TWI669390B - Multiplex slide plate device having storage tank - Google Patents

Abstract

The invention provides a multiplex test piece device, which comprises a test piece, a sacrificial layer and a casing. The test strip has a plurality of reaction vessels arranged in an array. The sacrificial layer has micro-channels, and the micro-channels have interconnecting injection channels, main channels and terminal channels. The shell is used for accommodating the test piece and the sacrificial layer, and is composed of an upper cover and a chassis. The upper cover has an injection hole, a discharge hole and a storage slot. Inject the sample solution and oil from the injection hole into the injection flow channel, and use the oil to push the sample solution. When the sample solution flows through the main flow channel, it is loaded into each reaction vessel, and the excess waste liquid enters the storage tank from the discharge hole. The oil is covered and cannot return.

Description

Multiplex test piece device with storage tank

The present invention relates to a multiplexed test strip device for application in molecular biological detection, and in particular to a multiplexed test strip device for polymerase chain reaction (PCR) with a storage tank to store waste liquid.

In the field of molecular biological detection, it is often necessary to test multiple samples for a sample. For example, the polymerase chain reaction test can be used to test the genotypes of several single nucleotide polymorphisms (SNPs) in a sample, or the degree of performance of several genes in a sample. At this time, it is necessary to form a test panel by several DNA or RNA tests (assay). Each set of test reagents in the PCR test includes at least two specific DNA primer molecules (some PCR tests also include target-specific reporter probes). This pair of primers (primer pairs) must be correctly matched with It is necessary to mix the extracted DNA template in the test sample (sample) in order to check whether the specific DNA target is present in the sample or in what amount.

Traditionally, primer pairs and samples are placed in the same reaction vessel for PCR. The general placement method is usually to pipette a pair of primers, enzyme and dNTP mixtures and buffer reagents and samples stored in a single bottle by pipette to pipette into the reaction container manually. The most common carrier container is a 96-well tray. Using the aforementioned placement method, PCR detection requires at least two rounds (two rounds) of pipette pipetting, one of which adds the sample to the reaction vessel, and the other adds the primer pair to the reaction vessel. For example, assuming that a test kit is used to check 36 targets in a sample, at least 36 pipetting operations are required to add each primer pair to 36 different reaction vessels. In addition, 36 pipetting operations are required to Add samples to each of the above reaction vessels. This type of operation is not only complicated and error-prone, but also requires a lot of manpower.

If primer pairs are pre-filled in individual reaction vessels, the experiment operator only needs to add samples to the pre-filled vessels. For the aforementioned example, to test 36 targets for a sample, it would only take 36 pipetting operations to add the sample to the 36 pre-filled reaction vessels. In addition, the volume of the reaction vessel can be reduced to the nano-liter range at the same time to save the amount of detection reagents used. Under this improvement, the 96-well plate that is commonly used as a loading container will be changed to a microtiter plate similar to a test piece.

However, the size and volume of the reaction vessels (also called microwells or nanowells) of microtiter plates are too small to manually fill the primer pairs or samples used while avoiding cross-contamination between adjacent reaction vessels (i.e. Primer pairs dissipate from one well to other wells), therefore, special microfluidic dispensing technology is required. In more detail, a primer pair is provided to each nanowell in advance and the primer is fixed to the surface of the nanowell. After that, the user can add a sample to each reaction vessel through a single pipetting operation or through a single microfluidic channel without worrying about the primer dissipating from one well to other wells, minimizing cross contamination between wells.

For subsequent sample testing, each reaction vessel must be filled with a predetermined amount of sample. The traditional method is to use a pipette or needle dispenser to add samples one by one to the reaction well. However, as the volume of the reaction vessel becomes smaller, the closer the distance between the wells, designing a dispenser or path with a special mechanical mechanism, and individually transferring to each reaction vessel is complicated and time-consuming. If a special test strip device is designed to achieve a single pipetting operation or add a sample to each reaction vessel through a single microfluidic channel, the manual operation required for the preparation of detection reagents by the polymerase chain reaction can be greatly simplified and increased The convenience of filling samples.

