one transparent or translucent tubing. The top of the support has an inlet, the number of which matches the number of tubing, and the lower end of the inlet is connected to and communicates with the top of the tubing. The tubing is used to inject nucleic acid amplification reaction solution, and the bottom of the tubing has a seal. The support also includes sealing caps, the number of which matches the number of inlets, for sealing the inlets.

[0009] During PCR amplification, the accuracy and uniformity of temperature control are crucial. Inaccurate heating temperatures can lead to problems in PCR amplification, such as low amplification efficiency, absence of amplified bands, the appearance of nonspecific amplified bands, and the formation of smeared bands, making subsequent analysis impossible and even requiring resampling. [Summary of the Invention]

[0010] In one aspect of the invention, a sample tray for a temperature-sensitive reaction includes a plurality of sample slots and a frame. The plurality of sample slots are independent of each other along the length of the frame, and each sample slot is connected to the frame along the width of the frame via two floating ribs located on either side of the sample slot.

[0011] In one embodiment, each floating rib is stepped and has a first rib segment and a second rib segment connected to each other, wherein the first rib segment connects to the corresponding sample slot and the second rib segment, the second rib segment connects to the first rib segment and the frame, and the thickness of the first rib segment is greater than the thickness of the second rib segment.

[0012] In one embodiment, the first rib gradually tapers away from the sample cell.

[0013] In one embodiment, the second rib gradually tapers away from the sample well.

[0014] In one embodiment, the two stepped floating ribs allow the sample groove to be displaced in the vertical direction as well as in the length and width directions.

[0015] In one embodiment, the wall thickness of each sample cell is between 0.15 mm and 0.25 mm.

[0016] In one embodiment, the heating or cooling rate of the plurality of sample cells is equal to or greater than 5°C / sec.

[0017] In one embodiment, the sample tray further includes a support base that connects to and supports the frame.

[0018] In one embodiment, the sample tray is integrally formed.

[0019] In one embodiment, the border is rectangular.

[0020] In one embodiment, the border is annular, with the length direction being the circumferential direction of the border and the width direction being the radial direction of the border.

Implementation Method

[0021] The present invention will now be described with reference to the accompanying drawings and various embodiments. Although the present invention has been described in accordance with the best mode for carrying out the objectives of the invention, those skilled in the art will understand that variations in embodiments based on these teachings may be implemented without departing from the concept or scope of the invention.

[0022] Reference is made to the bioanalytical systems including detection systems disclosed in U.S. Patent Nos. 8,778,155, 8,784,626, and 11,531,004, and U.S. Patent Application Publications US20150338347A1 and US20230330680A1, the entire contents of which are incorporated herein by reference and are considered a part of this specification. These patents and patent applications are collectively assigned to the applicant and patentee of this invention, BiOptic, Inc. In particular, these patents disclose a simplified, low-cost, high-efficiency, high-sensitivity, high-analytical-volume bioseparation system (e.g., a capillary electrophoresis (CE) system). The bioseparation system includes an instrument configured to work with a single-channel or multi-channel capillary cartridge and is provided with a detection configuration including optical elements for applying incident radiation along a detection zone of a separation channel and detecting output radiation from that detection zone. Optical elements detect radiation emitted by the sample analyte (e.g., photoexcited fluorescence), but precise alignment of the optical elements with the separation column is not required. The instrument is configured to automate bioseparation within the separation channel of the bioseparation cartridge. The CE system features a simpler optical detection mechanism, reducing costs while offering ease of operation, rapid analysis, high efficiency, high sensitivity, and large analytical capacity. US20150338347A1 further discloses two-color fluorescence detection. US20230330680A1 further discloses a heating control module. This invention employs and modifies these systems to include improvements to the system as disclosed below (including a thermal cycling module for PCR).

[0023] To illustrate the principles of the invention and not to limit it, the present invention will be described below using an example of capillary electrophoresis with a single capillary separation column. Furthermore, the invention will be described in conjunction with photoexcited fluorescence detection (e.g., using a laser or LED light source for excitation), but is not limited thereto.

