Chip for detecting residual plasmids in in-vitro synthesized circular RNA (Ribonucleic Acid)

Abstract

The utility model discloses a chip for detecting residual plasmids in circular RNA (Ribonucleic Acid) synthesized in vitro. The chip is composed of a substrate and a cover plate. The substrate is provided with a test solution cavity, a DNA diluent cavity, a qPCR reaction solution preparation cavity, a qPCR detection cavity, a plasmid standard sample solution mother solution preparation tank, a plasmid standard sample solution mother solution finished product tank, a plasmid standard sample solution gradient solution preparation tank, a plasmid standard sample solution gradient solution finished product tank and a standard recovery solution preparation tank; a standard recovery solution finished product tank, a qPCR MIX reaction liquid preparation tank and a qPCR MIX reaction liquid finished product tank are arranged. According to the detection chip provided by the utility model, the whole detection process of the residual plasmids in the in-vitro synthesized circular RNA can be completed on a single chip, and the reliability and the traceability of the detection of the residual plasmids in the in-vitro synthesized circular RNA are ensured.

Description

Technical Field

[0001] The utility model relates to the technical field of biomedical metrology, in particular to a detection chip for residual plasmids in in vitro synthesized circular RNA and a use method thereof. Background Art

[0002] Circular RNA (circRNA) exists in many organisms and has the characteristics of rich species, stable structure, conserved sequence and cell or tissue specific expression. It plays an important biological function and is one of the functional biomarkers and therapeutic targets for many diseases such as human diabetes, nervous system diseases, cardiovascular diseases and cancer. With the development of science and technology, circular RNA can be synthesized in vitro and applied or gradually applied to biomedicine and medical devices and other important life and health fields that are closely related to the national economy and people's livelihood.

[0003] Plasmids are needed in the process of synthesizing circular RNA in vitro. Therefore, the content of plasmid residues in circular RNA synthesized in vitro is an important quality control standard for circular RNA drugs. Because in addition to the risk of immunogenicity caused by residual plasmids in circular RNA synthesized in vitro as production impurities, there is also a biosafety risk that it may cause infection or gene mutation after entering the human body with circular RNA drugs. For example, residual plasmids carry dominant oncogenes, which may cause some receptor cells to differentiate into tumor cells; in addition, residual plasmids may be integrated into the genome of receptor cells, causing gene mutations or abnormal expression. Therefore, drug regulatory authorities in various countries will have very strict requirements on the reliability of plasmid residue detection in the registration application of circular RNA drugs.

[0004] Since no in vitro synthesized circular RNA drugs have been officially launched at home and abroad, there is currently no unified method for detecting plasmid residues. Most research and development institutions use the method of multiple reaction solution configurations to detect plasmid residues in in vitro synthesized circular RNA. However, due to the cumbersome operation process, there are often significant differences between different batches of reaction solutions configured by quality inspectors and they are difficult to trace, which often leads to reviewers repeatedly questioning the reliability and traceability of the residual plasmid detection data submitted for registration applications for in vitro synthesized circular RNA drug clinical trial projects.

[0005] In view of this, the purpose of the utility model is to provide a detection chip for residual plasmids in in vitro synthesized circular RNA, which optimizes the detection process of residual plasmids in in vitro synthesized circular RNA by integrating the operational process of residual plasmid detection in in vitro synthesized circular RNA, improves the detection efficiency of residual plasmids in in vitro synthesized circular RNA, and enhances the detection reliability of residual plasmids in in vitro synthesized circular RNA. Utility Model Content

[0006] The technical problem to be solved by the utility model is to overcome the deficiencies of the prior art in detecting residual plasmids in in vitro synthesized circular RNA, and to provide a detection chip for residual plasmids in in vitro synthesized circular RNA that can overcome the above problems or partially solve the above problems, integrating the preparation of test solution, preparation of plasmid standard sample solution, preparation of spiked recovery solution, preparation of qPCR MIX reaction solution, preparation of qPCR reaction solution, and qPCR detection. This provides a strong guarantee for the majority of in vitro synthesized circular RNA drug research and development institutions to efficiently, accurately, reliably and traceably implement the detection of residual plasmids in in vitro synthesized circular RNA.

