Limited orifice thermal cycling device

Abstract

A limited well thermal cycler for sample preparation and real-time fluorescence detection is described. The limited well thermal cycler includes a heating block having a sample well and at least one reaction well and an optical block including corresponding means for measuring real-time fluorescence in each reaction well. The limited well thermal cycler includes means for efficient heating and cooling of the reaction well and sample well for real-time fluorescence detection. The structure of the heating block provides means for efficient heating and cooling by raising each of the sample well and at least one reaction well above the heating block base such that the sample well and at least one reaction well are not enclosed by the metal heating block.

Description

[0001] This application claims priority to provisional application US63 / 130073 entitled “Limited Wellthermal Cycling Device”, filed on December 23, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to a finite-hole thermal circulation device. Background Technology

[0003] Thermal cycling devices are laboratory equipment that amplify nucleic acid fragments (DNA, RNA) via PCR by providing cyclic heating and cooling according to polymerase chain reaction (PCR) protocols. These thermal cyclers are used in various applications, from detecting human gene mutations to pathogen detection, in research and hospital settings. These thermal cyclers can be real-time (detecting fluorescence from the reaction in real time) or amplification devices that amplify nucleic acids using only PCR cycling protocols or by isothermal amplification. In the absence of a standard thermal cycler (non-real-time machine) for simultaneous detection, the amplified products are detected by other methods, such as by loading a gel and staining it with an insertion dye after electrophoresis.

[0004] Numerous conventional thermal cycling devices exist, such as U.S. Patents 5,333,675, 5,475,610, and 5,656,493, which involve single-tube nucleic acid amplification and amplification in 96-well plates. Additionally, another type of conventional thermal cycling device includes nucleic acid amplification by separating individual sample tubes for separate heating and cooling control using microfluidics, as described in U.S. Patent 6,521,447, which obviously does not use a thermal cycling block for cyclic heating and cooling. Furthermore, U.S. Patent 7,238,321 teaches conventional devices including rapid PCR via thermal cycling in a block using one or more thin-walled glass capillaries. All these conventional devices involve the amplification and detection of nucleic acids using a single sample (therefore the device has a single reaction well) or simultaneously using multiple samples (therefore the device has multiple reaction wells, such as a 96-well plate). These conventional devices address the efficiency factors of thermal cycling, and particularly speed and temperature control.

[0005] Conventional devices such as the apparatus in U.S. Patent No. 7,238,321 teach that conversion thermal cyclers also use fluorescence detection to detect amplification in real time using fluorescent dyes that include intercalation dyes. For example, conventional devices in U.S. Patent Nos. 6,171,785 and 6,814,934 detect fluorescence from one or more reaction vessels, where amplification and detection occur in the same reaction well. These conventional devices all share the key feature of a hot block, which comprises a metal block with machined holes serving as reaction wells, enabling typical reaction tubes (reaction vessels) found in standard laboratories to be used for both amplification and reaction detection.

[0006] These standard reaction tubes or containers can be single reaction tubes (ranging from small capillary tubes to 0.2 mL capped PCR tubes) or multi-well tubes (such as tubes for 8-well or 0.2 mL strips) to 96-well, 384-well, or 1536-well microplates. All of these conventional thermal cycling devices mentioned here accept all these reaction formats, and many conventional devices are designed to accept one or two of the most abundant available reaction containers. Because the heating and cooling of the hot block controls the heating and cooling of the sample in the reaction container, the shape of the reaction wells in these thermal cycling devices must be designed to fit the reaction container tightly so that heat transfer is efficient.

[0007] To accommodate these standard reaction tubes, conventional apparatus includes a reaction orifice with a tapered bottom to fit snugly into the corresponding tapered reaction tube. As used herein, “tapered” refers to a “V” or “U” shape at the bottom of the reaction tube and / or the reaction orifice, with the “V” shape being the most common. These conventional apparatuses lack the ability to accept non-standard reaction tubes, such as those with flat bottoms.

[0008] In addition, other conventional devices provide sample preparation followed by nucleic acid amplification and detection within the same device. For example, U.S. Patent No. 5,863,502 performs this amplification and detection in parallel via fluidic motion, while application WO2007106579A2 provides amplification and detection via microfluidics in a fully integrated manner (WO2007106579A2). Both of these devices require significant fluidic motion and control between the chamber and the reaction tube. Thermal cycling is achieved in these devices via simple heating and cooling systems. Similarly, these conventional devices also employ unique methods of detecting fluorescence by combining appropriate optics (such as image capture as seen in application WO2007106579A2) or waveguides as shown in U.S. Patent No. 5,832,165. These conventional devices have the disadvantage of requiring separate sample preparation chambers and separate amplification chambers, thus necessitating significant fluidic motion and control of this motion between the chamber, reaction tube, and reaction well.