[0024] System Overview

[0025] The miniaturization and automation of analytical instruments offer many advantages over traditional labor-intensive techniques (i.e., manual slab-gel electrophoresis). These advantages include improved data accuracy and reproducibility, shorter analysis time, minimal sample consumption, increased automation, and integration of complex workflows.

[0026] In one embodiment, a fully automated bioseparation system is a CE instrument (hereinafter referred to as PCR-CE instrument or system) incorporating a thermal cycling module / thermal cycler to provide direct terminal PCR testing with rapid amplification, high-speed separation, and fluorescence detection in clinical applications. Figure 1 is a schematic diagram of a PCR-CE system including a thermal cycler / thermal cycling module according to an embodiment of the present invention. The PCR-CE system 100 includes the detection configuration shown in the schematic diagram of Figure 1. The PCR-CE system 100 typically includes a capillary column 10 (external diameter, for example, 200-500 μm) defining an internal separation channel 12 (internal diameter, for example, 25-200 μm; for simplicity, only one separation channel / capillary column is illustrated). The capillary column 10 may be made of fused silica, glass, polyimide, or other ceramic / glass materials. The inner wall of the capillary column 10 (i.e., the wall defining the separation channel 12) may be coated with a material capable of accumulating electrostatic charge to promote electrophoresis and / or electrokinetic migration of sample components. The separation channel 12 can be filled with a separation support medium, which can be a running buffer or a sieving gel matrix (linear or nonlinear polymer composition) known in the art.

[0027] One end of the capillary column 10 is connected to a reservoir 14 for electrophoresis buffer. The other end of the capillary column 10 is connected to another reservoir 16, which may alternately contain the sample (to be injected into the separation channel 12) and the running buffer (after sample injection for separation). A power source 18 provides a high voltage to the reservoirs 14 and 16 through electrodes 20 and 22. The capillary column 10 is supported by a cartridge C disclosed in U.S. Patent No. 8,778,155, which is incorporated herein by reference and will be discussed further below.

[0028] The mechanisms of electrophoresis and photoexcitation fluorescence, when considered individually, are not within the scope of this invention. For completeness, the operation of the PCR-CE system 100 is briefly described below. In operation, a biological sample labeled with at least one known fluorophore can be introduced into the distal end of a capillary column away from the detection region by any method not part of this invention (e.g., electrokinetic injection from a sample reservoir or physical pressure injection using a syringe pump). When a DC potential (e.g., 1-30 kV) is applied to electrodes 20 and 22 by power source 18, the sample migrates along separation channel 12 in direction 24 at the applied potential (e.g., negatively charged sample flows towards positive electrode 22) and separates into multiple sample component bands. The degree of separation and the distance traveled along separation channel 12 depend on many factors, such as the migration rate 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 separation channel 12 can be electrophoresis, pressure, or electroosmotic flow (EOF).

[0029] When the sample reaches the detection area 32, the excitation light is directed to the detection area 32 along direction 35 via the excitation fiber 34. The sample components emit fluorescence, the intensity of which is proportional to the concentration of the individual sample components (and proportional to the amount of fluorescently labeled material). The detector 42 detects the intensity of the fluorescence emitted along direction 37 via the emission fiber 36 at one or more wavelengths different from the incident light wavelength. The detected emitted light can be analyzed by a multicolor (e.g., dual-color) detection structure (for further details, refer to US20150338347A1, incorporated herein by reference).

[0030] The automated system includes a controller 26 with a processor (e.g., in the form of an external notebook or desktop computer, or a computing unit integrated into the instrument or system) to control the operation of various components such as capillary electrophoresis separation and data collection, as well as control of other functions discussed below. The controller 26 may provide a user interface and programming for experimental / test settings and parameters. The controller includes necessary application software routines, and may also include data reduction applications. The controller 26 may be an integrated part of the instrument 100 (e.g., as part of a system board with ASIC-coded applications), or it may be a standalone unit coupled / interfaced to the PCR-CE instrument 100. An embodiment of Figure 1 shows a PCR-CE system 100 with a separation controller 26.