[0007] In order to achieve the above object, the technical solution of the utility model is:

[0008] A detection chip for residual plasmids in in vitro synthesized circular RNA, the detection chip consists of a substrate and a cover plate, the substrate is provided with a test solution chamber, a DNA dilution chamber, a qPCR reaction solution preparation chamber, a qPCR detection chamber, a plasmid standard sample solution mother solution preparation tank, a plasmid standard sample solution mother solution finished product tank, a plasmid standard sample solution gradient solution preparation tank, a plasmid standard sample solution gradient solution finished product tank, a spiked recovery solution preparation tank, a spiked recovery solution finished product tank, a qPCR MIX reaction solution preparation tank, and a qPCR MIX reaction solution finished product tank, and the entire detection process of residual plasmids in in vitro synthesized circular RNA can be completed on a single chip. Compared with the current detection technology solutions for residual plasmids in in vitro synthesized circular RNA, the detection chip for residual plasmids in in vitro synthesized circular RNA provided by the utility model can not only improve the integration and automation of the detection of residual plasmids in in vitro synthesized circular RNA, but also reduce the complexity of the operation process and the difference level between different batches of reaction solutions configured by quality inspectors, thereby ensuring the reliability and traceability of the detection of residual plasmids in in vitro synthesized circular RNA.

[0009] According to the embodiments of the present invention, the present invention can be further optimized. The following is the technical solution formed after optimization:

[0010] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA comprises a substrate and a cover plate that are bonded to each other.

[0011] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the substrate and cover of the detection chip are manufactured by thermocompression bonding packaging.

[0012] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the substrate and cover of the detection chip are manufactured by plasma bonding packaging.

[0013] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the substrate and cover of the detection chip are packaged by surface mounting technology.

[0014] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the base plate and the cover plate of the detection chip are packaged by injection packaging technology.

[0015] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the substrate and cover of the detection chip are packaged by coating packaging technology.

[0016] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the cover plate of the detection chip is made of a PC composite material, wherein the PC mass content in the PC composite material is 99.9%-90%, and the borosilicate content is 0.1%-10%.

[0017] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the cover plate of the detection chip is made of a PMMA composite material, wherein the mass content of PMMA in the PMMA composite material is 99.5%-87%, and the content of borosilicate is 0.5%-13%.

[0018] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein a substrate of the detection chip is made of a silica composite material, wherein the mass content of silica in the silica composite material is 70%-98%, and the mass content of PDMS is 2%-30%.

[0019] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with a test solution chamber, a DNA dilution chamber, a qPCR reaction solution preparation chamber, a qPCR detection chamber, a plasmid standard sample solution mother solution preparation tank, a plasmid standard sample solution mother solution finished tank, a plasmid standard sample solution gradient solution preparation tank, a plasmid standard sample solution gradient solution finished tank, a spiked recovery solution preparation tank, a spiked recovery solution finished tank, a qPCR MIX reaction solution preparation tank, and a qPCR MIX reaction solution finished tank.

[0020] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the substrate of the detection chip is respectively provided with injection pipelines connecting the test solution chamber, the standard sample solution mother solution preparation tank, the DNA dilution liquid chamber, the qPCR MIX reaction solution preparation tank, and the qPCR reaction solution preparation chamber.

[0021] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the substrate of the detection chip is respectively provided with a DNA dilution liquid chamber, a plasmid standard sample solution mother solution preparation tank, a test solution chamber, a qPCR MIX reaction solution preparation tank, an injection port of the qPCR reaction solution preparation chamber, and an injection port protective plug.

[0022] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with an injection pipeline port connected to the qPCR reaction solution preparation cavity, including an injection pipeline port for a template-free control solution, an output qPCR MIX reaction solution injection pipeline port of a qPCR MIX reaction solution finished tank, an output test solution injection pipeline port of a test solution cavity, an output spiked recovery solution injection pipeline port of a spiked recovery solution finished tank, and an output standard sample gradient solution injection pipeline port of a plasmid standard sample gradient solution finished tank.

[0023] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline port of the qPCRMIX reaction solution preparation tank of the detection chip includes an injection pipeline port for qPCR reaction buffer and an injection pipeline port for plasmid primers or a mixture of probes.

[0024] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline port of the plasmid standard sample solution mother solution preparation tank of the detection chip includes an injection pipeline port for plasmid standard samples and an output DNA dilution injection pipeline port of the DNA dilution liquid chamber.