[0009] Therefore, a thermal cycling device that provides efficient heating and cooling with reduced hot block material is desired. Further, a thermal cycling device that provides sample preparation, amplification, and real-time detection within the same apparatus is desired, without requiring significant fluid transport or control. Finally, a thermal cycling device that provides non-standard reaction tubes is desired. Summary of the Invention

[0010] In some aspects, a limited-well thermal cycling device for preparing biological samples for amplification and detection of an analyte is disclosed, the limited-well thermal cycling device comprising: a heating block configured for preparing the biological sample and amplifying the analyte, the heating block comprising: a sample well having a flat bottom and configured for receiving and preparing the biological sample for amplification and detection, the sample well extending from a top of a heating block base; a reaction well having a flat bottom and configured for receiving and amplifying the analyte for detection of the prepared biological sample, the reaction well extending from the top of the heating block base, wherein the reaction well has an excitation orifice, an emission orifice, and a cooling orifice, wherein the excitation orifice and the emission orifice are aligned at 90 degrees; and a heating block alignment hole for detachably connecting an optical block to the heating block base, the heating block alignment hole being located on the top of the heating block base; the optical block being configured for detecting the analyte of the amplified and prepared biological sample, wherein the optical block is detachably connected to the heating block, the optical block comprising: an internal... An internal excitation aperture and an external excitation aperture, wherein the internal excitation aperture has a smaller diameter than the external excitation aperture, wherein the internal and external excitation apertures are continuously aligned through the central portion of the optical block, and wherein the centers of the internal and external excitation apertures are linearly aligned with the center of the excitation aperture of the reaction aperture; an LED plate having an LED detachably connected to the optical block, wherein the LED is received by the external excitation aperture; a photodiode plate having a photodiode detachably connected to the optical block, wherein the photodiode is received by the external emission aperture; an excitation filter receiving the external excitation aperture by a flange formed by the continuous alignment between the internal and external excitation apertures; an emission filter receiving the external emission aperture by a flange formed by the continuous alignment between the internal and external emission apertures; and a heating element in heating communication with the heating block to provide heat transfer to the sample aperture and the reaction aperture.

[0011] In one aspect, a limited-pore thermal cycling apparatus for preparing biological samples for amplification and detection of an analyte is disclosed, the limited-pore thermal cycling apparatus comprising: a heating block configured for preparing the biological sample and amplifying the analyte, the heating block comprising: a sample well having a flat bottom and configured for receiving and preparing the biological sample for amplification and detection, the sample well extending from a top of a base of the heating block; and a first reaction well having a flat bottom and configured for receiving and amplifying a first analyte for detection of the prepared biological sample, the first reaction well extending from the top of the base of the heating block, wherein the first reaction well has a first excitation... The optical block includes an aperture, a first emission aperture, and a first cooling aperture, wherein the first excitation aperture and the first emission aperture are aligned at 90 degrees; and a heating block alignment aperture for detachably connecting an optical block to the heating block base, the heating block alignment aperture being located on the top of the heating block base; a second reaction aperture having a flat bottom and configured to receive and amplify a second analyte for detection of the prepared biological sample, the second reaction aperture extending from the top of the heating block base, wherein the second reaction aperture has a second excitation aperture, a second emission aperture, and a second cooling aperture, wherein the second excitation aperture and the second emission aperture are aligned at 90 degrees; and a heating block alignment aperture. It is used to detachably connect the optical block to the heating block base, the heating block alignment hole being on the top of the heating block base; the optical block, configured for detecting the first and second analytes of the amplified and prepared biological sample, wherein the optical block is detachably connected to the heating block, the optical block comprising: a first internal excitation aperture and a first external excitation aperture, the first internal excitation aperture having a smaller diameter than the first external excitation aperture, wherein the first internal excitation aperture and the first external excitation aperture are continuously aligned through the central portion of the optical block, wherein the centers of the first internal excitation aperture and the first external excitation aperture are aligned with the first... The center of the excitation aperture of a reaction aperture is linearly aligned; a second internal excitation aperture and a second external excitation aperture, the second internal excitation aperture having a smaller diameter than the second external excitation aperture, wherein the second internal excitation aperture and the second external excitation aperture are continuously aligned through the central portion of the optical block, wherein the centers of the second internal excitation aperture and the second external excitation aperture are linearly aligned with the center of the excitation aperture of the second reaction aperture; an LED plate having a first LED and a second LED, the LED plate being detachably connected to the optical block, wherein the first LED is received by the first external excitation aperture, and the second LED is received by the second external excitation aperture;A photodiode board having a first photodiode and a second photodiode, the photodiode board being detachably connected to the optical block, wherein the first photodiode is received by a first external emission aperture, and the second photodiode is received by a second external emission aperture; a first excitation filter, the first excitation filter being received by a flange of the first external excitation aperture, the flange being formed by a continuous alignment between a first internal excitation aperture and the first external excitation aperture; a second excitation filter, the second excitation filter being received by a flange of the second external excitation aperture, the flange being formed by a continuous alignment between a second internal excitation aperture and the second external excitation aperture; a first emission filter, the first emission filter being received by a flange of the first external emission aperture, the flange being formed by a continuous alignment between a first internal emission aperture and the first external emission aperture; a second emission filter, the second emission filter being received by a flange of the second external emission aperture, the flange being formed by a continuous alignment between a second internal emission aperture and the second external emission aperture; and a heating element, which is in thermal communication with the heating block to provide heat transfer to the sample aperture and the first and second reaction apertures.