[0031] In the embodiment shown in FIG. 1, the controller 26 is located outside the housing of the PCR-CE system 100 and is coupled to the PCR-CE system 100 via a system board (e.g., via a USB interface) in the form of a desktop or notebook computer. The external controller 26 may include a mass storage device, a display, a keyboard, etc., or some of these user components may be configured to be integrated with the PCR-CE system (e.g., integrated with the display and keyboard of the front housing of the PCR-CE system). Alternatively, the system board may be integrated as part of the external controller 26 without departing from the scope and spirit of the invention. Specific implementations of the controller 26 based on the disclosure herein are within the knowledge of those skilled in the art.

[0032] Figure 2A is a perspective view of a capillary electrophoresis system integrating a thermal cycler / thermal cycler module according to an embodiment of the present invention. Figure 2B is a cross-sectional view of Figure 2A along the A-A direction. Referring to Figures 2A and 2B, the cartridge C is vertically supported by a capillary column 10, which is suspended from one end to receive reagents in the sample slots 62 of the sample tray 6 and the reagent slots 45 of the reagent tray 4, which are supported on a plurality of first heating columns 5. The sample trays 3 and 4 are carried by a transfer mechanism (not shown) (e.g., by a stepper motor in the XZ (horizontal and vertical) biaxial or XY(r)-Z (horizontal and vertical) triaxial directions). Further details can be found in the patent applications incorporated herein by reference. It is noteworthy that the sample slot 62 of the sample tray 6 corresponds to the reservoir 16 of Figure 1. The reagent tray 4 includes a reagent slot 45 for containing, for example, a buffer solution, for access by the cartridge C. In the illustrated embodiment, the reagent tray 4 is provided with two additional smaller reagent slots 45 for additional reagents that may be required for certain CE procedures, such as alignment markers (AM) and size markers (SM).

[0033] As an example, using the PCR-CE system 100, a complete assay of SARS-CoV-2 can be completed within 90 minutes due to rapid, small-volume (1-20 μL) thermal cycling and integrated high-speed electrophoretic separation and detection. The detection limit of the PCR-CE system of this invention for multiplex amplification of genomic DNA is as low as 10-20 copies. The preferred technology of disposable gel cartridges is a simple yet very robust design suitable for high-volume manufacturing of easy-to-operate capillary gel electrophoresis (CGE) instruments, which significantly reduces background noise, thereby improving the signal-to-noise ratio (S / N) and sensitivity of biomolecules (e.g., pathogens) at a very low cost per sample run.

[0034] Gel cartridge C

[0035] The single-channel pen-shaped gel cartridge C is reusable and can be easily plugged and played in a robust injection-molded body with an integrated gel reservoir. This design incorporates a microfluidic glass capillary column 10 (external diameter, e.g., 20-100µm) with an effective separation length of 11cm. The shortened capillary length allows for a lower operating voltage (1-15 kV) and eliminates the need for expensive cooling systems, such as circulating coolers. The design includes top and bottom electrodes (anode and cathode), exposed detection areas, and an embedded RFID chip / tag to provide an ID for the gel cartridge type and track the number of runs for each cartridge. Each reagent cartridge contains a linear gel matrix, allowing analysis of 100-300 samples per run in just 2 minutes, with sample consumption as low as 1 pl directly from the PCR sample well after polymerase chain reaction. The pen-shaped cartridge design with narrow-pore capillary facilitates sample injection from a directly accessible PCR sample well with an integrated thermal cycling module. This is an open system where the sample tray 6, supported by multiple first heating columns 5, has no cap on its sample slot 62. The amplified PCR can then be directly injected into a gel-filled capillary containing a fluorescent dye (gel matrix) for electrophoretic analysis. The reusable / disposable gel cartridge is a simple yet very robust design suitable for high-volume manufacturing of easy-to-operate capillary gel electrophoresis (CGE) instruments. It significantly reduces background noise, thereby improving the signal-to-noise ratio (S / N) and sensitivity for detecting biomolecules (e.g., pathogens) at a very low cost per sample run.

