CN110819505B

CN110819505B - Multiplex test piece device with storage tank

Info

Publication number: CN110819505B
Application number: CN201811097967.0A
Authority: CN
Inventors: 味正唯; 张家豪
Original assignee: Quark Biosciences Inc
Current assignee: Quark Biosciences Inc
Priority date: 2018-08-08
Filing date: 2018-09-20
Publication date: 2023-03-14
Anticipated expiration: 2038-09-20
Also published as: TW202007771A; TWI669390B; CN110819505A

Abstract

The invention provides a multiplex test piece device, which comprises a test piece, a sacrificial layer and a shell. The test strip has a plurality of reaction vessels arranged in an array. The sacrificial layer is provided with a micro-channel, and the micro-channel is provided with an injection channel, a main channel and a tail end channel which are communicated with each other. The housing is used for accommodating the test piece and the sacrificial layer, and comprises an upper cover and a bottom plate, wherein the upper cover is provided with an injection hole, a discharge hole and a storage tank. The sample solution and oil are injected into the injection flow channel from the injection hole, the oil is used for pushing the sample solution, the sample solution is loaded into each reaction container when flowing through the main flow channel, and the redundant waste liquid enters the storage tank from the discharge hole and is covered by the oil and cannot flow back.

Description

Multi-function test piece device with storage tank

Technical Field

The present invention relates to a multiplex test strip device for molecular biological detection, and more particularly, to a multiplex test strip device for Polymerase Chain Reaction (PCR) with a storage tank for storing waste liquid.

Background

In the field of molecular biological detection, it is often necessary to test multiple targets against a single sample. For example, the genotype of several Single Nucleotide Polymorphisms (SNPs) in a sample is examined or the degree of expression of several genes in a sample is examined by polymerase chain reaction detection. In this case, several DNA or RNA tests (assay) are required to form a test set (panel). In PCR assays, each set of assay reagents contains at least two specific DNA primer molecules (some PCR assays additionally contain target-specific reporter probes), and the pair of primers (primer pair) must be properly mixed with the DNA template extracted from the sample (sample) to be tested to determine the presence or quantity of the specific DNA target in the sample.

Traditionally, primer pairs and samples are placed in the same reaction vessel for PCR. The general placement is usually performed by pipetting the primer pair, enzyme and dNTP mixture and buffer reagents and sample stored in a single vial into the reaction vessel by a pipette. The most common carrier container is a 96-well plate. With the above-described placement, PCR assays require at least two rounds (two rounds) of pipette pipetting, one of which adds the sample to the reaction vessel and the other of which adds the primer pair to the reaction vessel. For example, assuming that 36 targets in one sample are inspected by one test kit, at least 36 pipetting operations are required to add respective primer pairs to 36 different reaction vessels, and in addition, 36 pipetting operations are required to add samples to the respective reaction vessels. This 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 laboratory operator only needs to add the sample to the already pre-filled vessels. With the foregoing example, testing 36 targets for one sample would require only 36 pipetting operations to add the sample to 36 reaction vessels that were pre-filled. In addition, the volume of the reaction container can be reduced to be in the range of about nano-liter (nano-liter) at the same time, so that the using amount of the detection reagent is saved. Under this modification, a 96-well plate, which is commonly used as a carrier, is modified to a microtiter plate similar to a test strip.

However, the reaction vessels (also known as microwells or nanowells) of a microtiter plate are too small in size and volume to be manually filled with the primer pairs or samples used while avoiding cross-contamination between adjacent reaction vessels (i.e., primer pairs escaping from one well to another), and therefore, require special microfluidic dispensing techniques. In more detail, a primer pair is provided to each nano-well in advance and a primer is fixed on the surface of the nano-well. The user can then add samples to each reaction vessel in a single pipetting operation or through a single microfluidic channel without fear of primers escaping from one well to other wells, minimizing cross-contamination from well to well.

