TWI708057B - Centrifugal reaction microtube, centrifugal reaction device and centrifugal inspection method thereof - Google Patents

Abstract

This application provides a centrifugal reaction microtube, a centrifugal reaction device and a centrifugal inspection method thereof. Through the control of centrifugal force and the use of a one-way valve in the centrifugal reaction microtube, simple and fast operation, safe, energy-saving, and accurate It is economical, and avoids pollution.

Description

Centrifugal reaction microtube, centrifugal reaction device and centrifugal inspection method thereof

This case is about a reaction microtube and reaction device for experiments, especially a reaction microtube and reaction device that completes molecular biological detection through one-click.

With the development of the field of biotechnology, the demand for detection through biochemical and molecular biological methods is increasing. Although there are already polymerase chain reaction (PCR) instruments that can perform nucleic acid amplification and analysis at the same time on the market, many instruments are still required, such as: electric pipettes, nucleic acid hybridization reactors, cleaning instruments, signal interpretation instruments, etc. Only then can all detection steps be completed. Therefore, the detection process is often complicated and time-consuming, and there is also a risk of contamination during the pipetting process.

When the nucleic acid or sample to be analyzed is extremely complex, the current commercially available products cannot complete all molecular and biological detections with one click, and cannot balance qualitative and quantitative analysis at the same time.

In view of the above-mentioned problems of the prior art, an object of the present invention is to provide a centrifugal reaction microtube and a reaction device. Through the design of the reaction microtube, extraction, reaction, cleaning and cleaning can be performed in the same reaction microtube by centrifugation. / Or detect the signal to achieve the effect of reducing manpower demand and pollution.

According to an objective of the present invention, an embodiment of the present invention provides a centrifugal reaction microtube, including: The first reaction zone is used to contain the reaction mixture for the first reaction; and The first one-way valve is located in the centrifugal direction of the first reaction zone, wherein the first one-way valve is opened by the first centrifugal force to allow the reaction mixture to leave the first reaction zone.

Preferably, the centrifugal reaction microtube further includes: The second reaction zone is separated from the first reaction zone through the first one-way valve, and the reaction mixture is moved to the second reaction zone by the first centrifugal force for the second reaction; and The second one-way valve is arranged on the opposite side of the first one-way valve in the second reaction zone, wherein the second one-way valve is opened by the second centrifugal force to make the reaction mixture leave the second reaction zone.

Preferably, the second centrifugal force is greater than the first centrifugal force.

Preferably, the first centrifugal force and the second centrifugal force may be 1 to 80,000 g, respectively, and the second centrifugal force is greater than the first centrifugal force.

Preferably, the opening and closing of the first one-way valve and the second one-way valve can be controlled electronically or magnetically.

Preferably, the centrifugal reaction microtube may further include a biochip arranged in the second reaction zone, on which biomolecules including antibodies, aptamers, peptides or nucleic acids are coated.

Preferably, the centrifugal reaction microtube may further include a waste liquid zone, which is separated from the second reaction zone by a second one-way valve.

Preferably, the first reaction zone can perform a polymerase chain reaction and/or the second reaction zone can perform a nucleic acid hybridization reaction.

Preferably, the centrifugal reaction microtube further includes: The third reaction zone is used to contain the reaction mixture for the third reaction; and The third check valve is arranged between the third reaction zone and the first reaction zone, and the reaction mixture is moved to the first reaction zone by the third centrifugal force.

Preferably, the centrifugal reaction microtube can be substantially made of optical materials. More preferably, the optical material may include quartz, glass or plastic.

According to another objective of the present invention, an embodiment of the present invention provides a centrifugal reaction device, including: The reaction centrifugal disc is inclined upwardly from the shaft center, and the reaction centrifugal disc is provided with a plurality of fixed racks arranged symmetrically for accommodating a plurality of centrifugal reaction microtubes.

Preferably, the slope may be about 1 to 89 degrees.

Preferably, the centrifugal reaction device may further include a signal detection module for detecting the reaction signal of the centrifugal reaction microtube.

Preferably, the centrifugal reaction device may further include a liquid separation module, and reaction reagents and/or samples are added to the centrifugal reaction microtubes respectively.

Preferably, the centrifugal reaction device may further include a temperature control module for controlling the reaction temperature of the centrifugal reaction microtube.

Preferably, the centrifugal reaction device may further include a waste liquid collection module for collecting waste liquid leaving from the centrifugal reaction microtube.

According to another object of the present invention, an embodiment of the present invention provides a centrifugal detection method, including: Provide centrifugal reaction microtubes; and The sample and/or reaction reagent are added to the first reaction zone to form a reaction mixture, and the centrifugal reaction microtube is centrifuged to perform the first reaction.

Preferably, the method may further include adding a cleaning solution and intermittently centrifuging the centrifugal reaction microtube.

Preferably, the method may further include detecting the reaction signal of the centrifugal reaction microtube.

Preferably, the method may further include controlling the reaction temperature of the centrifugal reaction microtube.

Preferably, the method may further include collecting waste liquid leaving from the centrifugal reaction microtube.

Preferably, the method may further include: The centrifugal reaction microtube is used for the first centrifugal force to move the reaction mixture through the first one-way valve to the second reaction zone for the second reaction.

Preferably, the method may further include: The centrifugal reaction microtube is used to make the reaction mixture leave the second reaction zone through the second one-way valve through the second centrifugal force.