[0012] In some aspects, a limited-well thermal cycling apparatus for preparing biological samples for amplification and detection of an analyte is disclosed, the limited-well thermal cycling apparatus comprising: a heating block configured for the preparation of the biological sample and the amplification of the analyte, the heating block comprising: a sample well having means for receiving a sample tube having a flat bottom, the sample well extending from a top of a heating block base; a reaction well configured for amplifying an analyte for detection of the prepared biological sample, the reaction well extending from the top of the heating block base, wherein the reaction well has an excitation orifice, an emission orifice, and a cooling orifice, wherein the excitation orifice and the emission orifice are aligned at 90 degrees; and a heating block alignment hole for detachably connecting an optical block to the heating block base, the heating block alignment hole being located on the top of the heating block base; the optical block having means for measuring the real-time fluorescence of the analyte in the sample; and a heating element in thermal communication with the heating block to provide heat transfer to the sample well and the reaction well.

[0013] In some aspects, a limited-well thermal cycling apparatus for preparing biological samples for amplification and detection of an analyte is disclosed, the limited-well thermal cycling apparatus comprising: a heating block configured for preparing the biological sample and amplifying the analyte, the heating block comprising: a sample orifice having a tapered bottom and configured for receiving and preparing the biological sample for amplification and detection, the sample orifice extending from a top of a heating block base; a reaction orifice having a tapered bottom and configured for receiving and amplifying the analyte for detection of the prepared biological sample, the reaction orifice extending from the top of the heating block base, wherein the reaction orifice has an excitation orifice, an emission orifice, and a cooling orifice, wherein the excitation orifice and the emission orifice are aligned at 90 degrees; and a heating block alignment orifice for detachably connecting an optical block to the heating block base, the heating block alignment orifice being located on the top of the heating block base; the optical block being configured for detecting the analyte of the amplified and prepared biological sample, wherein the optical block is detachably connected to the heating block, the optical block comprising: an internal... An internal excitation aperture and an external excitation aperture, wherein the internal excitation aperture has a smaller diameter than the external excitation aperture, wherein the internal and external excitation apertures are continuously aligned through the central portion of the optical block, and wherein the centers of the internal and external excitation apertures are linearly aligned with the center of the excitation aperture of the reaction aperture; an LED plate having an LED detachably connected to the optical block, wherein the LED is received by the external excitation aperture; a photodiode plate having a photodiode detachably connected to the optical block, wherein the photodiode is received by the external emission aperture; an excitation filter receiving the external excitation aperture by a flange formed by the continuous alignment between the internal and external excitation apertures; an emission filter receiving the external emission aperture by a flange formed by the continuous alignment between the internal and external emission apertures; and a heating element in heating communication with the heating block to provide heat transfer to the sample aperture and the reaction aperture. Attached Figure Description

[0014] Figure 1 The heating block of a finite-hole thermal circulation device with two reaction orifices is shown.

[0015] Figure 2a The heating block of a finite-hole thermal circulation device with two reaction orifices is shown.

[0016] Figure 2b A front view of the heating block of a finite-hole thermal circulation device with two reaction orifices is shown.

[0017] Figure 3aAn optical block of a finite-hole thermal cycling device with two reaction holes is shown.

[0018] Figure 3b A front view of a finite-hole thermal cycle apparatus with two reaction orifices is shown.

[0019] Figure 3c A side view of a finite-hole thermal cycle apparatus with two reaction orifices is shown.

[0020] Figure 4 The heating block and optical block of a finite-hole thermal circulation device with two reaction holes are shown.

[0021] Figure 5 A finite-hole thermal cycle device with two reaction orifices is shown.

[0022] Figure 6 A heating block of a finite-hole thermal circulation device with a single reaction orifice is shown.

[0023] Figure 7 A finite-hole thermal circulation device with a single reaction hole (heating block with optical block) is shown. Detailed Implementation

[0024] As used in this article, "limited orifice" refers to a thermal cycling device with one to four reaction orifices.

[0025] As used herein, a "sample" refers to a nucleic acid sample that may contain one or more sequences of interest (analytes), one or more nucleic acid primers for detecting the nucleic acid sequences of interest, and PCR components for performing the PCR reaction. Each nucleic acid primer may be labeled with a fluorophore, each with unique absorption and emission properties for detection by fluorescence emission. For example, detection of an analyte by fluorescence emission of a fluorophore can be performed by primer extension of a probe due to the use of labeled nucleotides, by molecular beacons or similar fluorophores, and by primers based on quenchers or other means.

[0026] A limited-orifice thermal cycling device for sample preparation and real-time fluorescence detection is described. The device includes a heating block having a sample orifice and at least one reaction orifice, and an optical block including corresponding means for measuring real-time fluorescence in each reaction orifice.

[0027] This limited-orifice thermal cycling device includes means for efficient heating and cooling of the reaction orifice and sample orifice for real-time fluorescence detection. The structure of the heating block provides means for efficient heating and cooling by raising each of the sample orifice and at least one reaction orifice above the base of the heating block, so that the sample orifice and at least one reaction orifice are not surrounded by the metal heating block.