When the subsequent sample detection is performed, each reaction vessel must be filled with a predetermined amount of sample. Conventionally, samples are added one by one to the reaction well using a pipette or needle-like dispenser. However, as the reaction vessels become smaller and closer to each other, the design of a special mechanical mechanism of the dispenser or path for the wells becomes complicated and time consuming to transfer to each reaction vessel individually. If a single pipetting operation is achieved by a special test strip device design or a sample is added to each reaction vessel through a single microfluidic channel, the manual operation required for preparing a detection reagent by polymerase chain reaction can be greatly simplified, and the convenience of filling the sample is increased.

Disclosure of Invention

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 to instant polymerase chain reaction. By the multiplex test piece device, samples can be quickly and uniformly loaded into each reaction container of the test piece, all the reaction containers can be filled in a short time by single liquid return operation, redundant waste liquid can flow to the storage tank for storage, and the storage tank also has a positioning and fool-proofing function.

The invention provides a multiplex test piece device, which comprises a test piece, a sacrificial layer and a shell. The test strip 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 a micro flow channel, the micro flow channel has an injection flow channel, a main flow channel and a terminal flow channel which are mutually communicated, the sacrificial layer is suitable for being assembled with the test piece, and the main flow channel is assembled facing the opening part. The casing is used for accommodating the test piece and the sacrificial layer and consists of an upper cover and a chassis, the upper cover is suitable for being assembled with the chassis, and the upper cover is provided with a second injection hole, a second discharge hole and a storage tank. And sequentially injecting the sample solution and the oil into the injection flow channel from the second injection hole and the first injection hole, and pushing the sample solution by using the oil so that the sample solution and the oil flow through the main flow channel from the injection flow channel to the tail end flow channel. The sample solution is loaded into each reaction vessel while flowing through the main flow channel. The oil, while flowing through the main flow channel, excludes the sample solution not loaded in the reaction vessel. The excess waste liquid flows from the end flow passage through the first discharge hole and the second discharge hole into the storage tank, and is covered with the oil so as not to flow back.

In an embodiment of the present invention, the multi-function test strip device further comprises a panel member, adapted to be assembled with the upper cover and cover the storage groove, and having a label or different colors for identifying different detection samples, labels or functions.

In an embodiment of the invention, the multiplex test strip device further includes a cover member adapted to be assembled with the upper cover and the panel member, and the cover member is used to cover the second injection hole after the sample solution and the oil are injected from the second injection hole and the first injection hole in sequence, so as to prevent the sample solution and the oil from splashing and prevent the contamination of biochemical reaction.

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

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

In an embodiment of the present invention, the material of the test strip includes a light transmissive material.

In one embodiment of the present invention, the light transmissive material includes polycarbonate.

In one embodiment of the invention, the material of the sacrificial layer comprises wax.

In one embodiment of the invention, the oil comprises mineral oil or silicone oil.

In one embodiment of the present invention, during the polymerase chain reaction experiment, heat is applied to melt the sacrificial layer, and the melted sacrificial layer is mixed with oil.

In view of the above, the present invention provides a multi-functional test strip device with storage tanks, which allows a sample to be rapidly and uniformly loaded into each reaction vessel of a test strip while flowing through a main flow channel of a sacrificial layer, and then the sample solution 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, thereby simplifying the experimental operation and saving time. In addition, the redundant waste liquid can flow through the first discharge hole and the second discharge hole from the tail end flow channel to enter the storage tank and is covered by the oil, so that the waste liquid can not flow back, no extra step is needed to remove the waste liquid, the operation is more convenient and time-saving, and the storage tank also has the function of positioning and fool-proofing.

In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below.

Drawings

FIG. 1 is a schematic structural diagram of a multi-function test strip device according to an embodiment of the present invention.

Fig. 2 is a schematic structural diagram of a test strip according to an embodiment of the present invention.