Preferably, the second centrifugal force is greater than the first centrifugal force.

Preferably, the first centrifugal force and the second centrifugal force may be 1 to 80,000 g, respectively, and the second centrifugal force is greater than the first centrifugal force.

Preferably, the opening and closing of the first one-way valve and the second one-way valve can be controlled by electronic control or magnetic control.

Preferably, the first reaction zone can perform a polymerase chain reaction and/or the second reaction zone can perform a nucleic acid hybridization reaction.

The above and other objects, features, and advantages of the present invention will become apparent with reference to the following detailed description, preferred embodiments, and drawings attached to the text.

The following describes the embodiments in detail with reference to related drawings. However, these embodiments can be implemented in different forms, but this is not the only way to implement or use the specific embodiments of the invention requested in this case, and therefore should not be construed as limiting the above embodiments. The implementation manners cover the characteristics of a number of specific embodiments and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps. On the contrary, these embodiments are provided so that this specification can be thoroughly and completely disclosed, so as to fully express the spirit of the present invention to those with ordinary knowledge in the technical field of the present invention. Similar component symbols in the drawings refer to similar components. In the following description, the conventional functions or structures will not be described in detail, so as not to repeat unnecessary details in the embodiments.

Unless otherwise defined, all technical terms and terms used herein have the same meaning as commonly understood by those with ordinary knowledge in the technical field to which this creation belongs. In case of conflict, the present specification including definitions shall prevail.

Without conflict with the context, the singular nouns used in this specification cover the plural nouns; and the plural nouns also cover the singular nouns. In addition, in this specification and the scope of the patent application, expressions such as "at least one" and "one or more" have the same meaning, and both of them mean that one, two, three or more are included.

The conjunction "consisting essentially of" (consisting essentially of) is used to define a composition, method or device, which includes materials, steps, features, components or elements other than those stated in the text, and its limitations The condition is that these additional materials, steps, features, components or components will not significantly affect the basic and novel features of the claimed creation. The term "consisting essentially of" resides in the middle ground between "comprising" and "consisting of".

Although the numerical ranges and parameters used to define the wider range of the present invention are approximate numerical values, the relevant numerical values in the specific embodiments are presented here as accurately as possible. However, any value inherently inevitably contains the standard deviation due to individual test methods. Here, "about" usually means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Or, the word "about" means that the actual value falls within the acceptable standard error of the average value, depending on the consideration of a person with ordinary knowledge in the technical field of the present invention. In addition to the examples, or unless otherwise clearly stated, all ranges, quantities, values, and percentages used herein (for example, used to describe the amount of material, length of time, temperature, operating conditions, quantity ratios and other similar Those) have been modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the scope of the patent application are approximate values and can be changed according to requirements. At least these numerical parameters should be understood as the indicated effective number of digits and the value obtained by applying the general carry method. Here, the numerical range is expressed from one end point to another point or between two end points; unless otherwise specified, the numerical range described here includes the end points.

In one embodiment, a centrifugal reaction microtube is provided, which includes: at least one reaction zone for containing a reaction mixture including a reaction reagent, a cleaning solution and/or a sample for reaction; and at least one one-way valve, respectively located at In the centrifugal direction of the reaction zone, the reaction mixture flows away from the axis by the centrifugal force, and the one-way valve is opened when the centrifugal speed or centrifugal force exceeds a threshold to make the reaction mixture leave the reaction zone, and when the centrifugal speed or centrifugal force is lower than one When the threshold is set, the one-way valve is closed to prevent the reaction mixture from flowing back to the original reaction zone. In other words, by setting a one-way valve that can be selectively opened or closed repeatedly, the reaction microtubules can be partitioned into at least one reaction zone for the purification, amplification and analysis of analytes (such as proteins, nucleic acids and other biological molecules) .

In this application, the "one-way valve" means that the reaction reagent, cleaning solution, and/or sample can be controlled to stay in a reaction zone when it is not centrifuged, and the reaction reagent, cleaning solution and/or sample can be allowed to remain in a reaction zone during centrifugation. A mechanism in which the body passes through the "one-way valve" in one direction to another reaction zone without backflow.

There are many ways to operate the one-way valve body. For example, a mechanical one-way valve is composed of springs with different elastic constants and balls with different weights. The material can be metal or non-metal. Different sizes of centrifugal force can correspond to valve bodies with different elastic constants, allowing different valve bodies in the reaction microtube to open or close under different sizes of centrifugal force. That is, with the use of centrifugal force, the check valve can control the retention or elimination of the reaction mixture. In addition, the one-way valve can also be controlled by electric or magnetic control to open and close the one-way valve.

For example, if the one-way valve is opened and closed by a compression spring, the compression spring is fully extended under the condition of static, no centrifugal force, or centrifugal force that does not reach the compression stress of the spring, and the one-way valve is completely sealed. When the centrifugal force reaches the compression stress of the spring, as the centrifugal force gradually increases, the compression spring will gradually become shorter due to compression, thereby opening the one-way valve so that the reaction mixture can move through the one-way valve in the direction away from the rotation axis.

All the waste liquid generated in the reaction process can be collected in a closed or vacuum, depending on whether the reaction microtube has an opening and the amount of waste liquid. In one embodiment, the waste liquid is collected airtightly, that is, the centrifugal reaction microtube may further include a waste liquid area, which is separated from the reaction area by a one-way valve, so that the reaction mixture or cleaning liquid after the reaction in the reaction area is separated. Centrifugation can leave the reaction zone to the waste liquid zone without refluxing.