[0036] The pen-shaped gel cartridge integrates a gel reservoir directly coupled to a modular air compressor pump (external to the instrument). The air compressor (or nitrogen chamber) provides the required pressure to fill the capillary (microfluidic channel) with the separating gel / buffer (dynamic coating of the capillary). Depending on the viscosity of the separating gel / buffer, pressures up to 60 PSI are applied to the glass capillary through the top buffer / gel reservoir. Each cartridge's reservoir is equipped with a built-in electrode (anode) that automatically connects to the high-voltage power supply used for electrophoresis when installed inside the instrument. The anode is embedded in the gel reservoir to capture DNA fragments separated from the gel reservoir, preventing them from being reinjected into the capillary column during the purging phase between runs. The test sample (coated with mineral oil) is introduced directly into the separating capillary (microfluidic channel) from the PCR sample tray via electrokinetic injection. A high-voltage power supply provides an electric field of 0-20 kV to the capillary for electrokinetic injection and separation of biomolecules. An excitation LED light source with wideband (FWHM = 50 nm) light and a 100-degree viewing angle is coupled to a large-core excitation fiber (100-1000 μm) at a flat input end (polished or split end). A line filter (bandpass line filter with FWHM = 2-50 nm) is used in front of the LED, and then a 500 μm ball-end excitation fiber is used to couple the light to a 300 μm core to reduce background noise. Then, a ball-end fiber (large-core fiber with a 500 μm ball end) is used to collect the fluorescence emission signal generated by the separated analytes in the detection region of the capillary, and transmits it to the detection module (using a photomultiplier tube PMT, silicon photomultiplier tube SiPMT, or CCD) through a built-in emission filter (bandpass filter) for monochromatic or dual-color type detection.

[0037] Heat circulation module / heat circulation device

[0038] The PCR-CE system 100 has an integrated thermal cycling module. For PCR amplification of DNA / RNA samples, the thermal cycling module heats, cools, and / or cycles the samples in the form of a thermal cycler. Notably, in some embodiments, the thermal cycling module may be used as a standalone thermal cycler without being integrated with the CE separation system.

[0039] Figures 3A to 3E are perspective views, top views, front views, left side views, and bottom views of a sample tray 6 according to an embodiment of the present invention. As shown in Figures 3A to 3E, the entire sample tray 6 is preferably made of plastic material by injection molding. The material used is a polymer with good physical strength, uniformity, and smoothness—for example, polyurethane, polypropylene (PP), PE, PTE, PTFE, etc. The integrally molded sample tray 6 includes a frame 61, a plurality of sample slots 62, a support base 63, a plurality of floating ribs 64, etc. Each sample slot 62 corresponds to two floating ribs 64 and can hold, for example, a fluid sample with a volume of 2-100 μL. In the illustrated embodiment, the number of sample slots 62 is 8, but other numbers are also possible. The frame 61 is preferably rectangular, but it can also be rectangular, track and field shaped, ring-shaped, or polygonal. If the frame 61 is ring-shaped, 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. Multiple sample slots 62 are independent of each other (not connected) in the length direction L of the frame 61, while each sample slot 62 is connected to the frame 61 in the width direction W through two floating ribs 64 located on both sides of the sample slot 62. In a preferred embodiment, the floating ribs 64 are stepped, having a first rib segment 64A and a second rib segment 64B connected to each other. The first rib segment 64A connects the sample slot 62 and the second rib segment 64B, and the second rib segment 64B connects the first rib segment 64A and the frame 61. The thickness H1 of the first rib segment 64A is greater than the thickness H2 of the second rib segment 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 of a thermal cycler / thermal cycle module according to an embodiment of the invention in different directions. Referring to Figures 2C and 2D, in the illustrated embodiment, an openable top cover 8 of the thermal cycler / thermal cycle module can contact the first rib 64A to apply consistent pressure to the top of each sample cell 62. This ensures good contact between the sample cells 62 and the first heating column 5 for better heat conduction. The first rib 64A tapers upwards along the width direction W towards the frame (opposite to the sample cells 62), and the second rib 64B tapers upwards along the width direction W towards the frame (opposite to the sample cells 62). In the illustrated embodiment, the support base 63 has four support feet, located approximately below the four corners of the frame 61. The design of the support base 63 allows the sample tray 6 to be placed directly on a table without the need for a test tube rack. Figure 7 is a top view of the sample tray 6 according to another embodiment of the present invention. The difference between the sample tray 6 shown in Figures 3A to 3E is that the frame 61 is annular. In this embodiment, the length direction is defined as the circumferential direction of the annular frame 61, and the width direction is defined as the radial direction of the annular frame 61.The details of sample tray 6 in this embodiment are the same as those of sample tray 6 shown in Figures 3A to 3E, and will not be repeated here.

