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CN-115806874-B - Biological reaction chip, centrifugal microfluidic system and amplification method

CN115806874BCN 115806874 BCN115806874 BCN 115806874BCN-115806874-B

Abstract

The invention discloses a biological reaction chip, a centrifugal microfluidic system and an amplification method, wherein the biological reaction chip comprises a chip main body, a sample storage cavity, a pre-amplification cavity and a detection cavity which are communicated with each other are sequentially arranged on a first surface of the chip main body, a reaction liquid storage cavity and a reaction liquid distribution cavity which are communicated with each other are sequentially arranged on a second surface of the chip main body, the reaction liquid distribution cavity is communicated with the detection cavity through a first through hole formed in the chip main body, a first sealing body seals the first surface of the chip main body, and a second sealing body seals the second surface of the chip main body. According to the invention, through realizing twice amplification under the sealed environment by the centrifugal effect, the risk of being polluted is avoided. In addition, the first-step nucleic acid amplification and the second-step nucleic acid amplification are independently carried out in the equipartition cavity, so that the problems of cross interference among primers or probes of different amplification targets in the multiple amplification method are avoided.

Inventors

  • WANG HUILI
  • YIN SHUANG
  • XU YOUCHUN
  • PAN LIANGBIN

Assignees

  • 北京博奥晶典生物技术有限公司
  • 四川高性能医疗器械国家研究院有限公司

Dates

Publication Date
20260512
Application Date
20230110

Claims (11)