In one embodiment, a biochip can be arranged in the reaction zone of the centrifugal reaction microtube, on which biomolecules are coated for hybridization with the reaction mixture. Biomolecules may include antibodies, aptamers, peptides or nucleic acids, and the like.

As shown in FIG. 1, in the first embodiment, the centrifugal reaction microtube 1 includes a reaction zone 11 and a one-way valve 12. For example, the nucleic acid molecules amplified by PCR can react with the biochip pre-coated with target nucleic acid in the reaction zone 11. Part of the complementary nucleic acid molecules will be attached to the biochip, and then centrifuged The unattached nucleic acid molecules are removed through the one-way valve 12.

In the second embodiment, as shown in Figure 2, the centrifugal reaction microtube 2 not only includes a reaction zone 11 and a one-way valve 12, but also a waste liquid zone 13 for collecting the waste liquid after the reaction.

In the third embodiment, as shown in FIG. 3, the centrifugal reaction microtube 3 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, and a second check valve 122. The first reaction zone 111 is for the reaction mixture to perform the first reaction; the first check valve 121 is located in the centrifugal direction of the first reaction zone 111; the second reaction zone 112 is separated from the first reaction zone 111 through the first check valve 121; The second one-way valve 122 is arranged on the opposite side of the first one-way valve 121 in the second reaction zone 112.

In the third embodiment, since there are two reaction zones, two reactions can be performed sequentially in the same centrifugal reaction microtube 3. For example, the polymerase chain reaction can be carried out in the first reaction zone 111 and/or nucleic acid hybridization reaction can be carried out in the second reaction zone 112; or nucleic acid extraction can be carried out in the first reaction zone 111 and/or nucleic acid can be carried out in the second reaction zone 112 Hybridization reaction.

In this embodiment, those skilled in the art to which the present invention pertains can select springs with different or the same spring constant according to their needs to control the opening and closing of the springs of the first check valve 121 and the second check valve 122 under the same centrifugal condition. Open or close under different centrifugal conditions. In a preferred embodiment, when springs with the same spring constant are used, balls of different weights can be used, that is, a heavier ball is used for the first check valve 121, and a ball with a heavier weight is used for the second check valve 122. Since the ball is lighter in weight, the centrifugal force required to open the first check valve 121 is smaller than the centrifugal force required to open the second check valve 122. Therefore, at a lower speed, only the first one-way valve 121 will open, and the second one-way valve 122 will be closed. In another preferred embodiment, when springs with different elastic constants are used, in order to prevent the reaction mixture in the first reaction zone 111 from passing through the first one-way valve 121 and the second one-way valve 122 at the same time when it is In the second reaction zone 112, the spring constant of the second one-way valve 122 is greater than the spring constant of the first one-way valve 121. In this case, compared with the first one-way valve 121, a larger centrifugal force is required to open the second one-way valve 122, that is, a higher rotation speed is required to open the second one-way valve 122. Therefore, at a lower speed, only the first one-way valve 121 will open, and the second one-way valve 122 will be closed.

In a preferred embodiment, the first centrifugal force and the second centrifugal force may be 1 to 80,000 g, respectively, and the second centrifugal force is greater than the first centrifugal force.

In the 3A embodiment, as shown in FIG. 4, the centrifugal reaction microtube includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, and a second check valve 122. The difference from the third embodiment is that the first check valve 121 and the second check valve 122 are controlled by a single spring. As shown in part (A) of Figure 4, the first check valve 121, the second reaction zone 112, and the second check valve 122 together form a sliding member that can slide in the direction of centrifugal force during centrifugation. Wherein, the sliding piece can be connected with a spring, so that the sliding piece can return to the static state after centrifugation. In a preferred embodiment, the spring can be a compression spring and is arranged on the right side of the sliding member in the figure; or the spring can be a tension spring and is arranged on the left side of the sliding member.

For example, the part (A) of Fig. 4 shows the unprocessed centrifugal reaction microtube, the first reaction zone 111 and the second reaction zone 112 are not connected, and the sliding member is in the first position of the static state. When the first centrifugal force is used for centrifugation, the sliding member moves to the second position shown in part (B), so that the through hole is connected to the first check valve 121, and the reaction reagent, cleaning solution and/or sample will be automatically The first reaction zone 111 passes through the first one-way valve 121 to the second reaction zone 112, and the second one-way valve 122 has not yet communicated with the through hole, so the reaction reagent, cleaning solution and/or sample will not pass through the second one-way valve. Valve 122. When the second centrifugal force is greater than the first centrifugal force for centrifugation, the sliding member further moves to the third position shown in part (C), so that the through hole communicates with the second check valve 122, and the through hole is not A one-way valve 121 is connected, so the reaction reagent, cleaning solution, and/or sample will be removed from the second reaction zone 112 through the second one-way valve 122 and will not flow back to the first reaction zone 111.

In the fourth embodiment, as shown in part (A) of Figure 5, the centrifugal reaction microtube 4 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, and a second check valve. In addition to 122, it also includes the waste liquid area 13, which can be used to collect the waste liquid after the reaction.