[0028] The reaction orifice and sample orifice of the limited-orifice thermal cycling device may include means for receiving a flat-bottomed sample container and a reaction container, such that the flat-bottomed container design is used for real-time fluorescence detection. The structure of the sample orifice and at least one reaction orifice provides means for receiving the flat-bottomed sample container and reaction container, as each of the sample orifice and at least one reaction orifice has a flat bottom for receiving the sample container and reaction container. This flat-bottom design enables efficient heating of the sample by providing a larger surface area at the bottom of the sample tube to contact the reaction orifice closest to the heating block. The reaction orifice and sample orifice of the limited-orifice thermal cycling device may be tapered to receive a standard reaction tube.

[0029] Figure 7 A limited-orifice thermal cycling device 700 with a single reaction orifice is shown. The limited-orifice thermal cycling device 700 includes a heating block 100 and an optical block 300. The heating block 100 is shown in diagonal shading to distinguish it from the optical block 300 located on top of the heating block 100. The heating block 100 includes a sample orifice 107 and a reaction orifice 102.

[0030] The heating block 100 has a base 110, which has a height (thickness) of 0.5 mm to 3 mm. 207 (Ref.) Figure 2b Preferably, the base height 207 of the heating block 100 is 1 mm. The heating block has a width 105 of 20 mm to 30 mm, and preferably, the width 105 of the heating block 100 is 25 mm. The heating block has a length 106 of 60 mm to 100 mm, and preferably, the length 106 is 80 mm. The heating block 100 also includes a heating block alignment hole 101, which provides alignment of the optical block 300 for detachable connections such as those via screws. The heating block 100 can be made of any thermally conductive metal, metal alloy, or composite material. Preferably, the heating block 100 is aluminum or anodized aluminum. The optical block material can be polyoxymethylene resin. Preferably, the optical block material is selected from the group consisting of acetal copolymers and homopolymers.

[0031] Heating of the heating block 100 is accomplished by a Peltier thermoelectric device, resistance heating element, or any similar conventional heating element placed below the heating block 100. Cooling is accomplished by blowing air into and around the block and the three holes using a conventional fan. For example, a conventional fan may be placed on the side of the heating block 100, or in another location that facilitates cooling of the reaction holes 102.

[0032] The sample orifice 107 of the heating block 100 can be configured to receive a sample container with a flat bottom for sample preparation. The sample orifice 107 of the heating block 100 can be tapered to receive a sample container with a standard reaction tube shape. The sample orifice 107 includes a sample orifice circumference 108 ranging from 10 mm to 14 mm, preferably 12 mm. The sample orifice 107 includes a sample orifice width 103 ranging from 10 mm to 14 mm, preferably 12 mm. The sample orifice 107 includes a sample orifice height 208 ranging from 10 mm to 14 mm, preferably 12.2 mm.

[0033] When the sample orifice 107 is configured to receive a sample container with a flat bottom, the interior of the sample orifice 107 is circular with a diameter of 6 mm to 12 mm, preferably 8.2 mm. The interior of the sample orifice also has a flat bottom to receive sample containers made of plastic, glass, or other thermally conductive materials with flat bottoms. When the sample orifice 107 is tapered to receive a sample container with a standard reaction tube shape, the interior of the sample orifice 107 is tapered, with a diameter of 6 mm to 12 mm at the top (closest to the opening) of the sample orifice 107.

[0034] The sample hole 107 can be formed from the base 110 of the heating block 100 and is made of any thermally conductive material such as metal, metal alloy or composite material. Preferably, the sample hole 107 is aluminum or anodized aluminum.

[0035] Sample well 107 receives a sample container in which the sample can be heated and cooled to a desired temperature for a desired duration to prepare a sample for further analysis (i.e., amplification and detection) in reaction well 102. For example, a dissolution buffer is added to the sample, which is then heated to lyse cells and release nucleic acids.

[0036] The reaction wells 102 of the heating block 100 are configured for measuring the real-time fluorescence of a sample. Real-time fluorescence is measured as the sample is heated and cooled to the desired temperature for the duration required for sample amplification. For example, components required for amplification are added to the sample in the reaction wells 102, wherein, in the case of PCR amplification, these components include fluorescently labeled DNA primers, free nucleotides (ddNTPs), and DNA polymerase. Furthermore, for example, a polymerase chain reaction (PCR) protocol can be used to cycle the sample through heating and cooling to amplify the desired analyte while performing real-time fluorescence detection. The reaction wells 102 have a well width 103 ranging from 6 mm to 10 mm, preferably 8 mm. The reaction wells 102 have a well diameter length 103 ranging from 6 mm to 10 mm, preferably 8 mm. The reaction wells 102 have a well height 208 ranging from 10 mm to 14 mm, preferably 12.2 mm.

[0037] When the reaction orifice 102 is configured to receive a non-standard sample container with a flat bottom, the interior of the reaction orifice 102 is circular with a diameter of 4 mm to 8 mm, preferably 5.82 mm. The interior of the reaction orifice 102 has a flat bottom to accommodate a reaction container with a flat bottom made of heat-resistant plastic, glass, borosilicate glass, or other thermally conductive materials. When the reaction orifice 102 is tapered to receive a sample container with a standard reaction tube shape, the interior of the reaction orifice 102 is tapered, with a diameter of 4 mm to 8 mm at the top (closest to the opening) of the reaction orifice 102.