[0040] Figures 4A to 4C are a perspective view, a top view, and a right-side view of a thermal circulator / thermal circulation module according to an embodiment of the present invention. Referring to Figures 4A to 4C and Figures 2A and 2B, the thermal circulator / thermal circulation module includes a heating platform P, a thermoelectric module E located below the heating platform P, and a heat sink (not shown) located below the thermoelectric module E. As is known in the art, the thermoelectric module E can operate according to the Peltier effect, which generates a temperature difference by applying a voltage to two electrical contacts, causing heat to transfer between the two electrical contacts. Therefore, the thermoelectric module E adjusts / controls the temperature of the heating platform P according to the desired heating / cooling temperature distribution and dissipates heat through the heat sink.

[0041] The heating platform P has a base B and a plurality of first heating columns 5 and a plurality of second heating columns 7 thermally supported by the base B. The number of first heating columns 5 corresponds to the number of sample tanks 62, receiving and thermally supporting the corresponding sample tanks 62, thereby heating or cooling the fluid sample within the sample tanks 62. In the embodiment shown in FIG2A, there is one thermoelectric module E, which heats / cools the first heating column 5 and / or the second heating column 7 according to the desired heating / cooling temperature. In another embodiment shown in FIG4A, the thermoelectric module E has a separately controllable first thermoelectric module E1 and a second thermoelectric module E2, wherein the temperature of the plurality of first heating columns 5 is individually controlled by the first thermoelectric module E1, and the temperature of the plurality of second heating columns 7 is individually controlled by the second thermoelectric module E2. The base B, the plurality of first heating columns 5, and the plurality of second heating columns 7 are made of metal (e.g., aluminum) or alloy. The base, the plurality of first heating columns 5, and the plurality of second heating columns 7 may be individual structures thermally coupled together, or may be a single integral structure. In the illustrated embodiment, the eight first heating columns 7 are connected to each other in the length direction L, but in another embodiment, the eight first heating columns 7 may not be connected to each other in the length direction.

[0042] As shown in FIG2A, the plurality of first heating columns 5 and the plurality of second heating columns 7 may include one or more first temperature sensors (e.g., thermocouples, not shown) to provide temperature feedback, thereby controlling the temperature curve of the thermoelectric module E shown in FIG1. ​​Alternatively, as shown in FIG4A, the plurality of first heating columns 5 may have one or more first temperature sensors (e.g., thermocouples, not shown) to provide temperature feedback, thereby controlling the temperature curve of the corresponding first thermoelectric module E1. The plurality of second heating columns 7 may have one or more second temperature sensors (e.g., thermocouples, not shown) to provide temperature feedback, thereby controlling the temperature curve of the corresponding second thermoelectric module E2. The outer surface 621 of each sample slot 62 has a tapered profile, and each first heating column 5 has a tapered hole 51 that conforms to the profile of the outer surface 621 of the sample slot 62. Furthermore, the stepped floating ribs 64 and the sample slots 62 that are not connected in the length direction allow each sample slot 62 to have limited displacement in the vertical direction and in the length and width directions. For example, when the sample slot 62 is inserted downward into the conical hole 51 corresponding to the first heating column 5, each sample slot 62 is allowed to move in the vertical direction as well as in the length and width directions. This can further ensure that each first heating column 5 is in close contact with the outer surface 621 of the corresponding sample slot 62, thereby improving the heat conduction efficiency between the two.

[0043] In conventional technologies, such as TW201623938A and CN118580940A, multiple sample holders (e.g., 8-tube reaction tubes) are connected to each other along their length. In practice, it has been found that the fit between the 8-tube reaction tubes and the heating block may be compromised due to accumulated tolerances in the components, resulting in poor PCR amplification efficiency. In some cases, one or more of the 8 amplified samples may not be suitable for subsequent CE separation and analysis. Experiments have shown that when the same 8 samples are subjected to the same PCR thermal cycle using the sample tray of this invention and conventional 8-tube reaction tubes, the PCR amplification samples using the sample tray of this invention do not exhibit the problem of poor heating efficiency caused by accumulated tolerances.