In the 4A embodiment, as shown in part (B) of Figure 5, the 4A embodiment is compared with the fourth embodiment in that the first check valve 121 and the second check valve 122 in the centrifugal reaction microtube The system is designed in a slope shape, and the slope of the second one-way valve 122 is greater than that of the first one-way valve 121. In this embodiment, when it is not centrifuged, the reaction reagent, the cleaning solution and/or the sample are isolated by the first one-way valve 121 and remain in the first reaction zone 111. When the first centrifugal force is used for centrifugation, the reaction reagent, cleaning solution and/or sample will pass from the first reaction zone 111 through the first one-way valve 121 to the second reaction zone 112, and due to the second one-way valve 122 The slope is greater than the first one-way valve 121, so the reaction reagent, cleaning solution and/or sample will not pass through the second one-way valve 122. When the second centrifugal force is greater than the first centrifugal force for centrifugation, the reaction reagent, the cleaning solution and/or the sample will pass from the second reaction zone 112 through the second one-way valve 122 with a larger slope without backflow To the first reaction zone 111.

As shown in Figure 6, in the fifth embodiment, it is similar to the third embodiment, except that the centrifugal reaction microtube 5 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, and a In addition to the second one-way valve 122, it further includes a third reaction zone 113 and a third one-way valve 123. The third reaction zone 113 can contain the reaction mixture for the third reaction, and the third one-way valve 123 is arranged between the third reaction zone 113 and the first reaction zone 111, and the reaction mixture is moved to the first reaction by the third centrifugal force区111.

In the fifth embodiment, since there are three reaction zones, three reactions can be performed sequentially in the same centrifugal reaction microtube 5. For example, a nucleic acid extraction reaction can be carried out in the third reaction zone 113, a polymerase chain reaction can be carried out in the first reaction zone 111 and/or a nucleic acid hybridization reaction can be carried out in the second reaction zone 112.

In a preferred embodiment, as mentioned above, in order to prevent the reaction mixture in the third reaction zone 113 from passing through the first one-way valve 121, the second one-way valve 122 and the third one-way valve 123 at one time during centrifugation, In the first reaction zone 111, or to prevent the reaction mixture in the first reaction zone 111 from passing through the first one-way valve 121 and the second one-way valve 122 while not staying in the second reaction zone 112 during centrifugation, these one-way The elastic constant of the valve selects the second one-way valve 122, the first one-way valve 121, and the third one-way valve 123 in order from large to small. Therefore, at the lowest speed, only the third one-way valve 123 will open, while the first one-way valve 121 and the second one-way valve 122 are closed; at the speed suitable for the first one-way valve 121 to open Next, the first one-way valve 121 and the third one-way valve 123 will be opened, and the second one-way valve 122 will remain closed.

In the sixth embodiment, as shown in Figure 7, the centrifugal reaction microtube 6 includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, a second check valve 122, and a third reaction zone 111. In addition to the reaction zone 113 and the third one-way valve 123, it also includes a waste liquid zone 13, which can be used to collect the waste liquid after the reaction.

It is worth noting that, since it is necessary to perform various reactions in the centrifugal reaction microtube, such as polymerase chain reaction and nucleic acid hybridization, and to detect the reaction product qualitatively or quantitatively, the centrifugal reaction microtube is preferably heat-resistant optical Made of materials. "Heat resistance" means that the centrifugal reaction microtube does not change its properties, such as optical properties, at a certain temperature. Preferably, the specific temperature may be at least 120 degrees. "Optical material" means that the material has the characteristics of low dispersion, low chromatic aberration, wide wavelength range of light penetration, small refractive index, and high transmittance. More preferably, the optical material may include quartz, glass or plastic.

In a preferred embodiment, a centrifugal reaction microtube is provided. As shown in FIG. 8, it includes a first reaction zone 111, a first check valve 121, a second reaction zone 112, a second check valve 122 and a waste liquid zone 13. After adding the reaction reagent or sample to the reaction microtube, the mouth of the tube can be capped to prevent the reagent or sample from splashing. The length of the centrifugal reaction microtube can be 60 mm, but those skilled in the art to which the present invention pertains can freely adjust its length according to actual needs and the centrifugal device used. For example, when the centrifugal reaction microtube has only one reaction zone and one check valve as shown in the first embodiment, the length of the centrifugal reaction microtube may be 40 mm; or when the centrifugal reaction microtube is As shown in the embodiment 5, with three reaction zones and three one-way valves, the length of the centrifugal reaction microtube can be 100 mm.

A heating module 14 may be adjacent to the outside of the first reaction zone 111, which can increase the temperature of the first reaction zone 111 by means of substantial contact heating. Preferably, the heating module 14 may be U-shaped to effectively surround the first reaction zone 111, thereby increasing the heating speed of the first reaction zone 111. Similarly, heating modules can also be installed outside the first reaction zone 111 and the second reaction zone 112 at the same time to independently control the reaction temperature of the first reaction zone 111 and the second reaction zone 112.