Fig. 3 is a schematic cross-sectional view of a reaction vessel of a test strip according to an embodiment of the present invention.

FIG. 4 is a schematic view of a multi-purpose test strip device for loading a sample solution according to an embodiment of the present invention.

Fig. 5A to 5C are schematic views illustrating an operation method of a multiplexed test strip device according to the present invention.

FIGS. 6A to 6C are partial schematic views illustrating the operation of the reservoir of the multi-functional test strip device according to the present invention.

[ notation ] to show

10: test piece

12. 42: injection hole

14. 44: discharge hole

20: sacrificial layer

22: injection runner

24: main runner

26: end runner

28: micro flow channel

30: shell body

40: upper cover

46: storage tank

50: chassis

60: panel member

62: cover member

70a, 70b: sample solution

80: oil

100: detection area

102. 160a, 160b, 160c, 160d, 160e, 160f: reaction vessel

102a: opening part

102b: bottom part

Detailed Description

The invention provides a multiplex test piece device which can be widely applied to different types of reaction tests. Hereinafter, the terms used in the specification are defined and explained.

"reagent" may refer to a formulation or preparation of several ingredients for one particular test/assay. For example, in assays that employ polymerase chain reaction, detection reagents include a pair of primers, enzymes, dNTPs, fluorescent reporters and salts, and the like. In an application, different primer pairs and fluorescent reporters may be added to the reaction vessel first, followed by mixing the sample with enzymes, dNTPs and other additives and then adding to the reaction vessel.

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

"analysis" or "testing" may refer to one or more experiments or test items performed on the same sample. For example, 300 single nucleotide polymorphism (300 SNP) genotypes are detected for a nucleic acid sample using PCR, which includes several PCR detection items by examining each genotype (A, T, C, G) for each Single Nucleotide Polymorphism (SNP). For example, the amount of nucleic acid of a particular sequence is determined using instant fluorescent quantitative PCR.

The "sample solution" refers to a mixture or a mixed solution of the above-mentioned "sample" and a mixing reagent (master mix).

"reaction vessel" may refer to an individual tube of a tube tray, an individual reaction tube, a well or well of a microtiter plate, or a well/well of a test strip or array plate. As used herein, a "test strip", "test strip plate", "detection array plate" or "detection plate" can all refer to the same base or substrate that holds the aforementioned reaction vessels.

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 surfaces rather than by gravity. If the volume of the liquid in the container is only a few nanoliters, the liquid has a high surface adhesion and sticks to the container (the well), i.e., the liquid can be regarded as an adhesive-like stable attachment to the bottom of the container or to the wall of the container.

Preferably, the reaction vessel can be a single reaction well or well of a test strip or a detection array plate. As discussed above, it is preferred to use reaction vessels of smaller volume, for example, ranging in size from a few nanoliters to hundreds of nanoliters.

FIG. 1 is a schematic diagram of a multi-function test strip device according to an embodiment of the present invention.

Referring to fig. 1, the multiplex 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 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 fig. 2 and 3.

Referring to fig. 2, the test strip 10 has a detection region 100, and the area of the detection region 100 is 22.5mm × 22.5mm, for example. A plurality of reaction vessels 102 are contained in the detection region 100, and the reaction vessels 102 are arranged in an n × n array. The test piece 10 further has an injection hole 12 and a discharge hole 14. In this embodiment, the test piece 10 has dimensions of, for example, 36mm × 36mm × 0.8mm. 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 to fig. 2 again, each reaction container 102 has a wider opening 102a and a narrower bottom 102b when viewed from the cross-sectional view (the right part of fig. 2) of the test strip 10. In the present embodiment, the depth of each reaction vessel 102 is, for example, 200 μm (d 1), the size of the opening portion 102a is, for example, 410 μm (L1). Times.410 μm (W1), and the size of the bottom portion 102b is, for example, 220 μm (L2). Times.220 μm (W2). The pitch (P1) between the reaction vessels 102 ranges, for example, from 27 μm to 45 μm. 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 may hold, for example, 21.65nL of sample solution.