[0038] The reaction orifice 102 also includes a cooling orifice 205 extending from the interior to the outer periphery to facilitate sample cooling by a fan. The diameter of the cooling orifice 205 ranges from 0.5 mm to 2 mm, and is preferably 1 mm. The reaction orifice 102 can be formed from the base 110 of the heating block 100 and is made of any thermally conductive material such as metal, metal alloy, or composite material. Preferably, the reaction orifice 102 is aluminum or anodized aluminum.

[0039] The reaction well 102 includes an excitation orifice 203 and an emission orifice 204 of at least one reaction well, which allows fluorescence excitation and emission of the sample to be measured in real time via optical block 300. Figure 7 A reaction aperture 102 with two emission apertures 204 is shown, but the reaction aperture 102 may have a single emission aperture 204. The emission aperture 204 of each reaction aperture extends from the interior to the outer periphery of the reaction aperture 102, allowing fluorescence from the sample to reach the photodiode 201. The emission aperture 204 of each reaction aperture is linearly aligned with the corresponding photodiode 201 of the optical block, as well as the inner emission aperture 304 and the outer emission aperture 312. The emission aperture 204 of each reaction aperture is aligned at a 90-degree angle with the excitation aperture 203 of the reaction aperture. The diameter of the emission aperture 204 of each reaction aperture ranges from 0.5 mm to 2 mm, and is preferably 1 mm.

[0040] The excitation aperture 203 of the reaction aperture extends from the interior to the outer periphery of the reaction aperture 102 to allow light from the LED 202 to reach the sample. The excitation aperture 203 of the reaction aperture is linearly aligned with the LED 202, the inner excitation aperture 303, and the outer excitation aperture 311, respectively. The diameter of the excitation aperture 203 of the reaction aperture ranges from 0.5 mm to 2 mm, and is preferably 1 mm.

[0041] The optical block 300 of the limited-orifice thermal cycling device 700 provides a means for measuring the real-time fluorescence of a sample received in the sample orifice 102. The means for measuring the real-time fluorescence of the sample in the optical block 300 includes an internal excitation orifice 303, an external excitation orifice 311, an excitation filter 301, at least one internal emission orifice 304, at least one external emission orifice 312, at least one emission filter 302, an LED plate 502, and at least one photodiode plate 501. The optical block also includes a central portion 310 having an LED mounting hole 305 and a photodiode mounting hole 306. The optical block 300 also includes a foot 309 having an alignment hole 307.

[0042] The optical block 300 is detachably connected to the heating block 100 via alignment holes 101 on the heating block and alignment holes 307 on the legs 309, such as via screws. As further explained herein, this alignment provides proper alignment of at least one reaction hole 102 with the apparatus for measuring real-time fluorescence. The optical block has a width from 20 mm to 30 mm, and preferably 25 mm. The optical block 300 has a length from 60 mm to 100 mm, and preferably 80 mm. The width and length of the optical block are equal to the width and length of the heating block 100.

[0043] The support leg 309 of the optical block 300 extends from the central portion 310 and includes an alignment hole 307 for detachable connection to the heating block 100. The support leg 309 has a height less than that of the central portion 310.

[0044] The central portion 310 has a height 314 ranging from 12 mm to 20 mm, and preferably 16 mm. The central portion of the optical block 300 rises from the foot 309 at elevation point 308 (e.g., Figure 3a (As shown). The central portion 310 of the optical block 300 includes an internal excitation aperture 303, an external excitation aperture 311, an excitation filter 301, at least one internal emission aperture 304, at least one external emission aperture 312, and at least one emission filter 302 for a device for real-time fluorescence detection. The central portion also includes: an LED mounting hole 305 providing a detachable connection to an LED board 502 and a photodiode mounting hole 306 providing a detachable connection to at least one photodiode board 501.

[0045] An external excitation aperture 311 extends from the outer periphery of the optical block 300 to the internal excitation aperture 303. The external excitation aperture 311 has a diameter ranging from 4 mm to 8 mm, and preferably 5.5 mm. The external excitation aperture 311 has a depth ranging from 5 mm to 10 mm, and preferably 7 mm. The external excitation aperture 311 is linearly aligned with the excitation aperture 203 of the reaction aperture. The external excitation aperture 311 houses the excitation filter 301.

[0046] An internal excitation aperture 303 extends from an external excitation aperture 311 to the inner periphery of the optical block 300. The internal excitation aperture 303 has a diameter ranging from 2.5 mm to 5 mm, and preferably 3.5 mm. The depth of the internal excitation aperture 303 ranges from 0.5 mm to 4 mm, and preferably 1 mm. The internal excitation aperture is linearly aligned with the excitation aperture 203 of the reaction aperture. The diameter change from the internal excitation aperture 303 to the external excitation aperture 311 forms a ledge, in which the excitation filter is housed by the external excitation aperture 311 at the location closest to the internal excitation aperture 303.

[0047] Excitation filter 301 provides the wavelength for filtering light from LED 202 to excite the fluorescent dye in the sample. For example, when LED 202 produces wavelengths in the range of 465 nm to 485 nm, excitation filter 301 can be an optical filter that filters light in the range of 475 nm to 495 nm. This excitation filter 301 is sufficient for many commonly used fluorescent dyes such as fluorescein, 6-FAM (6-carboxyfluorescein), and Atto 488.