In one embodiment, the present application provides a centrifugal reaction device, including: a reaction centrifuge disk, which is inclined upwardly from the shaft center, and the reaction centrifuge disk has a plurality of fixed racks arranged symmetrically for accommodating a plurality of centrifugal reactions Microtubules. In other words, the reaction centrifuge disc presents a disc-like structure (that is, the center is low, the edges are high), and the spin axis is inclined upward and outward, so that the reaction microtubes will naturally tilt after being placed in the reaction centrifuge disc, which helps the reaction microtubes The liquid inside flows naturally in the direction of the axis of the reaction centrifugal disc due to gravity. That is, the larger the inclination angle of the reaction centrifuge disc, the faster the reflux speed of the reaction mixture after being thrown out, which can accelerate the reaction and achieve the purpose of energy saving. The liquid thrown out due to centrifugal action, after the centrifugation is terminated or the centrifugal force becomes smaller, it will flow to a lower place by gravity, that is, backflow toward the rotation axis of the reaction centrifugal plate. By intermittent centrifugation, it can be removed without any additional In the case of pipetting equipment, the purpose of rapid liquid exchange or mixing is achieved. Preferably, the inclination of the reaction centrifuge disk from the axis of rotation to the edge can be about 1 to 89 degrees, preferably 10 to 80 degrees, 20 to 70 degrees, 30 to 60 degrees, 40 to 50 degrees, or any degree in between. . Optionally, the reaction centrifuge disc can also be designed with a horizontal angle, and only need to match the reaction microtubes with inclined inner surfaces to achieve the above purpose.

In a preferred embodiment, in order to achieve high reaction throughput, the reaction microtubes can be designed to be arranged on the reaction centrifuge disk in an eight-row manner, as shown in FIG. 9. In other words, using 8 reaction microtubes as a group is beneficial to increase the speed of adding reaction reagents or specimens. However, those with ordinary knowledge in the technical field to which the present invention pertains can freely adjust the number of reaction microtubes in a set of reaction microtubes, such as 12 rows, 8 rows, 6 rows, and 4-row or 2-row, etc., not limited to this. In addition, in Figure 9, the reaction microtubes are arranged in 6 groups and 8 rows in the reaction centrifuge disk. Those with ordinary knowledge in the technical field of the present invention can also use the reaction centrifuge disk according to actual needs and matching. And adjust the number of 8-row groups that can be set on the reaction centrifuge disk, such as 8, 6, 4, or 2 groups.

In one embodiment, the centrifugal reaction device may further include a liquid separation module, and the reaction reagent and/or the sample are respectively added to the centrifugal reaction microtube. The centrifugal reaction microtube is placed with the nozzle (such as the left side of the centrifugal reaction microtube 1 in Figure 1) facing the direction of the rotation axis of the reaction centrifuge disc, which facilitates the dispensing module when adding reagents and/or samples , There is no need to move in a large area, resulting in longer time required for dispensing.

In one embodiment, the centrifugal reaction device may further include a temperature control module for controlling the reaction temperature of the centrifugal reaction microtubes, including through heating block, liquid (such as hot water), gas (such as hot air) , Far infrared, etc. Preferably, the heating block and liquid heating surround the centrifugal reaction microtubes in a U-shaped or O-shaped manner to increase the efficiency of temperature control.

For example, when the polymerase chain reaction is carried out in the reaction zone, the temperature of the reaction zone must be controlled to be 0-100°C, preferably 4-95°C. For example, during the denaturation phase, control the temperature of the reaction zone at 96°C to open the double-stranded DNA; during the conjugation phase, control the temperature of the reaction zone at about 60°C to allow the primers to pair with the template DNA ; And during the extension phase, control the temperature of the reaction zone at 72°C to extend the DNA and repeat the open cycle several times.

In one embodiment, the centrifugal reaction device may further include a signal detection module for detecting the reaction signal of the centrifugal reaction microtube. For example, the signal detection module can be a fluorescent, luminescent or visible light camera sensing system. After the reaction in the reaction zone of the centrifugal reaction microtube is completed, biomolecules (such as antibodies, aptamers, peptides or nucleic acids) labeled with fluorescent, luminescence or color signals can be added to the reaction zone to detect The signal is a qualitative or quantitative reaction product.

Preferably, the centrifugal reaction device may further include a waste liquid collection module for collecting waste liquid leaving from the centrifugal reaction microtube. For example, in the first embodiment, the third embodiment and the fifth embodiment of the centrifugal reaction microtubes, none of the centrifugal reaction microtubes 1, 3, and 5 have the waste liquid area 13, which means that they cannot be used in the centrifugal reaction microtubes. Pipes 1, 3, and 5 collect the reaction waste liquid. In this case, the waste liquid leaving from the centrifugal reaction microtube can be collected by installing a waste liquid collection module in the centrifugal reaction device. In a preferred embodiment, the waste liquid collection module is a vacuum suction device arranged on the inner wall of the centrifugal reaction device. When the waste liquid leaves the centrifugal reaction microtubes 1, 3, 5 through the one-way valve, the vacuum suction The device will suck out the waste liquid directly without splashing back into the open one-way valve, causing reaction pollution.

In another embodiment, the present case provides a centrifugal detection method, including: providing a centrifugal reaction microtube; and adding a sample and/or reaction reagent to the reaction zone to form a reaction mixture, and centrifuging the centrifugal reaction microtube to perform reaction.