The invention provides a multiplex test piece device with a storage tank, which is suitable for molecular biological detection; in particular, for polymerase chain reaction; and more specifically, for instant polymerase chain reaction. With the multiplexed test strip device of the present invention, the sample can be quickly and evenly loaded into each reaction vessel of the test strip, and all reaction vessels can be filled in a short time with a single pipetting operation. The liquid can flow to the storage tank for storage, and the storage tank has the function of positioning foolproof.

The invention provides a multiplex test piece device, which includes a test piece, a sacrificial layer and a casing. The test piece has a plurality of reaction vessels, a first injection hole and a first discharge hole. The reaction vessels are arranged in an array, and each reaction vessel has an opening and a bottom. The sacrificial layer has micro-channels. The micro-channels have an interconnecting injection channel, main channel, and terminal channel. The sacrificial layer is suitable for assembly with the test piece, and the main channel is assembled facing the opening. The shell is used to accommodate the test piece and the sacrificial layer, and is composed of an upper cover and a bottom plate. The upper cover is suitable for assembly with the bottom plate. The upper cover has a second injection hole, a second discharge hole, and a storage slot. Inject the sample solution and oil into the injection flow channel from the second injection hole and the first injection hole in sequence, and use the oil to push the sample solution, so that the sample solution and oil flow from the injection flow channel through the main flow channel to the terminal flow channel. The sample solution is loaded into each reaction vessel as it flows through the main flow channel. When the oil flows through the main flow path, the sample solution not loaded in the reaction vessel is excluded. Excess waste liquid flows from the end flow channel through the first discharge hole and the second discharge hole into the storage tank, which is covered with oil and cannot return.

In an embodiment of the invention, the multiplex test strip device further includes a panel member, which is suitable for being assembled with the upper cover and covering the storage tank, and has a label or a different appearance color to identify different test samples, targets, or functions.

In an embodiment of the present invention, the multiplex test piece device further includes a cover member, which is suitable for assembly with the upper cover and the panel member. After sequentially injecting the sample solution and oil from the second injection hole and the first injection hole, the seal is used The cover member covers the second injection hole to avoid splashing of the sample solution and oil and to prevent contamination by biochemical reactions.

In an embodiment of the invention, the material of the housing includes a thermally conductive material.

In an embodiment of the invention, the chassis has a groove for accommodating the test piece and the sacrificial layer.

In an embodiment of the invention, the material of the test piece includes a light-transmitting material.

In an embodiment of the invention, the light-transmitting material includes polycarbonate.

In an embodiment of the invention, the material of the sacrificial layer includes wax.

In an embodiment of the invention, the oil includes mineral oil or silicone oil.

In an embodiment of the present invention, during the experiment of the polymerase chain reaction, heating is performed to melt the sacrificial layer, and the melted sacrificial layer is mixed with oil.

Based on the above, the present invention provides a multiplexed test piece device with a storage tank, so that when the sample flows through the main flow channel of the sacrificial layer, it is quickly and evenly loaded into each reaction vessel of the test piece, and then Exclude sample solutions that are not loaded in the reaction vessel. In this way, all reaction vessels can be filled in a short time with a single pipetting operation, therefore, the experimental operation can be simplified and time can be saved. In addition, excess waste liquid can flow from the end flow channel through the first discharge hole and the second discharge hole into the storage tank, which is covered with oil and cannot be returned. Therefore, no additional steps are required to remove the waste liquid. For convenience and time saving, the storage tank also has a foolproof positioning function.

In order to make the above-mentioned features and advantages of the present invention more obvious and understandable, the embodiments are specifically described below in conjunction with the accompanying drawings for detailed description as follows.

The invention provides a multiplex test piece device, which can be widely applied to different types of reaction tests. In the following, we first define and explain the terms used in the description.

"Reagent" may refer to a formulation or preparation of several ingredients used for a particular test/inspection. For example, in a test using polymerase chain reaction, the detection reagents include a pair of primers, enzymes, dNTPs, fluorescent reporters and salts. In the application, different primer pairs and fluorescent reporters may be added to the reaction vessel first, followed by the sample mixed with enzymes, dNTPs, and other additives before being added to the reaction vessel.

"Sample" refers to the nucleic acid sample being tested. For example, the sample may be nucleic acid fragments (including DNA or RNA, etc.) extracted from blood, tissue, saliva and other sources.