[0025] In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with 5 plasmid standard sample solution gradient solution preparation slots, and correspondingly, the substrate of the detection chip is provided with 5 plasmid standard sample solution gradient solution finished slots. Among them, the injection pipeline port of the first plasmid standard sample solution gradient solution preparation slot includes the output plasmid standard sample solution mother solution injection pipeline port of the plasmid standard sample mother solution finished slot, and the output DNA dilution injection pipeline port of the DNA dilution liquid chamber; the injection pipeline port of the second plasmid standard sample solution gradient solution preparation slot includes the output plasmid standard sample solution gradient solution injection pipeline port of the first plasmid standard sample solution gradient concentration solution finished slot, and the output DNA dilution injection pipeline port of the DNA dilution liquid chamber; the injection pipeline port of the third plasmid standard sample solution gradient solution preparation slot includes the output plasmid standard sample solution gradient solution injection pipeline port of the second plasmid standard sample solution gradient concentration solution finished slot, and the output DNA dilution injection pipeline port of the DNA dilution liquid chamber. The output plasmid standard sample solution gradient solution injection pipeline port and the output DNA dilution injection pipeline port of the DNA dilution liquid cavity; the injection pipeline port of the fourth gradient solution preparation tank of the plasmid standard sample solution includes the output plasmid standard sample solution gradient solution injection pipeline port of the third gradient concentration solution finished tank of the plasmid standard sample solution and the output DNA dilution injection pipeline port of the DNA dilution liquid cavity; the injection pipeline port of the fifth gradient solution preparation tank of the plasmid standard sample solution includes the output plasmid standard sample solution gradient solution injection pipeline port of the fourth gradient concentration solution finished tank of the plasmid standard sample solution and the output DNA dilution injection pipeline port of the DNA dilution liquid cavity.

[0026] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with N or more (where N is greater than 5) plasmid standard sample solution gradient solution preparation slots. Correspondingly, the substrate of the detection chip is provided with N plasmid standard sample solution gradient solution finished slots. Among them, the injection pipeline ports of the N or more (where N is greater than 5) plasmid standard sample solution gradient solution preparation slots include the output plasmid standard sample solution gradient solution injection pipeline port of the N-1th gradient concentration solution finished slot of the plasmid standard sample solution, and the output DNA dilution injection pipeline port of the DNA dilution liquid cavity. Furthermore, the substrate of the detection chip is provided with an injection pipeline port of the spiked recovery solution preparation slot, including the output plasmid standard sample solution (N+1) / 2th gradient solution finished slot of the plasmid standard sample solution, and the output test solution injection pipeline port of the test solution cavity.

[0027] In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with 6-14 plasmid standard sample solution gradient solution preparation slots, and each plasmid standard sample solution gradient solution preparation slot is provided with a plasmid standard sample solution gradient solution finished slot. In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with 7 plasmid standard sample solution gradient solution preparation slots. In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with 9 plasmid standard sample solution gradient solution preparation slots. In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with 11 plasmid standard sample solution gradient solution preparation slots. In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with 13 plasmid standard sample solution gradient solution preparation slots.

[0028] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, when the number of plasmid standard sample solution gradient solution preparation slots of the detection chip is 5, the plasmid standard sample solution gradient solution injection pipeline of the spiked recovery solution preparation slot of the detection chip is connected to the third plasmid standard sample solution gradient solution finished slot.

[0029] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, when the number of plasmid standard sample solution gradient solution preparation slots of the detection chip is 7, the substrate of the detection chip is provided with a plasmid standard sample solution gradient solution injection pipeline of the spiked recovery solution preparation slot connected to the fourth plasmid standard sample solution gradient solution finished slot.

[0030] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, when the number of plasmid standard sample solution gradient solution preparation slots of the detection chip is 9, the substrate of the detection chip is provided with a plasmid standard sample solution gradient solution injection pipeline of the spiked recovery solution preparation slot connected to the fifth plasmid standard sample solution gradient solution finished slot.

[0031] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, when the number of plasmid standard sample solution gradient solution preparation slots of the detection chip is 11, the substrate of the detection chip is provided with a plasmid standard sample solution gradient solution injection pipeline of the spiked recovery solution preparation slot connected to the sixth plasmid standard sample solution gradient solution finished slot.