Hereinafter, a centrifugal detection method according to an embodiment of the present invention will be described with reference to FIG. 3. First, the sample and/or reaction reagents are sequentially added to the centrifugal reaction microtube 3 to form a reaction mixture. The centrifugal reaction microtube 3 is centrifuged to perform the first reaction in the first reaction zone 111. Wherein, the centrifugal speed or centrifugal force can be controlled, so that the first reaction is performed by oscillating the reaction mixture when the first one-way valve 121 and the second one-way valve 122 are closed. Then, the centrifugal reaction microtube 3 is centrifuged at a speed with the first centrifugal force, and the reaction mixture passes through the opened first check valve 121 to the second reaction zone 112 for the second reaction. The centrifugal reaction microtube 3 is centrifuged at a speed with a second centrifugal force, so that the reaction mixture leaves the second reaction zone 112 through the opened second one-way valve 122. Optionally, during the reaction process, the cleaning solution can be added to the centrifugal reaction microtube 3 through the liquid separation module, and the centrifugal reaction microtube 3 can be centrifuged at a speed lower than the first centrifugal force and the second centrifugal force to shake The cleaning is performed in the first reaction zone 111 or the second reaction zone 112.

Specific examples of centrifugal detection methods:

(1) The nucleic acid sample and reaction reagents are added to the centrifugal reaction microtube with the liquid separation module . I. From the sample tank (such as a 1.5 mL centrifuge tube or 96-well plate), take 1-20 μL of the sample to be tested and add it to the centrifugal reaction microtube; II. Take 1 from the reagent tank (such as a 1.5 mL centrifuge tube) Add -20 μL of reagent to the centrifugal reaction microtube; III. Take 20-30 μL of mineral oil from another reagent tank (such as 1.5 mL or 15 mL centrifuge tube) into the centrifugal reaction microtube; and IV. Low speed (such as 10-100 g centrifugal force) centrifugation, mix the sample and reaction reagents in the first reaction zone, the first one-way valve and the second one-way valve are still closed.

(2) PCR is performed in the first reaction zone by centrifugal mixing and temperature control to generate many amplified fragments ( Amplicon ). The PCR reaction is carried out in the temperature range of 37-60-95°C for about 40-45 cycles.

(3) The first signal detection, quantitative analysis of nucleic acid. The concentration of nucleic acid was detected with a spectrophotometer in a centrifugal reaction device.

(4) Centrifuge with the first centrifugal force to make the amplified fragments enter the second reaction zone containing the nucleic acid biochip through the first one-way valve. I. Preheat the second reaction zone to 50-60°C; II. Take 20 μL of denaturation solution (strong base) from the reagent tank (such as a 1.5 mL or 15 mL centrifuge tube), and add it to the centrifugal reaction microtube; III. Centrifuge at low speed (such as 10-100 g centrifugal force), let the denatured liquid pass through the mineral oil, and mix with the original reaction mixture, the first one-way valve and the second one-way valve are still closed; IV. Let stand for 30 minutes to allow DNA Denaturation, from double strands to single strands to facilitate the hybridization reaction; V. Take 100 μL of hybridization buffer (salt buffer) from the reagent tank (such as 1.5 mL or 15 mL centrifuge tube) and add it to the centrifuge reaction microtube; VI. Centrifuge at a low speed (such as 10-100 g centrifugal force) to mix the hybridization reaction buffer with the reaction mixture in the first reaction zone to facilitate the hybridization reaction. The first and second check valves are still closed; and VII. Centrifuge at a medium speed (such as 500-1000 g centrifugal force), so that only the first one-way valve is opened to allow the aforementioned mixture to move from the first reaction zone to the second reaction zone.

(5) Perform a nucleic acid hybridization reaction in the second reaction zone by centrifugal mixing and temperature control, and perform a washing step. I. Centrifuge intermittently at a low speed (such as 10-100 g of centrifugal force) at a certain temperature (such as 50°C) and time (such as 30-60 min) to allow the reaction mixture to react in the second reaction under the action of centrifugal force and gravity To facilitate the hybridization reaction II. Centrifuge at high speed (such as 1500-4000 g centrifugal force) to open the second one-way valve to remove all the reaction mixture from the second reaction zone to the waste liquid zone or The waste liquid collection module of the centrifugal reaction device; III. Take 100 μL of cleaning solution 1 (salt buffer) from the reagent tank (such as 1.5 mL or 15 mL centrifuge tube) and add it to the centrifuge reaction microtube. Such as centrifugal force of 500-1000 g) centrifugation, so that only the first one-way valve is opened, and the cleaning solution 1 is moved to the second reaction zone, and then the biochips are cleaned by centrifugation at an intermittent low speed (such as centrifugal force of 10-100 g) for 3 min ; IV. Centrifuge at high speed (such as 1500-4000 g centrifugal force) to open the second one-way valve to remove cleaning solution 1 from the second reaction zone; V. From the reagent tank (such as 1.5 mL or 15 mL centrifuge tubes) Take 100 μL of cleaning solution 2 (salt buffer) into the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000 g centrifugal force) to open only the first one-way valve, and let the cleaning solution 2 move to the first In the second reaction zone, the biochips are cleaned by centrifugation at an intermittent low speed (such as 10-100 g centrifugal force) for 3 minutes; VI. Centrifugation at a high speed (such as 1500-4000 g centrifugal force) to open the second one-way valve to Remove the cleaning solution 2 from the second reaction zone.