FIG. 3 is a schematic sectional view of a reaction vessel of a test strip according to an embodiment of the present invention.

Referring to fig. 3, the reaction vessel 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 strip 10 but do not penetrate the test strip 10. The

reaction vessels

160b, 160d and 160f have sloped sidewalls. The

reaction vessels

160c, 160d, 160e and 160f penetrate the test strip 10 and have two open ends on the top and bottom surfaces of the test strip 10. Due to capillary action, the sample liquid can be stably held in the

reaction vessels

160c, 160d, 160e, and 160 f. The reaction vessel 160d penetrates the test strip 10 and has two open ends on the top and bottom surfaces of the test strip 10 and two open ends connected by inclined side walls.

However, the structure diagrams shown in fig. 2 and 3 are only for illustrating the present embodiment, the shape, size or number of the reaction vessel of the present invention is not limited thereto, and the cross-sectional shape of the reaction vessel may be, for example, circular, square or polygonal.

In general, the primers are dissolved in an aqueous solvent or solution, and the test strip of the present invention may be designed such that the inner and bottom surfaces of the reaction vessels are hydrophilic and the reaction vessels are hydrophobic. The reagents or probes will only attach to the hydrophilic regions, that is, only to the inner walls and bottom surfaces 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 to fill a large number of reaction vessels within 10 seconds, significantly improving sample loading efficiency.

Referring back to fig. 1, the sacrificial layer 20 has a microchannel 28, and the microchannel 28 has an injection channel 22, a main channel 24 and a tail channel 26 connected to each other. In this embodiment, the material of the sacrificial layer 20 may include wax and, therefore, may melt when heated to about 60 ℃ in a polymerase chain reaction. However, the invention is not limited thereto, and any material having a fusible temperature in the range of from above room temperature to 60 ℃ may be used, and preferably a material having a fusible temperature of about 60 ℃. In more detail, the dimensions of the sacrificial layer 20 are, for example, 38mm × 38mm × 0.6mm, the depth of the micro flow channel 28 is, for example, 0.2mm, and the dimensions of the main flow channel 24 are, for example, 33mm × 33mm. Referring to fig. 1 and fig. 2, the sacrificial layer 20 is suitable for being assembled with the test strip 10, wherein the main flow channel 24 faces the opening 102a of the reaction container 102 in the test strip 10.

Referring to fig. 1, the housing 30 may include an upper cover 40 and a bottom plate 50, wherein the upper cover 40 is suitable for being assembled with the bottom plate 50, and the bottom plate 50 may have a groove for accommodating the test piece 10 and the sacrificial layer 20. More specifically, the dimensions of the upper cover 40 are, for example, 50mm × 50mm × 8mm, and the dimensions of the base 50 are, for example, 42mm × 42mm × 4.4mm. 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 fool-proofing, and the storage tank 46 has a convex structure, so that the multi-functional test piece device of the present invention can not be placed upside down during the operation, thereby achieving the positioning fool-proofing effect.

In the embodiment, the upper cover 40 and the chassis 50 are combined with each other by, for example, a tenon, and the combination manner has a sufficient downward pressure to achieve a better sealing effect and further prevent liquid from leaking, but the invention is not limited thereto, and the upper cover 40 and the chassis 50 may be combined with each other by using an adhesive or other fixing manner. The housing 30 has a function of conducting heat during the polymerase chain reaction, and the material of the housing may include a heat conducting material, such as a metal, e.g., aluminum or copper, graphite or a 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 to prevent the reaction from being affected.