[0044] Furthermore, in some embodiments, the wall thickness of each sample slot 62 of the sample tray 6 is between 0.15 mm and 0.25 mm, preferably 0.2 mm. The wall thickness of the sample slots in the previously designed sample tray is 0.5 mm. The thinner wall thickness not only improves the heat conduction rate but also gives the sample slot 62 better elasticity, allowing it to elastically fit into the conical hole corresponding to the first heating column 5, thereby improving the heat conduction efficiency. As shown in FIG2A, when the sample slots 62 of the sample tray 6 are respectively inserted into the holes corresponding to the first heating column 5, the support 63 can just abut against the upper surface of the base.

[0045] Heating / Cooling Performance Test:

[0046] In this test, a data logger and two K-type thermocouples were used to check the heating and cooling rates and uniformity of the combination of the first heating column 5 and the sample tray 6, as well as the combination of the conventional 8-tube reaction tube and the heating block. Figure 5A shows the heating / cooling curve of the conventional 8-tube reaction tube, and Figure 5B shows the heating / cooling curve of the sample cell of the present invention. As shown in Figures 5A and 5B, the maximum heating rate of the conventional 8-tube reaction tube is 3˚C / s, and the maximum cooling rate is 1.8˚C / s; the maximum heating rate of the sample cell of the present invention is 5˚C / s, and the maximum cooling rate is 5.5˚C / s. Furthermore, compared with the heating uniformity of the 8-tube reaction tube, the sample cell of the present invention, with its floating rib design, exhibits better heating uniformity between the two sample cells.

[0047] PCR test:

[0048] The PCR efficiency of a thermal cycler was evaluated using an aldehyde dehydrogenase (ALDH2) gene assay. Aldehyde dehydrogenase (ALDH2) is located on chromosome 12 and is responsible for the oxidation of acetaldehyde. A single nucleotide polymorphism (SNP) in the ALDH2 gene, a G-to-A substitution, results in the ALDH2 Glu504Lys allele, leading to three distinct genotypes: G / G homozygote (wild type, WT), G / A heterozygote (heterotype), and A / A homozygote (mutant type, MT). The G-to-A mutation alters the protein structure, leading to decreased acetaldehyde metabolism and reduced alcohol tolerance. The incidence of ALDH2 gene SNPs ranges from 35% to 57% in different regions of East Asia, which explains why Asians are more prone to facial flushing after drinking alcohol. The same test samples were subjected to multiplex polymerase chain reaction (PCR) using both a conventional 8-tube reaction tube and heating block combination and the first heating column 5 and sample tray 6 combination of the present invention. Figure 6A shows the relationship between migration time and relative fluorescence units (RFU) for the two wells in the conventional 8-tube reaction tube, and Figure 6B shows the relationship between migration time and relative fluorescence units (RFU) for the two sample slots 62 in the sample tray 6 of the present invention. In this test, the ratio of the mutant (MT) peak value to the wild-type (WT) peak value should be less than 1:5. As shown in Figure 6A, after multiplex PCR, the MT / WT ratio of one of the two wells in the conventional 8-tube reaction tube exceeded the stated ratio. As shown in Figure 6A, after multiplex PCR, the ratios of the two sample slots 62 in the sample tray 6 of the present invention were both less than the stated ratio. The experimental results show that the combination of the first heating column 5 and sample tray 6 of the present invention has good heating uniformity and does not have the problem of poor heating efficiency due to cumulative tolerance.

[0049] As shown in Figures 2A to 2B and Figures 4A to 4C, in the illustrated embodiments, a plurality of second heating columns 7 are used for sample pretreatment. Each second heating column 7 has a conical cavity for receiving a sample tube 71 with a conical bottom for heating or cooling the sample within the sample tube 71. For example, the primary function of the sample tube is to inactivate enzymes such as proteinase K. The prepared sample can then be transferred to the sample slot 62 in the sample tray 6 for PCR amplification.