"Analysis" or "test" may refer to one or more experiments or test items performed on the same sample. For example, PCR is used to detect 300 single nucleotide polymorphism (300 SNP) genotypes for nucleic acid samples. The test includes several PCR test items, by checking each of the single nucleotide polymorphism (SNP) Genotype (A, T, C, G). For example, real-time fluorescent quantitative PCR is used to determine the amount of nucleic acid for a specific sequence.

"Sample solution" refers to the mixture or mixed solution of the above "sample" and the master mix.

"Reaction vessel" may refer to each tube of the tube tray, each reaction tube, the hole or well of the microtiter plate, or the pit/hole of the test strip or array plate. As described herein, "test strip", "test strip plate", "detection array plate" or "detection plate" can all refer to the same base or substrate that houses the aforementioned reaction vessel.

When the volume of liquid in the container is reduced to a certain extent, the flow of liquid in the container is mainly controlled by the adhesion of the surface rather than gravity. If the volume of the liquid in the container is only a few nanoliters, the liquid has a high surface adhesion and will stick to the container (nai well), that is to say, the liquid can be regarded as an adhesive that is stably attached to the bottom of the container or On the container wall.

Preferably, the reaction container may be a single reaction hole or well of a test strip or a detection array plate. As previously discussed, it is preferred to use a smaller volume reaction vessel with dimensions ranging from a few nanoliters to hundreds of nanoliters, for example.

FIG. 1 is a schematic structural diagram of a multiplex test piece device according to an embodiment of the present invention.

Referring to FIG. 1, the multiplexed test strip device includes a test strip 10, a sacrificial layer 20, a housing 30, a panel member 60 and a cover member 62, wherein the housing 30 can be used to accommodate the test strip 10 and the sacrificial layer 20. Hereinafter, the structure of the test piece 10 will be described in detail with reference to FIGS. 2 and 3.

FIG. 2 is a schematic diagram of a test piece according to an embodiment of the present invention.

Please refer to FIG. 2, the test piece 10 has a detection area 100, and the area of the detection area 100 is, for example, 22.5 mm×22.5 mm. The detection area 100 includes a plurality of reaction vessels 102, and the reaction vessels 102 are arranged in an n×n array. In addition, the test piece 10 further has an injection hole 12 and a discharge hole 14. In this embodiment, the size of the test piece 10 is, for example, 36 mm×36 mm×0.8 mm. In more detail, the material of the test piece 10 may include a light-transmitting material, and the light-transmitting material is, for example, polycarbonate (PC), but the invention is not limited thereto.

Referring again to FIG. 2, from the cross-sectional view of the test piece 10 (the right part of FIG. 2 ), each reaction vessel 102 has a wider opening 102 a and a narrower bottom 102 b. In this embodiment, the depth of each reaction vessel 102 is, for example, 200 μm (d1), the size of the opening 102a is, for example, 410 μm (L1)×410 μm (W1), and the size of the bottom 102b is, for example, 220 μm ( L2)×220 μm(W2). The interval (P1) between the reaction vessels 102 ranges from 27 μm to 45 μm, for example. The angle θ of the inclined side wall of the reaction vessel 102 is, for example, 90 degrees to 180 degrees, preferably 110 degrees to 160 degrees, and more preferably 120 degrees to 140 degrees. Each reaction vessel 102 can contain, for example, 21.65 nL of sample solution.

3 is a schematic cross-sectional view of a reaction container of a test piece according to an embodiment of the present invention.

Please refer to FIG. 3, the reaction container of the test strip 10 can be designed to have different shapes or configurations. For example, the reaction vessels 160a and 160b are grooves formed in the test piece 10 but do not penetrate the test piece 10. The reaction vessels 160b, 160d, and 160f have inclined side walls. The reaction vessels 160c, 160d, 160e, and 160f penetrate the test piece 10 and have two open ends on the top and bottom surfaces of the test piece 10. Due to capillary action, the sample liquid can be stably held in the reaction vessels 160c, 160d, 160e, and 160f. The reaction container 160d penetrates the test piece 10 and has two open ends on the top and bottom surfaces of the test piece 10, and has two open ends connected by inclined side walls.

However, the structural diagrams shown in FIG. 2 and FIG. 3 are only used to illustrate the present embodiment. The shape, size or number of the reaction container of the present invention is not limited thereto, and the cross-sectional shape of the reaction container may also be, for example Round, square or polygonal.