Referring to fig. 1, the panel member 60 is adapted to be assembled with the upper cover 40 and cover the storage slot 46, and the panel member 60 may have a label or different colors for identifying different detection samples, labels or functions. More specifically, when the multi-functional test piece device of the present invention is applied to an instrument (e.g. a thermal cycler) equipped with a label reading device, the label reading device can read the label on the panel member 60 to identify different detection samples, wherein the label is, for example, a handwritten mark, a bar code or other marks, but the present invention is not limited thereto, and the label reading device can be selected to be suitable for use according to the requirement and the matched application. Alternatively, the panel member 60 can be designed with colors according to different operation requirements to identify different targets or functions, such as identification of different target (miRNA or mRNA detection) test strips, or distinguishing between blank test strip devices containing samples to be detected and blank test strip devices containing no samples to be detected during the loading process.

Referring to fig. 1, the cover member 62 is adapted to be assembled with the upper cover 40 and the panel member 60. In the operation process, the cover member 62 is opened first, the sample solution and the oil are injected from the injection hole 42 and the injection hole 12 in sequence, and then the cover member 62 is closed, so that the injection hole 42 can be covered by the cover member 62 to prevent the sample solution and the oil from splashing and prevent the contamination of biochemical reaction.

FIG. 4 is a schematic view of a multi-functional test strip device according to an embodiment of the present invention applied to a sample solution loading. FIGS. 5A to 5C are schematic views illustrating an operation method of a multi-functional test strip device according to the present invention. FIGS. 6A to 6C are partial views illustrating the operation of the storage well of the multi-functional test strip device according to the present invention. Next, the structure of the multi-functional test strip device according to the embodiment of the present invention and the application of the multi-functional test strip device in loading a sample solution will be described below with reference to fig. 1, 4, 5A to 5C, and 6A to 6C.

Referring to fig. 1 and 4, the cover member 62 can be opened first, and sample solution and oil can be sequentially injected into the injection channel 22 from the injection hole 42 of the upper cover 40 and the injection hole 12 of the test strip 10 by pipetting (pipetting) or other suitable liquid dispenser, and the sample solution is pushed by the oil, so that the sample solution and the oil can flow through the main channel 24 to the end channel 26 from the injection channel 22. In more detail, the total amount of the sample solution added is, for example, 60. Mu.L, and the oil is, for example, a mineral oil or a silicone oil.

Referring to fig. 1, fig. 4 and fig. 5A, the

sample solutions

70a and 70b are loaded into each reaction container 102 of the test strip 10 while flowing through the main flow channel 24 (the flowing direction of the sample solution is shown by the dashed arrow in fig. 4). Referring to fig. 1, 4, 5B and 5C, the oil flows through the main channel 24 to remove the sample solution 70B not loaded in the reaction vessel 102 (the flow direction of the oil is shown by the dotted arrow in fig. 4). Referring to fig. 1, 4 and 6A to 6C, the excessive waste liquid such as the sample solution 70b not loaded in the reaction vessel 102 flows from the end flow path 26 through the discharge hole 14 and the discharge hole 44 into the storage tank 46, and is covered with the oil 80 and cannot flow back.

In fig. 1, the filling hole 42 and the discharge hole 44 of the upper cover 40 and the filling hole 12 and the discharge hole 14 of the test piece 10 are diagonally arranged, but the present invention is not limited thereto. The arrangement of the injection hole 12 and the discharge hole 14 may be adjusted according to the arrangement of the injection flow path 22 and the end flow path 26 of the sacrificial layer 20, as long as the sample solution can be sufficiently spread during the flow. The arrangement of the injection hole 42 and the discharge hole 44 of the upper cover 40 depends on the arrangement of the injection hole 12 and the discharge hole 14 of the test piece 10, and thus can be adjusted according to the arrangement of the injection flow path 22 and the end flow path 26 of the sacrificial layer 20.