[0048] LED board 502 is an electronic control board (e.g., including a printed circuit board) that includes LED 202 and LED board connection hole 503, and turns the LED on and off. LED 202 excites a fluorescent dye in the sample. For example, LED 202 may be a blue LED that produces wavelengths in the range of 465 nm to 485 nm. LED 202 is received by an external excitation aperture 311. LED board connection hole 503 provides a detachable connection between LED board 502 and optical block 300, such as via screws, at LED mounting hole 305.

[0049] At least one of the external emission apertures 312 extends from the outer periphery of the optical block 300 to a respective internal emission aperture 304. Each of the external emission apertures 312 has a diameter from 4 mm to 8 mm, and preferably 5.5 mm. Each of the external emission apertures 312 has a depth from 4 mm to 8 mm, and preferably 5.5 mm. Each of the external emission apertures 312 is linearly aligned with the emission aperture 204 of its respective reaction aperture. Each of the external emission apertures 312 houses its respective emission filter 302.

[0050] Each of the internal emission apertures 304 extends from its external emission aperture 312 to the inner periphery of the optical block 300. Each of the internal emission apertures 304 has a diameter ranging from 2.5 mm to 5 mm, and preferably 3.5 mm. The depth of the internal emission aperture 304 ranges from 0.5 mm to 4 mm, and preferably 1 mm. Each of the internal emission apertures 304 is linearly aligned with the emission aperture 204 of its respective reaction aperture. The diameter variation from the internal emission aperture 304 to the external emission aperture 312 forms a flange, in which the emission filter 302 is received by the external emission aperture 312 at the position closest to the internal emission aperture 304.

[0051] Before the photodiode 201 measures fluorescence, each emission filter 302 provides filtering of the fluorescence from the sample. The emission filter 302 directly corresponds to the fluorescent dye used in the sample. For example, the emission filter 302 can be an optical filter that filters light in the 517 nm to 537 nm range. When the fluorescent dye of the sample is 6-FAM (6-carboxyfluorescein), this would be a sufficient emission filter, as 6-FAM has maximum absorbance at 495 nm and maximum emissivity at 520 nm. Each emission filter 302 can be the same filter, or it can be different filters to provide filtering of light at different wavelengths.

[0052] Each photodiode board 501 is an electronic control board (e.g., including a printed circuit board) that includes a photodiode 201 and a photodiode board connection hole 504. Each photodiode 201 detects fluorescence emitted from a sample after passing through a respective emission filter 302. For example, the photodiode 201 includes a photosensitive region specific for detecting wavelengths from 320 nm to 1100 nm. Each photodiode 201 is received by its respective external emission aperture 312. The photodiode board connection hole 504 provides a detachable connection, such as via screws, to the optical block 300 at its respective photodiode mounting hole 306.

[0053] The internal excitation aperture 303 and the external excitation aperture 311 are oriented at a 90-degree angle to each other with respect to the internal emission aperture 304 and the external emission aperture 312 when oriented from the center of each aperture. This orientation provides that the LED 202 and the photodiode 201 are oriented at a 90-degree angle to each other. Figure 7 The internal emission aperture 304 and external emission aperture 312, as well as the emission filter 302, are shown at positions oriented at 90 degrees to the internal excitation aperture 303 and the external excitation aperture 311.

[0054] A finite-orifice thermal cycling device with one reaction orifice 102 may include emission ports 204 for two reaction orifices and two photodiodes 201, emission ports 304 and 312 for two internal and external reaction orifices, and two emission filters 302 corresponding to them respectively. Assuming that each reaction vessel receives the same sample, this arrangement provides real-time detection of two separate analytes within the same sample. In this case, each photodiode 201 and emission filter 302 can be configured to detect and filter the unique fluorescence corresponding to the two fluorescent dyes within the sample.

[0055] Figure 6 The heating block 100 of the limited-orifice thermal circulation device 700 is shown. This view of the heating block 100 shows the emission orifice 204 of the reaction orifice, the excitation orifice 203 of the reaction orifice, and the cooling orifice 205.

[0056] Figure 5 A limited-orifice thermal cycling device 700 with two reaction orifices 102 is shown. This thermal cycling device 700 with two reaction orifices 102 correspondingly has two internal excitation orifices 303 and two external excitation orifices 311, two excitation filters 301, and two LEDs 202 on an LED plate 502. Furthermore, the thermal cycling device 700 has two photodiode plates 501, each with a photodiode 201, wherein the photodiode plates 501 are positioned such that the photodiode 201 and thus its respective emission orifice 204 are at a 90-degree angle to the excitation orifice 203 of the corresponding reaction orifice 102. Assuming that each reaction vessel receives the same sample, this arrangement provides real-time detection of two separate analytes within the same sample. In this case, each photodiode 201 and emission filter 302 can be configured to detect and filter the unique fluorescence corresponding to the two fluorescent dyes within the sample.

[0057] Figure 4 It shows Figure 5 The figure shows the heating block 100 and optical block 300 of the limited-orifice thermal circulation device 700. The figure also shows the emission orifice 204 and excitation orifice 203 of the reaction orifice, aligned at 90 degrees. Furthermore, the figure shows the cooling orifice 205 of the reaction orifice.