[0032] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, when the number of plasmid standard sample solution gradient solution preparation slots of the detection chip is 13, the substrate of the detection chip is provided with a plasmid standard sample solution gradient solution injection pipeline of the spiked recovery solution ERC preparation slot connected to the sixth plasmid standard sample solution gradient solution finished slot.

[0033] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the qPCR detection chamber of the detection chip is connected to a safety pipeline, and the safety pipeline is provided with a safety outlet and a safety outlet protective plug.

[0034] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein a microfluidic channel is provided between a plasmid standard sample solution mother liquor preparation tank and a plasmid standard sample solution mother liquor finished product tank of the detection chip; the microfluidic channel is provided with a check valve and an atomizing nozzle with a check function.

[0035] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein a microfluidic channel is provided between a plasmid standard sample solution gradient solution preparation tank and a plasmid standard sample solution gradient solution finished tank of the detection chip; the microfluidic channel is provided with a check valve and an atomizing nozzle with a check function.

[0036] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein a microfluidic channel is provided between a preparation tank for a spiked recovery solution and a finished tank for a spiked recovery solution of the detection chip; the microfluidic channel is provided with a check valve and an atomizing nozzle with a check function.

[0037] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein a microfluidic channel is provided between the qPCR MIX reaction solution preparation tank and the qPCR MIX reaction solution finished product tank of the detection chip; the microfluidic channel is provided with a check valve and an atomizing nozzle with a check function.

[0038] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline of the qPCR reaction solution preparation chamber includes a template-free control solution injection pipeline, and the template-free control solution injection pipeline is provided with a check valve.

[0039] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline of the qPCR reaction solution preparation chamber includes an output test solution injection pipeline of the test solution chamber, and the output test solution injection pipeline is provided with a quantitative sampling valve and a check valve with a non-return function.

[0040] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline of the qPCR reaction solution preparation chamber includes an output plasmid standard product solution gradient solution injection pipeline of a plasmid standard product solution gradient solution finished tank, and the output plasmid standard product solution gradient solution injection pipeline of the plasmid standard product solution gradient solution finished tank is provided with a quantitative sampling valve and a check valve with a check function.

[0041] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline of the qPCR reaction solution preparation chamber includes an output qPCR MIX reaction solution injection pipeline of the qPCR MIX reaction solution finished product tank, and the output qPCR MIX reaction solution injection pipeline of the qPCR MIX reaction solution finished product tank is provided with a quantitative sampling valve and a check valve with a check function.

[0042] In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline of the qPCR reaction solution preparation chamber includes an output spiked recovery solution injection pipeline of a spiked recovery solution finished product tank, and the output spiked recovery solution injection pipeline of the spiked recovery solution finished product tank is provided with a quantitative sampling valve and a check valve with a check function.

[0043] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein the substrate of the detection chip is provided with a DNA dilution liquid output pipeline connected to the DNA dilution liquid chamber, and the DNA dilution liquid output pipeline is provided with a quantitative sampling valve and a check valve with a check function.

[0044] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein a microfluidic channel is provided between the qPCR reaction solution preparation chamber and the qPCR detection chamber; and the microfluidic channel is provided with a check valve and an atomizing nozzle with a check function.

[0045] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the qPCR MIX reaction solution preparation tank is provided with an injection pipeline for qPCR reaction buffer, and the injection pipeline for qPCR reaction buffer is provided with a check valve with a check function.

[0046] In one of the preferred embodiments, a detection chip for residual plasmids in in vitro synthesized circular RNA, the qPCR MIX reaction solution preparation tank is provided with an injection pipeline for plasmid primers or a mixture of probes, and the injection pipeline for the plasmid primers or a mixture of probes is provided with a check valve with a check function.

[0047] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the standard solution mother liquor preparation tank is provided with an injection pipeline for plasmid standard samples, and the injection pipeline for plasmid standard samples is provided with a check valve with a check function.

[0048] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the injection pipeline of the plasmid standard sample solution mother solution preparation tank includes an injection pipeline for outputting DNA dilution liquid from a DNA dilution liquid chamber, and the injection pipeline for outputting DNA dilution liquid from the DNA dilution liquid chamber is provided with a quantitative sampling valve and a check valve with a check function.

[0049] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the DNA diluent chamber is provided with an injection pipeline for the DNA diluent, and the injection pipeline for the DNA diluent is provided with a check valve.