During the experiment of the polymerase chain reaction, heat is applied to melt the sacrificial layer, and the melted sacrificial layer is mixed with oil, wherein the temperature at which the sacrificial layer is heated to melt is, for example, about 60 ℃. It should be noted that the test piece of the present invention is spaced from the sacrificial layer by a distance of at least about 100 μm (e.g., 100 μm to 400 μm), and the sacrificial layer has a certain thickness (e.g., 200 μm to 500 μm). Therefore, when the melted sacrificial layer is mixed with oil, the distance between the test piece and the base plate can be about 600 μm, so that the reaction can be smoothly performed. Because the specific distance between the test piece and the bottom plate can be maintained without adding excessive samples, the method has the function of saving the adding amount of the samples.

In summary, the present invention provides a multiplex test strip device suitable for molecular biological detection, which allows a sample solution to be rapidly and uniformly loaded into each reaction container of a test strip while flowing through a main flow channel of a sacrificial layer, and then the sample solution not loaded in the reaction container is removed by oil. In this way, all reaction vessels can be filled in a short time with a single pipetting operation, thereby simplifying the experimental operation and saving time. In addition, the invention can maintain a specific distance between the test piece and the bottom plate without adding excessive samples, thereby saving the sample addition. On the other hand, the redundant waste liquid can also enter the storage tank from the tail end flow channel discharge hole and is covered by the oil, so that the waste liquid can not flow back, no extra step is needed for removing the waste liquid, the operation is more convenient and time-saving, and the storage tank also has the positioning and fool-proof functions.

Although the present invention has been described with reference to the above embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. A multi-purpose test strip device for polymerase chain reaction, comprising:

a test strip having a plurality of reaction vessels, a first injection hole and a first discharge hole, the reaction vessels being arranged in an array, each of the reaction vessels having an opening and a bottom;

a sacrificial layer having a microchannel, the microchannel having an injection channel, a main channel and a terminal channel which are communicated with each other, the sacrificial layer being adapted to be assembled with the test piece, the main channel being assembled facing the opening; and

a housing for accommodating the test piece and the sacrificial layer, which comprises an upper cover and a bottom plate, wherein the upper cover is suitable for being assembled with the bottom plate, the upper cover is provided with a second injection hole, a second discharge hole and a storage tank, the storage tank is provided with an outward convex structure,

wherein a sample solution and an oil are sequentially injected from the second injection hole and the first injection hole into the injection flow channel, the sample solution is pushed by the oil so that the sample solution and the oil flow from the injection flow channel through the main flow channel to the end flow channel, the sample solution is carried into each of the reaction vessels while flowing through the main flow channel, the oil excludes the sample solution not loaded in the reaction vessels while flowing through the main flow channel, and an excess waste liquid flows from the end flow channel through the first discharge hole and the second discharge hole into the reservoir tank, and is covered with the oil so as not to flow back.

2. The multi-functional test strip device of claim 1, further comprising a panel member adapted to be assembled with the upper cover and cover the storage groove, having a label or different colors for identifying different detection samples, labels or functions.

3. The multi-functional test piece device according to claim 2, further comprising a cover member adapted to be assembled with the upper cover and the panel member, wherein the cover member covers the second injection hole after the sample solution and the oil are sequentially injected from the second injection hole and the first injection hole, so as to prevent the sample solution and the oil from splashing and contamination of biochemical reaction.

4. The multi-function test strip device of claim 1, wherein the material of the housing comprises a thermally conductive material.

5. The multi-function test strip device according to claim 1, wherein the bottom plate has a groove for accommodating the test strip and the sacrificial layer.

6. The multi-function test strip device of claim 1, wherein the material of the test strip comprises a light transmissive material.

7. The multi-function test strip device according to claim 6, wherein the light transmissive material comprises polycarbonate.

8. The multi-function test strip device according to claim 1, wherein the material of the sacrificial layer comprises wax.

9. The multi-working test strip device according to claim 1, wherein the oil comprises mineral oil or silicone oil.

10. The multi-function test strip device of claim 1, wherein during the polymerase chain reaction experiment, heat is applied to melt the sacrificial layer, and the melted sacrificial layer is mixed with the oil.