[0058] Figure 3a It shows Figure 5 The optical block 300 of the limited-aperture thermal cycling device 700, wherein the display of photodiodes 201 and LEDs 202 is used only for representative positioning purposes. The figure shows the arrangement of the internal excitation aperture 303 and the external excitation aperture 311, and the excitation filter 301 in appropriate positions. Furthermore, the figure shows the internal emission aperture 304 and the external emission aperture 312, and the emission filter 302 in appropriate positions.

[0059] Figure 3b It shows Figure 3a A front view of the optical block 300. The figure shows the inner excitation aperture 303 and the outer excitation aperture 311, particularly the difference in their diameters. Furthermore, the figure shows the height difference from the support leg 309 to the central portion 310.

[0060] Figure 3c It shows Figure 3a A side view of the optical block 300. The figure shows the inner emission aperture 304 and the outer emission aperture 312, particularly the difference in their diameters. Furthermore, in this figure, the legs 309 are shaded to indicate that they protrude from the central portion 310.

[0061] Figure 1 and Figure 2a It shows Figure 5 A top view of the heating block 100. The figure shows the emission orifice 204 and the excitation orifice 203 of the reaction orifice aligned at 90 degrees. Additionally, the figure shows the cooling orifice 205 of the reaction orifice.

[0062] Figure 2b It shows Figure 1 and Figure 2a The figure shows a front view of the heating block 100. It illustrates the reaction hole rising from the base 110 of the heating block 100, showing that the reaction hole is not surrounded by the heating block. Furthermore, the figure shows the height from the heating block to the center 206 of the excitation orifice of the reaction hole, the thickness 207 of the heating block base 110, and the height 208 of the reaction hole.

Claims

1. A limited-well thermal cycling device for preparing biological samples for amplification and detection of analytes, the limited-well thermal cycling device comprising: A heating block configured for the preparation of the biological sample and the amplification of the analyte, the heating block comprising: The sample well, configured to receive and prepare the biological sample for amplification and detection, extends upward from the top of the heating block base and has a flat or tapered bottom; One to four reaction wells are configured to receive and amplify analytes for detection in the prepared biological sample. Each of the reaction wells extends upward from the top of the heating block base and has a flat or tapered bottom. Each of the aforementioned reaction orifices has an excitation orifice, an emission orifice, and a cooling orifice, wherein the excitation orifice and the emission orifice are aligned at a 90-degree angle; and A heating block alignment hole for detachably connecting an optical block to the heating block base, the heating block alignment hole being located on the top of the heating block base; The optical block, configured for detecting the analyte in the amplified and prepared biological sample, is detachably connected to the heating block, the optical block being located on top of the heating block base and comprising, for each reaction well: An internal excitation aperture and an external excitation aperture, wherein the internal excitation aperture has a smaller diameter than the external excitation aperture, wherein the internal excitation aperture and the external excitation aperture are continuously aligned through the central portion of the optical block, and wherein the centers of the internal excitation aperture and the external excitation aperture are linearly aligned with the center of the excitation aperture of the reaction aperture; An internal emission aperture and an external emission aperture, wherein the internal emission aperture has a smaller diameter than the external emission aperture, wherein the internal emission aperture and the external emission aperture are continuously aligned through the central portion of the optical block, and wherein the centers of the internal emission aperture and the external emission aperture are linearly aligned with the center of the emission aperture of the reaction aperture; An LED board having LEDs, the LED board being detachably connected to the optical block, wherein the LEDs are received by the external excitation aperture; A photodiode board having a photodiode, the photodiode board being detachably connected to the optical block, wherein the photodiode is received by the external emission aperture; An excitation filter is received by a flange of the external excitation aperture, the flange being formed by continuous alignment between the internal excitation aperture and the external excitation aperture; A transmission filter, the transmission filter being received by a flange of the external transmission aperture, the flange being formed by continuous alignment between the internal transmission aperture and the external transmission aperture; and A heating element, which is in heating communication with the heating block to provide heat transfer to the sample well and the reaction well.

2. The apparatus according to claim 1, further comprising: A reaction vessel having a flat bottom and configured to receive the reaction orifice, the reaction vessel being made of a material selected from the group consisting of glass and borosilicate glass.

3. The apparatus according to claim 1, further comprising: A reaction vessel having a flat bottom and configured to receive the reaction orifice, the reaction vessel being made of a heat-resistant plastic material.

4. The apparatus according to claim 1, wherein The external excitation orifice has a diameter ranging from 4 mm to 8 mm; The internal excitation orifice has a diameter ranging from 2.5 mm to 5 mm; The external emission port has a diameter ranging from 4 mm to 8 mm; The internal firing aperture has a diameter ranging from 2.5 mm to 5 mm.

5. The apparatus according to claim 4, wherein, The excitation orifice of the reaction pore has a diameter ranging from 0.5 mm to 2 mm; The emission orifice of the reaction orifice has a diameter ranging from 0.5 mm to 2 mm.