Generally, the primer is soluble in an aqueous solvent or solution. The test piece of the present invention can be designed such that the inner wall and bottom surface of the reaction vessel are hydrophilic, and the reaction vessels are hydrophobic. The reagent or probe will only attach to the hydrophilic area, that is, only to the inner wall and bottom surface of the reaction vessel. The size of each reaction vessel may be less than 1 millimeter (mm). With this size scale, a small amount of sample liquid can overflow and fill a large number of reaction vessels within 10 seconds, which significantly improves the sample loading efficiency.

Referring back to FIG. 1, the sacrificial layer 20 has micro-channels 28, and the micro-channels 28 have an injection channel 22, a main channel 24, and an end channel 26 that are interconnected. In the present embodiment, the material of the sacrificial layer 20 may include wax, so it can be melted when heated to about 60°C in the polymerase chain reaction. However, the present invention is not limited to this, and any material with a melting temperature ranging from room temperature to 60°C can also be used, and a material with a melting temperature of about 60°C is preferably used. In more detail, the size of the sacrificial layer 20 is, for example, 38 mm×38 mm×0.6 mm, the depth of the micro flow channel 28 is, for example, 0.2 mm, and the size of the main flow channel 24 is, for example, 33 mm×33 mm. 1 and FIG. 2 at the same time, the sacrificial layer 20 is suitable for assembling with the test piece 10, wherein the main flow channel 24 is assembled facing the opening 102a of the reaction container 102 in the test piece 10.

Referring to FIG. 1, the housing 30 may be composed of an upper cover 40 and a chassis 50. The upper cover 40 is suitable for assembly with the chassis 50. The chassis 50 may have a groove for accommodating the test piece 10 and the sacrificial layer 20. In more detail, the size of the upper cover 40 is, for example, 50 mm×50 mm×8 mm, and the size of the chassis 50 is, for example, 42 mm×42 mm×4.4 mm. The upper cover 40 has an injection hole 42, a discharge hole 44 and a storage tank 46. The storage tank 46 has functions of storing waste liquid and positioning foolproof. Since the storage tank 46 has a convex structure, the multiplex test piece device of the present invention can It will not be placed upside down when it is on the machine, so as to achieve the role of positioning foolproof.

In this embodiment, the upper cover 40 and the chassis 50 are combined with each other, for example, by a tenon. This combination has sufficient downforce to achieve a good sealing effect, thereby preventing liquid leakage, but the invention is not limited to this. The upper cover 40 and the chassis 50 can also be combined with each other by using glue or other fixing methods. The casing 30 has a heat conduction function in the polymerase chain reaction, and its material may include a heat conduction material, such as metal such as aluminum or copper, graphite or wafer, but the invention is not limited thereto. In addition, the housing 30 also has the function of isolating the test piece 10 and the sacrificial layer 20 from the outside, so as to prevent the reaction from being affected.

Referring to FIG. 1, the panel member 60 is suitable for assembling with the upper cover 40 and covering the storage tank 46, and the panel member 60 may have a label or a different appearance color to identify different test samples, targets, or functions. In more detail, when the multiplexed test strip device of the present invention is applied to an instrument (for example, a thermal cycler) provided with a label reading device, the label reading device can read the label on the panel member 60, and further Identify different test samples, where the labeling label is, for example, a handwritten mark, a bar code, or other labels, but the present invention is not limited to this, and a suitable labeling label can be selected according to the requirements and the label reading device used with it. On the other hand, the panel member 60 can also be designed with exterior colors according to different operating requirements to identify different test targets or functions, such as the identification of different test targets (miRNA or mRNA test) test strips, or the samples to be tested when they are on the machine Separated from the blank test strip device that does not contain the sample to be tested.

Referring to FIG. 1, the cover member 62 is suitable for assembly with the upper cover 40 and the panel member 60. During the operation, the cover member 62 can be opened first, and then the sample solution and oil are injected from the injection hole 42 and the injection hole 12 in sequence, and then the cover member 62 can be closed. The cover member 62 can be used to cover the injection hole 42 In order to avoid sample solution and oil splashing and to prevent biochemical reaction pollution.