[0050] In some embodiments of the utility model, a detection chip for residual plasmids in in vitro synthesized circular RNA, a cover plate that is not in contact with the substrate of the detection chip is marked with the words "test solution chamber, DNA dilution chamber, qPCR reaction solution preparation chamber, qPCR detection chamber, plasmid standard sample solution mother solution preparation tank, plasmid standard sample solution mother solution finished tank, plasmid standard sample solution gradient solution preparation tank, plasmid standard sample solution gradient solution finished tank, spiked recovery solution preparation tank, spiked recovery solution finished tank, qPCR MIX reaction solution preparation tank, and qPCR MIX reaction solution finished tank.

[0051] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, wherein the substrate of the detection chip is provided with injection pipelines or output pipelines connecting different cavities or grooves and the diameter of the safety pipeline is consistent or inconsistent.

[0052] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the substrate of the detection chip is provided with an injection line or output line connecting different cavities or grooves and a safety line with a diameter of 0.1 mm-1.5 mm. In one embodiment, the diameter of the injection line or output line connecting different cavities or grooves and the safety line is 0.11 mm. In certain embodiments, the diameter of the injection line or output line connecting different cavities or grooves and the safety line is 0.12 mm, 0.13 mm, 0.14 mm, 0.15 mm, 0.16 mm, 0.17 mm, 0.18 mm, 0.19 mm, 0.2 mm. In certain embodiments, the diameter of the injection line or output line connecting different cavities or grooves and the safety line is 0.21 mm-0.4 mm. In certain embodiments, the diameter of the injection line or output line connecting different cavities or grooves and the safety line is 0.41 mm-0.6 mm. In certain embodiments, the diameter of the injection pipeline or output pipeline and the safety pipeline connected to different cavities or grooves is 0.61 mm-0.8 mm. In certain embodiments, the diameter of the injection pipeline or output pipeline and the safety pipeline connected to different cavities or grooves is 0.81 mm-1 mm. In certain embodiments, the diameter of the injection pipeline or output pipeline and the safety pipeline connected to different cavities or grooves is 1.1 mm-1.2 mm. In certain embodiments, the diameter of the injection pipeline or output pipeline and the safety pipeline connected to different cavities or grooves is 1.21 mm-1.3 mm. In certain embodiments, the diameter of the injection pipeline or output pipeline and the safety pipeline connected to different cavities or grooves is 1.31 mm-1.4 mm. In certain embodiments, the diameter of the injection pipeline or output pipeline and the safety pipeline connected to different cavities or grooves is 1.41 mm-1.5 mm.

[0053] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the cross-sectional area of ​​the microfluidic channel interconnected between the test solution cavity, the spike recovery solution preparation tank, and the standard solution preparation tank is 1 / 5-2 / 3 of the cross-sectional area of ​​the test solution cavity injection pipeline channel. In one embodiment, the cross-sectional area of ​​the microfluidic channel is 1 / 4 of the cross-sectional area of ​​the test solution cavity injection pipeline channel. In certain embodiments, the cross-sectional area of ​​the microfluidic channel is 1 / 3 and 1 / 2 of the cross-sectional area of ​​the test solution cavity injection pipeline channel.

[0054] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the cross-sectional area of ​​the microfluidic channel interconnected between the test solution cavity, the spike recovery solution preparation tank, and the standard solution preparation tank is 1 / 5-2 / 3 of the cross-sectional area of ​​the test solution cavity injection pipeline channel. In one embodiment, the cross-sectional area of ​​the microfluidic channel is 1 / 4 of the cross-sectional area of ​​the test solution cavity injection pipeline channel. In certain embodiments, the cross-sectional area of ​​the microfluidic channel is 1 / 3 and 1 / 2 of the cross-sectional area of ​​the test solution cavity injection pipeline channel.

[0055] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single test solution cavity provided on the substrate of the detection chip is 0.1 microliters-50 microliters. In one embodiment, the volume of the single test solution cavity is 0.2 microliters. In certain embodiments, the volumes of the single test solution cavity are 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1.0 microliters. In certain embodiments, the volume of a single test solution cavity is 1.1-5 microliters. In certain embodiments, the volume of a single test solution cavity is 5.1-10 microliters. In certain embodiments, the volume of a single test solution cavity is 10.1-15 microliters. In certain embodiments, the volume of a single test solution cavity is 15.1-20 microliters. In certain embodiments, the volume of a single test solution cavity is 20.1-25 microliters. In certain embodiments, the volume of a single test solution chamber is 25.1-49.9 μl.