6. The apparatus according to claim 1, wherein, The heating block is made of anodized aluminum.

7. The apparatus according to claim 6, wherein, The optical block material is polyoxymethylene resin.

8. The apparatus according to claim 6, wherein, The optical block material is selected from the group consisting of acetal copolymers and homopolymers.

9. The apparatus according to claim 1, wherein, The sample well has a diameter ranging from 6 mm to 12 mm; and The reaction pores have diameters ranging from 4 mm to 8 mm.

10. The apparatus according to claim 9, wherein, The sample well is located at a distance of at least 5 mm from the reaction well.

11. The apparatus according to claim 1, The reaction pores include: A first reaction well having a flat bottom and configured to receive and amplify a first analyte for detection of a prepared biological sample, the first reaction well extending from the top of the heating block base, and having a first excitation orifice, a first emission orifice and a first cooling orifice, wherein the first excitation orifice and the first emission orifice are aligned at 90 degrees. as well as The second reaction well has a flat bottom and is configured to receive and amplify a second analyte for detection of the prepared biological sample. The second reaction well extends from the top of the heating block base and has a second excitation orifice, a second emission orifice, and a second cooling orifice, wherein the second excitation orifice and the second emission orifice are aligned at 90 degrees. The optical block is configured to detect the first and second analytes in the amplified and prepared biological sample, and the optical block includes: A first internal excitation aperture and a first external excitation aperture, wherein the first internal excitation aperture has a smaller diameter than the first external excitation aperture, wherein the first internal excitation aperture and the first external excitation aperture are continuously aligned through the central portion of the optical block, wherein the centers of the first internal excitation aperture and the first external excitation aperture are linearly aligned with the center of the excitation aperture of the first reaction aperture. A second internal excitation aperture and a second external excitation aperture, wherein the second internal excitation aperture has a smaller diameter than the second external excitation aperture, wherein the second internal excitation aperture and the second external excitation aperture are continuously aligned through the central portion of the optical block, wherein the centers of the second internal excitation aperture and the second external excitation aperture are linearly aligned with the center of the excitation aperture of the second reaction aperture; The LED plate having a first LED and a second LED is detachably connected to the optical block, wherein the first LED is received by a first external excitation aperture and the second LED is received by a second external excitation aperture; The photodiode board having a first photodiode and a second photodiode, the photodiode board being detachably connected to the optical block, wherein the first photodiode is received by the first external emission aperture and the second photodiode is received by the second external emission aperture; A first excitation filter is received by a flange of the first external excitation aperture, the flange being formed by continuous alignment between the first internal excitation aperture and the first external excitation aperture; The second excitation filter is received by the flange of the second external excitation aperture, the flange being formed by the continuous alignment between the second internal excitation aperture and the second external excitation aperture; A first emission filter, wherein the first emission filter is received by a flange of the first external emission aperture, the flange being formed by continuous alignment between the first internal emission aperture and the first external emission aperture; and The second emission filter is received by the flange of the second external emission aperture, the flange being formed by the continuous alignment between the second internal emission aperture and the second external emission aperture; The heating element is in heating communication with the heating block to provide heat transfer to the sample hole, the first reaction hole, and the second reaction hole.

12. The apparatus of claim 11, further comprising: A first reaction vessel having a flat bottom and configured to receive through the first reaction orifice, the first reaction vessel being made of a material selected from the group consisting of glass and borosilicate glass; The second reaction vessel has a flat bottom and is configured to receive the second reaction orifice. The second reaction vessel is made of a material selected from the group consisting of glass and borosilicate glass.

13. The apparatus of claim 11, further comprising: A first reaction vessel having a flat bottom and configured to receive through the first reaction orifice, the first reaction vessel being made of a heat-resistant plastic material; The second reaction vessel has a flat bottom and is configured to receive through the second reaction orifice, and the second reaction vessel is made of a heat-resistant plastic material.

14. The apparatus according to claim 11, wherein, The first external excitation aperture has a diameter ranging from 4 mm to 8 mm; The first internal excitation orifice has a diameter ranging from 2.5 mm to 5 mm; The first external emission aperture has a diameter ranging from 4 mm to 8 mm; The first internal emission aperture has a diameter ranging from 2.5 mm to 5 mm; The second external excitation aperture has a diameter ranging from 4 mm to 8 mm; The second internal excitation orifice has a diameter ranging from 2.5 mm to 5 mm; The second external emission aperture has a diameter ranging from 4 mm to 8 mm; The second internal emission aperture has a diameter ranging from 2.5 mm to 5 mm.

15. The apparatus according to claim 14, wherein, The excitation orifice of the first reaction orifice has a diameter ranging from 0.5 mm to 2 mm; The emission orifice of the first reaction orifice has a diameter ranging from 0.5 mm to 2 mm; The excitation orifice of the second reaction orifice has a diameter ranging from 0.5 mm to 2 mm; The emission orifice of the second reaction orifice has a diameter ranging from 0.5 mm to 2 mm.

16. The apparatus according to claim 11, wherein, The sample well has a diameter ranging from 6 mm to 12 mm; and The first reaction orifice has a diameter ranging from 4 mm to 8 mm; The second reaction orifice has a diameter ranging from 4 mm to 8 mm.

17. The apparatus according to claim 1, wherein, The sample well and the reaction well have flat bottoms.

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