4 is a schematic diagram of a multiplexed test strip device applied to loading a sample solution according to an embodiment of the present invention. 5A to 5C are schematic diagrams of the operation method of the multiplex test strip device according to the present invention. 6A to 6C are partial operation schematic diagrams of the storage tank of the multiplex test strip device according to the present invention. Next, in the following, the structure of the multiplexed test strip device according to an embodiment of the present invention and its operation application in loading a sample solution will be described with reference to FIGS. 1, 4, 5A to 5C, and 6A to 6C simultaneously.

Please refer to FIG. 1 and FIG. 4 at the same time, the cover member 62 can be opened first, and the injection hole 42 of the upper cover 40 and the injection of the test piece 10 can be sequentially injected by pipetting or other suitable liquid dispensers. The hole 12 injects the sample solution and oil into the injection flow channel 22, and the oil is used to push the sample solution, so that the sample solution and oil flow from the injection flow channel 22 through the main flow channel 24 to the terminal flow channel 26. In more detail, the total amount of sample solution added is, for example, 60 μL, and the oil is, for example, mineral oil or silicone oil.

Please refer to FIG. 1, FIG. 4 and FIG. 5A at the same time. When the sample solutions 70a and 70b flow through the main flow channel 24, they are loaded into each reaction vessel 102 of the test strip 10 (the flow direction of the sample solution is shown by the dotted line in FIG. 4) Arrow). Please refer to FIGS. 1, 4, 5B, and 5C at the same time. When the oil flows through the main flow channel 24, the sample solution 70b that is not loaded in the reaction vessel 102 is excluded (the flow direction of the oil is indicated by the dotted arrows in FIG. 4) Show). Please refer to FIG. 1, FIG. 4 and FIGS. 6A to 6C at the same time. Excess waste liquid such as sample solution 70b not loaded in the reaction vessel 102 flows from the end channel 26 through the discharge hole 14 and the discharge hole 44 into the storage tank 46. Covered with oil 80 and unable to return.

In FIG. 1, the injection hole 42 and the discharge hole 44 of the upper cover 40 and the injection hole 12 and the discharge hole 14 of the test piece 10 are arranged diagonally, respectively, but the invention is not limited thereto. The arrangement of the injection holes 12 and the discharge holes 14 can also be adjusted according to the arrangement of the injection channels 22 and the end channels 26 of the sacrificial layer 20, as long as the sample solution can be sufficiently extended during the flow. In addition, the arrangement of the injection holes 42 and the discharge holes 44 of the upper cover 40 depends on the arrangement of the injection holes 12 and the discharge holes 14 of the test piece 10, so it can also depend on the injection channel 22 and the end flow of the sacrificial layer 20 The arrangement of the channel 26 is adjusted.

During the experiment of the polymerase chain reaction, heating is performed to melt the sacrificial layer. The melted sacrificial layer is mixed with oil, and the temperature at which the sacrificial layer is heated to melt is, for example, about 60°C. It must be noted that the distance between the test piece of the present invention and the sacrificial layer is at least about 100 μm (for example, 100 μm to 400 μm), and the sacrificial layer has a certain thickness (for example, a thickness of 200 μm to 500 μm). Therefore, when the sacrificial layer after melting is mixed with oil, a distance of about 600 μm can be made between the test piece and the bottom plate, so that the reaction proceeds smoothly. Since there is no need to add an excessive amount of sample, a specific distance can be maintained between the test piece and the bottom plate, so it has the effect of saving the amount of sample added.

In summary, the present invention provides a multiplexed test strip device suitable for molecular biological detection, so that when the sample solution flows through the main flow channel of the sacrificial layer, it is quickly and evenly loaded into each reaction vessel of the test strip Then, the sample solution that is not loaded in the reaction vessel is removed by oil. In this way, all reaction vessels can be filled in a short time with a single pipetting operation, therefore, the experimental operation can be simplified and time can be saved. In addition, since there is no need to add an excessive amount of sample, a specific distance can be maintained between the test piece and the bottom plate. Therefore, the present invention has the effect of saving the amount of sample added. On the other hand, excess waste liquid can enter the storage tank through the discharge hole of the end flow channel, which is covered by oil and cannot be returned. Therefore, no additional steps are required to remove the waste liquid, which is more convenient and time-saving in operation, and storage The slot also has a foolproof positioning function.