[0056] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single DNA dilution liquid chamber provided on the substrate of the detection chip is 3.6 microliters-1500 microliters. In one embodiment, the volume of the single DNA dilution liquid chamber is 5.4 microliters. In certain embodiments, the volumes of the single DNA dilution liquid chambers are 7.2 microliters, 9 microliters, 10.8 microliters, 12.6 microliters, 14.4 microliters, 16.2 microliters, 18 microliters, and 19.8 microliters, respectively. In certain embodiments, the volume of the single DNA dilution liquid chamber is 20-50 microliters. In certain embodiments, the volume of the single DNA dilution liquid chamber is 51-100 microliters. In certain embodiments, the volume of the single DNA dilution liquid chamber is 101-150 microliters. In certain embodiments, the volume of the single DNA dilution liquid chamber is 151-200 microliters. In certain embodiments, the volume of the single DNA dilution liquid chamber is 201-300 microliters. In some embodiments, the volume of a single DNA dilution chamber is 301-450 microliters. In some embodiments, the volume of a single DNA dilution chamber is 451-600 microliters. In some embodiments, the volume of a single DNA dilution chamber is 601-900 microliters. In some embodiments, the volume of a single DNA dilution chamber is 901-1200 microliters. In some embodiments, the volume of a single DNA dilution chamber is 1201-1499 microliters.

[0057] In one preferred embodiment, a detection chip for residual plasmids in an in vitro synthesized circular RNA, the volume of a single standard solution preparation tank provided on the substrate of the detection chip is 0.1 microliters-375 microliters. In one embodiment, the volume of the single standard solution preparation tank is 0.2 microliters. In certain embodiments, the volumes of the single standard solution preparation tanks are 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the volume of the single standard solution preparation tank is 2-10 microliters. In certain embodiments, the volume of the single standard solution preparation tank is 11-20 microliters. In certain embodiments, the volume of the single standard solution preparation tank is 21-30 microliters. In certain embodiments, the volume of the single standard solution preparation tank is 31-40 microliters. In certain embodiments, the volume of the single standard solution preparation tank is 41-50 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 51-60 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 61-70 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 71-80 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 81-90 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 91-100 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 101-120 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 121-150 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 151-200 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 201-250 microliters. In certain embodiments, the volume of a single standard solution preparation tank is 251-300 microliters. In certain embodiments, the volume of a single standard solution preparation reservoir is 301-374 μL.

[0058] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single standard solution finished tank provided on the substrate of the detection chip is 0.1 microliter-375 microliters. In one embodiment, the volume of the single standard solution finished tank is 0.2 microliters. In certain embodiments, the volumes of the single standard solution finished tanks are 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the volume of the single standard solution finished tank is 2-10 microliters. In certain embodiments, the volume of the single standard solution finished tank is 11-20 microliters. In certain embodiments, the volume of the single standard solution finished tank is 21-30 microliters. In certain embodiments, the volume of the single standard solution finished tank is 31-40 microliters. In certain embodiments, the volume of the single standard solution finished tank is 41-50 microliters. In certain embodiments, the volume of a single standard solution finished tank is 51-60 microliters. In certain embodiments, the volume of a single standard solution finished tank is 61-70 microliters. In certain embodiments, the volume of a single standard solution finished tank is 71-80 microliters. In certain embodiments, the volume of a single standard solution finished tank is 81-90 microliters. In certain embodiments, the volume of a single standard solution finished tank is 91-100 microliters. In certain embodiments, the volume of a single standard solution finished tank is 101-120 microliters. In certain embodiments, the volume of a single standard solution finished tank is 121-150 microliters. In certain embodiments, the volume of a single standard solution finished tank is 151-200 microliters. In certain embodiments, the volume of a single standard solution finished tank is 201-250 microliters. In certain embodiments, the volume of a single standard solution finished tank is 251-300 microliters. In certain embodiments, the volume of a single standard solution finished tank is 301-374 μL.