(6) The second signal interpretation, qualitative analysis of nucleic acid. I. Take 50 μL of antibody-containing conjugate solution (salt buffer) from the reagent tank (such as a 1.5 mL or 15 mL centrifuge tube) and add it to the centrifugal reaction microtube at a medium speed (such as 500-1000 g centrifugal force) Centrifuge, make only the first one-way valve open, and move the conjugate solution to the second reaction zone, and then centrifuge at an intermittent low speed (such as a centrifugal force of 10-100 g) to make the conjugate solution react with the biochip for 20 minutes; II. Centrifuge at high speed (such as 1500-4000 g centrifugal force) to open the second check valve to remove the conjugate solution from the second reaction zone; III. Take 100 μL from the reagent tank (such as a 1.5 mL or 15 mL centrifuge tube) Add the cleaning solution 3 (salt-containing buffer) to the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000 g centrifugal force), so that only the first one-way valve is opened, and the cleaning solution 3 is moved to the second reaction zone , And then centrifuge to clean the biochips at intermittent low speed (such as 10-100 g centrifugal force) for 3 min; IV. Centrifugation at high speed (such as 1500-4000 g centrifugal force) to open the second check valve to get from the second reaction zone Remove the cleaning solution 3; V. Take 50 μL of the coloring agent (salt buffer) from the reagent tank (such as 1.5 mL or 15 mL centrifuge tube) and add it to the centrifugal reaction microtube at a medium speed (such as 500-1000 g Centrifugal force) centrifugation to open only the first one-way valve to move the color former to the second reaction zone, and then centrifuge at an intermittent low speed (such as a centrifugal force of 10-100 g) to make the color former react with the biochip for 15 minutes VI. Centrifuge at high speed (such as 1500-4000 g centrifugal force), open the second one-way valve to remove the coloring agent from the second reaction zone; VII. From the reagent tank (such as 1.5 mL or 15 mL centrifuge tube) Take 100 μL of cleaning solution 4 (salt buffer) into the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000 g centrifugal force) to open only the first one-way valve, and let the cleaning solution 4 move to the first In the second reaction zone, the biochips are cleaned by centrifugation at an intermittent low speed (such as 10-100 g centrifugal force) for 3 minutes; VIII. Centrifugation at a high speed (such as 1500-4000 g centrifugal force) to open the second check valve to In the second reaction zone, remove the cleaning solution 4; IX. Take 100 μL of the fixed terminator (salt buffer) from the reagent tank (such as 1.5 mL or 15 mL centrifuge tube) and add it to the centrifugal reaction microtube at a medium speed (such as 500 -1000 g centrifugal force) centrifugation, so that only the first one-way valve is opened, and the fixed terminator is moved to the second reaction zone, and then centrifuged at an intermittent low speed (such as 10-100 g centrifugal force) to make the fixed terminator and the biochip Reaction for 3 min; X. Centrifuge at high speed (such as 1500-4000 g centrifugal force) to open the second check valve to remove the fixed terminator from the second reaction zone; XI. From the reagent tank (such as 1.5 mL or 15 mL centrifuge tube) Take 100 μL of cleaning solution 5 (salt buffer) into the centrifugal reaction microtube, and centrifuge at a medium speed (such as 500-1000 g centrifugal force), so that only the first check valve is opened , Let the cleaning solution 5 move to the second reaction zone, and then centrifuge at an intermittent low speed (such as 10-100 g centrifugal force) to clean the biochip for 3 min; XII. Centrifuge at a high speed (such as 1500-4000 g centrifugal force) to make the second The one-way valve is opened to remove the cleaning solution 5 from the second reaction zone; XIII. Take the camera and record the samples in sequence according to the sample number on the biochip, then analyze the results by the software and output the report. For example, if the biochip corresponding to a patient's sample shows a color reaction in a specific test at a specific location, it means that a patient has a positive reaction to the chip analysis result of the specific test.

(7) Complete the inspection.

As mentioned above, through the centrifugal reaction microtube, the centrifugal reaction device and the centrifugal inspection method described in this case, human intervention can be avoided, and the goal of one-click molecular biological detection can be achieved. With the control of centrifugal force and the application of centrifugal reaction microtubes, the molecular biology detection machine can be miniaturized, which saves energy and space, and can greatly shorten the detection time. In addition, there is no need to transfer between different machines during the reaction process, thereby achieving many goals such as simple and fast operation, safe and energy-saving, accurate and economical, and avoiding pollution.

The above description is only exemplary and not restrictive. Any equivalent modifications or changes made to the present invention without departing from the spirit and scope of the present invention shall be included in the scope defined by the scope of the patent application.

1, 2, 3, 4, 5, 6: Centrifugal reaction microtubes 11: reaction zone 111: The first reaction zone 112: second reaction zone 113: The third reaction zone 12: Check valve 121: The first one-way valve 122: second check valve 123: The third check valve 13: Waste area 14: Heating module

In the following detailed description, in order to explain the present invention, many specific details are provided in order to thoroughly understand the disclosed embodiments. However, it is obvious that one or more embodiments may be implemented without the specific details. In other cases, in order to simplify the drawings, the conventional structure and process will be shown in a schematic manner.

Figure 1 is a schematic diagram of the centrifugal reaction microtube according to the first embodiment of the present application.

Figure 2 is a schematic diagram of the centrifugal reaction microtube according to the second embodiment of the present application.

Figure 3 is a schematic diagram of the centrifugal reaction microtube according to the third embodiment of the present application.

Figure 4 (A) ~ (C) is a schematic diagram of the use state of the centrifugal reaction microtube according to Example 3A of this case.

Parts (A) and (B) of Figure 5 are schematic diagrams of the centrifugal reaction microtubes according to Examples 4 and 4A of this case, respectively.

Figure 6 is a schematic diagram of the centrifugal reaction microtube according to the fifth embodiment of the present application.

Figure 7 is a schematic diagram of the centrifugal reaction microtube according to the sixth embodiment of the present application.