Although the present invention has been disclosed as above with examples, it is not intended to limit the present invention. Any person with ordinary knowledge in the technical field can make some changes and modifications without departing from the spirit and scope of the present invention. The scope of protection of the present invention shall be subject to the scope defined in the appended patent application.

10‧‧‧Sample

12, 42‧‧‧ Injection hole

14, 44‧‧‧ discharge hole

20‧‧‧Sacrifice

22‧‧‧Inject into the flow channel

24‧‧‧Main channel

26‧‧‧End channel

28‧‧‧Micro channel

30‧‧‧Housing

40‧‧‧Top cover

46‧‧‧Storage tank

50‧‧‧Chassis

60‧‧‧Panel components

62‧‧‧Capping member

70a, 70b ‧‧‧ sample solution

80‧‧‧ oil

100‧‧‧ detection area

102, 160a, 160b, 160c, 160d, 160e, 160f‧‧‧‧Reaction vessel

102a‧‧‧Opening

102b‧‧‧Bottom

FIG. 1 is a schematic structural diagram of a multiplex test piece device according to an embodiment of the present invention. FIG. 2 is a schematic diagram of a test piece according to an embodiment of the present invention. 3 is a schematic cross-sectional view of a reaction container of a test piece according to an embodiment of the present invention. 4 is a schematic diagram of a multiplexed test strip device applied to loading a sample solution according to an embodiment of the present invention. 5A to 5C are schematic diagrams of the operation method of the multiplex test strip device according to the present invention. 6A to 6C are partial operation schematic diagrams of the storage tank of the multiplex test strip device according to the present invention.

Claims (10)

A multiplex test piece device for polymerase chain reaction, comprising: a test piece, having a plurality of reaction vessels, a first injection hole and a first discharge hole, the reaction vessels are arranged in an array, each of the reaction vessels With an opening and a bottom; a sacrificial layer with a micro-channel, the micro-channel has an interconnected injection channel, main channel and end channel, and the sacrificial layer is suitable for assembly with the test piece, so The main flow channel is assembled facing the opening; and a housing for accommodating the test piece and the sacrificial layer is composed of an upper cover and a chassis, the upper cover is suitable for assembly with the chassis, the The upper cover has a second injection hole, a second discharge hole, and a storage tank, wherein the sample solution and oil are injected into the injection flow path from the second injection hole and the first injection hole in sequence, and the oil is used to push the The sample solution, so that the sample solution and the oil flow from the injection flow channel through the main flow channel to the terminal flow channel, and the sample solution is loaded when flowing through the main flow channel In each of the reaction vessels, when the oil flows through the main flow channel, the sample solution not loaded in the reaction vessel is excluded, and excess waste liquid flows from the terminal flow channel through the first A drain hole and the second drain hole enter the storage tank and are covered by the oil and cannot flow back. The multiplex test piece device as described in item 1 of the patent application scope further includes a panel member, suitable for assembling with the upper cover and covering the storage tank, with a label or a different appearance color to identify different test samples and target Or function. The multiplex test piece device as described in item 2 of the patent application scope further includes a cover member suitable for assembling with the upper cover and the panel member, sequentially from the second injection hole and the first injection After the sample solution and the oil are injected into the hole, the second injection hole is covered with the capping member to prevent the sample solution and the oil from splashing out and to prevent contamination by biochemical reactions. The multiplex test strip device as described in item 1 of the patent application range, wherein the material of the housing includes a thermally conductive material. The multiplex test piece device according to item 1 of the patent application scope, wherein the chassis has a groove for accommodating the test piece and the sacrificial layer. The multiplex test piece device as described in item 1 of the patent application range, wherein the material of the test piece includes a light-transmitting material. The multiplex test piece device according to item 6 of the patent application range, wherein the light-transmitting material includes polycarbonate. The multiplex test piece device as described in item 1 of the patent application range, wherein the material of the sacrificial layer includes wax. The multiplex test piece device as described in item 1 of the patent application range, wherein the oil includes mineral oil or silicone oil. The multiplex test piece device as described in item 1 of the patent application range, wherein during the experiment of the polymerase chain reaction, heating is performed to melt the sacrificial layer, and the melted sacrificial layer is mixed with the oil.