[0050] In another alternative embodiment, a plurality of first heating columns 5 and a plurality of second heating columns 7 are used to perform PCR thermal cycling, and up to 16 PCR product samples can be automatically analyzed using a single delivery mechanism.

[0051] It is worth noting that the thermal cycling module of the present invention is an open system that does not require a lid. The sample in the sample slot 62 of the sample tray 6 is covered with a thin layer of mineral oil to prevent evaporation and heat loss. When using mineral oil, there is no need to provide an individual lid for the sample slot 62. With this open system, CE can be performed with a smaller number of PCR cycles, thereby reducing the overall operation time.

[0052] It is worth noting that the thermal cycling module described in this invention, including the sample tray 6 therein, can be used for other temperature-sensitive reactions in addition to polymerase chain reaction (PCR), such as, but not limited to, restriction enzyme digestion or rapid diagnostics.

[0053] Although the present invention has been specifically shown and described with reference to preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit, scope and teachings of the invention. [Simplified Explanation of the Diagram]

[0054] Non-limiting and non-exhaustive embodiments of the disclosed technology, including preferred embodiments, will be described below with reference to the accompanying drawings, wherein, unless otherwise stated, the same symbols denote the same elements or parts in all views.

[0055] Figure 1 is a schematic diagram of a capillary electrophoresis system integrating a thermal cycler / thermal cycle module according to an embodiment of the present invention.

[0056] Figure 2A is a perspective view of a capillary electrophoresis system integrating a thermal cycler / thermal cycle module according to an embodiment of the present invention.

[0057] Figure 2B is a cross-sectional view of Figure 2A along the A-A direction.

[0058] Figures 2C and 2D are cross-sectional views of a thermal circulator / thermal circulator module according to an embodiment of the invention in different directions.

[0059] Figures 3A to 3E are a perspective view, a top view, a front view, a left side view, and a perspective view from the bottom of a sample tray according to an embodiment of the present invention.

[0060] Figures 4A to 4C are a perspective view, a top view, and a right side view of a thermal cycling module according to an embodiment of the present invention.

[0061] Figure 5A shows the heating / cooling curve of a conventional 8-tube reaction tube; Figure 5B shows the heating / cooling curve of the sample cell of the present invention.

[0062] Figure 6A is a graph showing the relationship between the migration time of two reaction wells in a conventional 8-tube reaction system and the relative fluorescence unit (RFU); Figure 6B is a graph showing the relationship between the migration time of two sample slots in the sample tray of the present invention and the relative fluorescence unit (RFU).

[0063] Figure 7 is a top view of the sample tray 6 according to another embodiment of the present invention.

Claims

1. A sample tray for temperature-sensitive reactions, comprising: Multiple sample cells; A border; And multiple floating ribs, with two floating ribs corresponding to each sample cell; The plurality of sample slots are not connected to each other along the length of the frame, and each sample slot is connected to the frame along the width of the frame through two floating ribs located on both sides of the sample slot.

2. The sample tray of claim 1, wherein each of the floating ribs is stepped and has a first rib segment and a second rib segment connected to each other, wherein the first rib segment connects to the corresponding sample slot and the second rib segment, the second rib segment connects to the first rib segment and the frame, and the thickness of the first rib segment is greater than the thickness of the second rib segment.

3. The sample tray as requested in item 2, wherein the first rib gradually tapers toward the border along the width direction.

4. The sample tray as requested in item 2, wherein the second rib gradually tapers toward the border along the width direction.

5. The sample tray as requested in claim 2, wherein the two floating ribs allow displacement of the sample slot in the vertical direction as well as in the length and width directions.

6. The sample tray as requested in item 1, wherein the wall thickness of each sample slot is between 0.15 mm and 0.25 mm.

7. The sample tray as requested in item 1, wherein the heating or cooling rate of the plurality of sample tanks is equal to or greater than 5°C / sec.

8. The sample tray of claim 1, wherein the sample tray further includes a support base connected to and supporting the frame.

9. The sample tray as requested in item 1, wherein the sample tray is integrally formed.

10. The sample tray as requested in item 1, wherein the border is rectangular.

11. The sample tray as requested in item 1, wherein the border is annular, the length direction is the circumferential direction of the border, and the width direction is the radial direction of the border.

Images