  1. 1. A bioreactor chip (1000), comprising: The chip comprises a chip main body (100), wherein a sample storage cavity (101), a pre-amplification cavity (102) and a detection cavity (103) which are communicated with each other are sequentially formed in a first surface of the chip main body (100) along a direction from a first end to a second end, sample liquid is stored in the sample storage cavity (101), a first primer required for pre-amplification is stored in the pre-amplification cavity (102), a second primer required for amplification reaction is stored in the detection cavity (103), a reaction liquid storage cavity (104) and a reaction liquid distribution cavity (105) which are communicated with each other are sequentially formed in a direction from the first end to the second end on a second surface of the chip main body (100) which is deviated from the first surface, the reaction liquid distribution cavity (105) is communicated with the detection cavity (103) through a first through hole (106) formed in the chip main body (100), and the reaction liquid storage cavity (104) stores reaction liquid; a first sealing body (200), the first sealing body (200) sealing a first face of the chip main body (100); A second sealing body (300), the second sealing body (300) sealing a second face of the chip body (100); Wherein the first end is closer to the rotational axis of the chip body (100) in centrifugal motion than the second end.
  2. 2. The biological reaction chip (1000) according to claim 1, wherein the number of the pre-amplification chambers (102) and the number of the detection chambers (103) are plural, and are arranged in a one-to-one correspondence manner, and adjacent pre-amplification chambers (102) are arranged in parallel, and adjacent detection chambers (103) are arranged in parallel; The number of the reaction liquid distribution cavities (105) and the number of the first through holes (106) are equal to the number of the detection cavities (103), the reaction liquid distribution cavities (105) are uniformly and correspondingly arranged, the adjacent reaction liquid distribution cavities (105) are arranged in parallel, and the adjacent first through holes (106) are arranged in parallel.
  3. 3. The biological reaction chip (1000) according to claim 2, wherein along the direction from the first end to the second end of the chip main body (100), a first distribution channel (107) is further formed on the first surface of the chip main body (100), and the first distribution channel (107) is disposed between the sample storage cavity (101) and the pre-amplification cavity (102), and a plurality of first communication channels (108) are disposed at intervals at one end of the first distribution channel (107) away from the sample storage cavity (101), and the first communication channels (108) are respectively in one-to-one correspondence and are communicated with the pre-amplification cavities (102); Along the direction of first end to the second end of chip main part (100), still set up on the second face of chip main part (100) place in reaction liquid storage chamber (104) with second distribution passageway (109) between reaction liquid distribution chamber (105), second distribution passageway (109) keep away from the one end interval of reaction liquid storage chamber (104) is provided with a plurality of second intercommunication passageway (110), second intercommunication passageway (110) respectively with reaction liquid distribution chamber (105) one-to-one and intercommunication.
  4. 4. A biological reaction chip (1000) according to claim 3, wherein, along the direction from the first end to the second end of the chip main body (100), the first surface of the chip main body (100) is further provided with a first buffer cavity (111) disposed between the sample storage cavity (101) and the first distribution channel (107), one end of the first buffer cavity (111) away from the sample storage cavity (101) is communicated with one end of the first distribution channel (107) facing the sample storage cavity (101) through a third communication channel (112), and one end of the first buffer cavity (111) facing the other end of the sample storage cavity (101) is communicated through a fourth communication channel (113); Along the direction of the first end to the second end of chip main part (100), second buffer chamber (114) between reaction solution storage chamber (104) and second distribution passageway (109) have still been seted up to the second face of chip main part (100), second buffer chamber (114) keep away from the one end of reaction solution storage chamber (104) with the one end of second distribution passageway (109) towards reaction solution storage chamber (104) is through fifth intercommunication passageway (115) intercommunication, second buffer chamber (114) with between the one end towards the other side of reaction solution storage chamber (104) through sixth intercommunication passageway (116) intercommunication.
  5. 5. The biological reaction chip (1000) according to claim 4, characterized in that the pre-amplification chamber (102) and the detection chamber (103) are communicated through a seventh communication channel (117), and an end of the seventh communication channel (117) communicating with the pre-amplification chamber (102) is located at a preset position of the pre-amplification chamber (102) along a direction from a first end to a second end of the chip body (100), and an end of the seventh communication channel (117) communicating with the detection chamber (103) is located at an end of the detection chamber (103) facing the pre-amplification chamber (102); One end of the reaction liquid distribution cavity (105) facing the first through hole (106) is communicated with the first through hole (106) through an eighth communication channel (118), and the first through hole (106) is communicated with the seventh communication channel (117); The third communication channel (112) and the fifth communication channel (115) are siphon channels, and first interface valves (119) are respectively arranged on the third communication channel (112), the fourth communication channel (113), the fifth communication channel (115) and the sixth communication channel (116).
  6. 6. The bioreactor chip (1000) of claim 4 or 5, further comprising a first venting structure (400) and a second venting structure (500); The first ventilation structure (400) is arranged on the first surface of the chip main body (100) and is used for gas communication between the cavities on the first surface of the chip main body (100); the second venting structure (500) is disposed on the second side of the chip body (100) for gas communication between the cavities on the second side of the chip body (100).
  7. 7. The bioreactor chip (1000) of claim 6, wherein the first venting structure (400) comprises a first venting channel (401) and a second venting channel (402), One end of the first ventilation pipeline (401) is communicated with one end of the sample storage cavity (101) far away from the first buffer cavity (111), the other end of the first ventilation pipeline (401) is communicated with one end of the first buffer cavity (111) facing the sample storage cavity (101), One end of the second ventilation pipeline (402) is communicated with one end of the first buffer cavity (111) facing away from the first distribution channel (107), and the other end of the second ventilation pipeline (402) is communicated with one end of the first distribution channel (107) facing towards the first buffer cavity (111); The second venting structure (500) comprises a third venting channel (501) and a fourth venting channel (502), One end of the third ventilation pipeline (501) is communicated with one end of the reaction liquid storage cavity (104) far away from the second buffer cavity (114), the other end of the third ventilation pipeline (501) is communicated with one end of the second buffer cavity (114) facing the reaction liquid storage cavity (104), One end of the fourth ventilation pipeline (502) is communicated with one end of the second buffer cavity (114) which faces away from the second distribution channel (109), and the other end of the fourth ventilation pipeline (502) is communicated with one end of the second distribution channel (109) which faces towards the second buffer cavity (114).
  8. 8. The bioreactor chip (1000) according to claim 7, wherein both ends of the second vent pipe (402) and both ends of the fourth vent pipe (502) are respectively provided with a second interface valve (600); A first ventilation through hole (120) communicated with the first ventilation pipeline (401) is formed in the chip main body (100); The chip main body (100) is provided with a second ventilation through hole (121) communicated with the third ventilation pipeline (501).
  9. 9. The biological reaction chip (1000) according to claim 8, wherein the first ventilation through hole (120) and the second ventilation through hole (121) are both formed on the second surface of the chip main body (100), and a sample inlet (122) and a liquid inlet (123) are respectively formed on the second surface of the chip main body (100); The sample adding port (122) is communicated with the sample storage cavity, and the liquid adding port (123) is communicated with the reaction liquid storage cavity (104); The biological reaction chip (1000) further comprises a third sealing body (700), the second sealing body (300) is provided with a second through hole (301) which is respectively communicated with the first ventilation through hole (120), the second ventilation through hole (121), the sample adding port (122) and the liquid adding port (123), and the third sealing body (700) is connected to the second sealing body (300) in a sealing way so as to seal the second through hole (301).
  10. 10. A centrifugal microfluidic system comprising a centrifugal drive mechanism and a bioreactor chip (1000) according to any one of claims 1-9; The biological reaction chip (1000) is fixed on the centrifugal driving mechanism, and the centrifugal driving mechanism is used for driving the biological reaction chip (1000) to perform centrifugal movement.
  11. 11. An amplification method, comprising the steps of, characterized by comprising the following steps: providing a bioreactor chip (1000) according to any one of claims 1-9; Injecting a sample into the sample storage cavity (101), injecting a reaction liquid into the reaction liquid storage cavity (104), sealing the biological reaction chip (1000), and placing the biological reaction chip (1000) on a centrifugal driving mechanism for heating, temperature control and centrifugal rotation; centrifuging the biological reaction chip (1000), centrifuging the sample liquid in the sample storage cavity (101) into the pre-amplification cavity (102), and completing the pre-amplification reaction, wherein the reaction liquid in the reaction liquid storage cavity (104) is centrifuged into the reaction liquid distribution cavity (105); After the reaction in the pre-amplification cavity (102) is finished, the biological reaction chip (1000) is subjected to centrifugal operation again, the amplification solution in the pre-amplification cavity (102) is transferred into the detection cavity (103), the reaction solution in the reaction solution distribution cavity (105) is transferred into the detection cavity (103) through the first through hole (106), and is mixed with the pre-amplification solution to perform re-amplification reaction, and meanwhile fluorescent signal acquisition is performed.