Figure 8 is a schematic diagram of a centrifugal reaction microtube according to a preferred embodiment of the present case.

Figure 9 is a schematic diagram of a reaction centrifuge disk according to an embodiment of the present case.

1: Centrifugal reaction microtube

11: reaction zone

12: Check valve

Claims (29)

A centrifugal reaction microtube includes: a first reaction zone for containing a reaction mixture for a first reaction; a first one-way valve located in a centrifugal direction of the first reaction zone, wherein the first one-way The valve is opened by a first centrifugal force to allow the reaction mixture to leave the first reaction zone; a second reaction zone is separated from the first reaction zone through the first one-way valve, and the reaction is caused by the first centrifugal force The mixture moves to the second reaction zone for a second reaction; and a second one-way valve is arranged on the opposite side of the first one-way valve in the second reaction zone, wherein the second one-way valve passes through a The second centrifugal force opens to cause the reaction mixture to leave the second reaction zone. The centrifugal reaction microtube described in item 1 of the scope of patent application, wherein the second centrifugal force is greater than the first centrifugal force. In the centrifugal reaction microtube as described in item 1 of the scope of patent application, the first centrifugal force and the second centrifugal force are respectively 1 to 80,000 g, and the second centrifugal force is greater than the first centrifugal force. For the centrifugal reaction microtube as described in item 1 of the scope of patent application, the opening and closing of the first one-way valve and the second one-way valve are controlled by electric control or magnetic control. The centrifugal reaction microtube described in item 1 of the scope of the patent application further includes a biochip disposed in the second reaction zone, and the biochip is coated with an antibody, an aptamer, a peptide or a nucleic acid Of biological molecules. The centrifugal reaction microtube as described in item 1 of the scope of the patent application further includes a waste liquid zone, which is separated from the second reaction zone by the second one-way valve. The centrifugal reaction microtube described in item 1 of the scope of patent application, wherein the first reaction zone performs a polymerase chain reaction. The centrifugal reaction microtube as described in item 7 of the scope of patent application, wherein the second reaction zone performs a nucleic acid hybridization reaction. The centrifugal reaction microtube described in item 1 of the scope of patent application further includes: a third reaction zone for containing the reaction mixture for a third reaction; and a third one-way valve arranged in the third reaction Between the zone and the first reaction zone, the reaction mixture is moved to the first reaction zone by a third centrifugal force. The centrifugal reaction microtube described in item 1 of the scope of the patent application, wherein the centrifugal reaction microtube is substantially made of an optical material. For the centrifugal reaction microtube described in claim 10, the optical material includes quartz, glass or plastic. A centrifugal reaction device includes: a reaction centrifugal disc, which inclines upward at an inclination from the shaft center; the reaction centrifugal disc is provided with a plurality of fixed racks arranged symmetrically for accommodating a plurality of fixed racks such as item 1 to The centrifugal reaction microtube according to any one of Item 11. The centrifugal reaction device described in item 12 of the scope of patent application, wherein the slope is about 1 to 89 degrees. For example, the centrifugal reaction device described in item 12 of the scope of patent application further includes a signal detection module to detect a reaction signal of the centrifugal reaction microtubes. For example, the centrifugal reaction device described in item 12 of the scope of the patent application further includes a liquid separation module for adding a reaction reagent and/or a sample to the centrifugal reaction microtubes. The centrifugal reaction device described in item 12 of the scope of patent application further includes a temperature control module to control the reaction temperature of one of the centrifugal reaction microtubes. The centrifugal reaction device described in item 12 of the scope of patent application further includes a waste liquid collection module to collect a waste liquid leaving from the centrifugal reaction microtubes. A centrifugal detection method, comprising: providing the centrifugal reaction microtube as described in item 1 of the scope of patent application; and adding a sample and/or a reaction reagent to the first reaction zone to form a reaction mixture, and centrifuging the centrifugation Type reaction microtube to perform a first reaction. The method described in item 18 of the scope of patent application further includes adding a cleaning solution and intermittently centrifuging the centrifugal reaction microtube. The method described in item 18 of the scope of patent application further includes detecting a reaction signal of the centrifugal reaction microtube. The method described in item 18 of the scope of patent application further includes controlling a reaction temperature of the centrifugal reaction microtube. The method described in item 18 of the patent application further includes collecting a waste liquid from the centrifugal reaction microtube. The method described in item 18 of the scope of patent application further includes: centrifuging the centrifugal reaction microtube, and moving the reaction mixture through the first one-way valve to a second reaction zone by a first centrifugal force. Two responses. The method described in item 23 of the scope of the patent application further includes: centrifuging the centrifugal reaction microtube, and using a second centrifugal force to make the reaction mixture leave the second reaction zone through a second one-way valve. The method described in item 24 of the scope of patent application, wherein the second centrifugal force is greater than the first centrifugal force. The method according to item 24 of the scope of patent application, wherein the first centrifugal force and the second centrifugal force are respectively 1~80,000 g, and the second centrifugal force is greater than the first centrifugal force. The method described in item 24 of the scope of patent application, wherein the opening and closing of the first one-way valve and the second one-way valve are controlled by electric control or magnetic control. The method described in item 23 of the scope of patent application, wherein the first reaction zone performs a polymerase chain reaction. The method described in claim 28, wherein the second reaction zone performs a nucleic acid hybridization reaction.

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