Description

Biological reaction chip, centrifugal microfluidic system and amplification method Technical Field The invention relates to the technical field of in-vitro diagnostic equipment, in particular to a biological reaction chip, a centrifugal microfluidic system and an amplification method. Background Nucleic acid amplification is a key technology for detecting current pathogens, and the existing common nucleic acid amplification technologies include PCR (polymerase chain reaction), LAMP (loop-mediated isothermal nucleic acid amplification), RPA (recombinase polymerase amplification technology), RCA (rolling circle amplification) and the like. However, the existing one-step nucleic acid amplification technology has the defects of sensitivity and specificity, and for this purpose, a nested amplification technology is proposed. Taking nested PCR as an example, two sets of PCR primers are used for carrying out two rounds of PCR amplification, firstly, the target DNA is subjected to a first round of amplification, then, a part of the target DNA is taken out of the first round of reaction products as a reaction template for carrying out a second round of amplification, the limitation of the single amplification stage effect is overcome, and the amplification multiple is improved, so that the sensitivity of the PCR is greatly improved. However, although the nested amplification technology has better sensitivity and specificity, the two-step operation involves liquid transfer, the operation is more complicated, and the exposure of the nucleic acid amplification product in the first step is involved, so that the product pollution is easy to generate. Thus, how to accomplish nested amplification in a closed device is a current problem that is highly desirable to those skilled in the art. Disclosure of Invention In view of the above, a first object of the present invention is to provide a bioreactor chip capable of achieving nested amplification in a closed device. A second object of the present invention is to provide a centrifugal microfluidic system. A third object of the present invention is to provide an amplification method. In order to achieve the first object, the present invention provides the following solutions: a bioreactor chip comprising: The chip comprises a chip main body, wherein a sample storage cavity, a pre-amplification cavity and a detection cavity which are communicated with each other are sequentially formed in a first surface of the chip main body along a direction from a first end to a second end, sample liquid is stored in the sample storage cavity, a first primer required for pre-amplification is stored in the pre-amplification cavity, a second primer required for amplification reaction is stored in the detection cavity, a reaction liquid storage cavity and a reaction liquid distribution cavity which are communicated with each other are sequentially formed in a second surface of the chip main body, which is away from the first surface, along a direction from the first end to the second end, the reaction liquid distribution cavity is communicated with the detection cavity through a first through hole formed in the chip main body, and the reaction liquid storage cavity is used for storing reaction liquid; A first sealing body sealing the first face of the chip body; a second sealing body sealing a second face of the chip main body; the first end is closer to the rotating shaft of the chip main body under centrifugal motion than the second end. In a specific embodiment, the number of the pre-amplification cavities and the number of the detection cavities are multiple, the pre-amplification cavities and the detection cavities are arranged in a one-to-one correspondence manner, the adjacent pre-amplification cavities are arranged in parallel, and the adjacent detection cavities are arranged in parallel; The number of the reaction liquid distribution cavities and the number of the first through holes are equal to that of the detection cavities, the reaction liquid distribution cavities and the first through holes are uniformly and correspondingly arranged, the adjacent reaction liquid distribution cavities are arranged in parallel, and the adjacent first through holes are arranged in parallel. In another specific embodiment, along the direction from the first end to the second end of the chip main body, the first surface of the chip main body is further provided with a first distribution channel arranged between the sample storage cavity and the pre-amplification cavity, one end of the first distribution channel away from the sample storage cavity is provided with a plurality of first communication channels at intervals, and the first communication channels are respectively in one-to-one correspondence and are communicated with the pre-amplification cavities; Along the direction of the first end to the second end of the chip main body, a second distribution channel arranged between the reaction liquid