[0059] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single addition recovery solution preparation tank provided on the substrate of the detection chip is 0.1 microliters-200 microliters. In one embodiment, the volume of the single addition recovery solution preparation tank is 0.2 microliters. In certain embodiments, the volumes of the single addition recovery solution preparation tanks are 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the volume of the single addition recovery solution preparation tank is 2-10 microliters. In certain embodiments, the volume of the single addition recovery solution preparation tank is 11-20 microliters. In certain embodiments, the volume of the single addition recovery solution preparation tank is 21-30 microliters. In certain embodiments, the volume of the single addition recovery solution preparation tank is 31-40 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 41-50 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 51-60 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 61-70 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 71-80 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 81-90 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 91-100 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 101-120 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 121-150 microliters. In certain embodiments, the volume of a single spike recovery solution preparation tank is 151-199 microliters.

[0060] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single finished tank for adding a spiked recovery solution provided on the substrate of the detection chip is 0.1 microliters to 200 microliters. In one embodiment, the volume of the finished tank for adding a single spiked recovery solution is 0.2 microliters. In certain embodiments, the volumes of the finished tanks for adding a single spiked recovery solution are 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the volume of the finished tank for adding a single spiked recovery solution is 2-10 microliters. In certain embodiments, the volume of the finished tank for adding a single spiked recovery solution is 11-20 microliters. In certain embodiments, the volume of the finished tank for adding a single spiked recovery solution is 21-30 microliters. In certain embodiments, the volume of the finished tank for adding a single spiked recovery solution is 31-40 microliters. In certain embodiments, the volume of the finished tank for adding a single spiked recovery solution is 41-50 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 51-60 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 61-70 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 71-80 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 81-90 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 91-100 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 101-120 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 121-150 microliters. In certain embodiments, the volume of a single spiked recovery solution finished tank is 151-199 microliters.

[0061] In one preferred embodiment, a detection chip for residual plasmids in an in vitro synthesized circular RNA, the volume of a single qPCR MIX reaction solution preparation tank provided on the substrate of the detection chip is 0.2 microliters-400 microliters. In one embodiment, the volume of the single qPCR MIX reaction solution preparation tank is 0.3 microliters. In certain embodiments, the volumes of the single qPCR MIX reaction solution preparation tanks are 0.4 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 2-10 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 11-20 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 21-30 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 31-40 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 41-50 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 51-60 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 61-70 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 71-80 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 81-90 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 91-100 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 101-120 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 121-150 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 151-200 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 201-250 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 251-300 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 301-350 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution preparation tank is 351-399 microliters.

[0062] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single qPCR MIX reaction solution finished tank provided on the substrate of the detection chip is 0.2 microliters-400 microliters. In one embodiment, the volume of the single qPCR MIX reaction solution finished tank is 0.3 microliters. In certain embodiments, the volumes of the single qPCR MIX reaction solution finished tanks are 0.4 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the volume of the single qPCR MIX reaction solution finished tank is 2-10 microliters. In certain embodiments, the volume of the single qPCR MIX reaction solution finished tank is 11-20 microliters. In certain embodiments, the volume of the single qPCR MIX reaction solution finished tank is 21-30 microliters. In certain embodiments, the volume of the single qPCR MIX reaction solution finished tank is 31-40 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 41-50 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 51-60 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 61-70 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 71-80 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 81-90 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 91-100 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 101-120 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 121-150 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 151-200 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 201-250 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 251-300 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 301-350 microliters. In certain embodiments, the volume of a single qPCR MIX reaction solution finished tank is 351-399 microliters.

[0063] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single qPCR reaction solution preparation chamber provided on the substrate of the detection chip is 1 microliter-4000 microliters. In one embodiment, the volume of the single qPCR reaction solution preparation chamber is 2 microliters. In certain embodiments, the volumes of the single qPCR reaction solution preparation chamber are 3 microliters, 4 microliters, 5 microliters, 6 microliters, 7 microliters, 8 microliters, 9 microliters, and 10 microliters, respectively. In certain embodiments, the volume of the single qPCR reaction solution preparation chamber is 11-50 microliters. In certain embodiments, the volume of the single qPCR reaction solution preparation chamber is 51-100 microliters. In certain embodiments, the volume of the single qPCR reaction solution preparation chamber is 101-150 microliters. In certain embodiments, the volume of the single qPCR reaction solution preparation chamber is 151-200 microliters. In certain embodiments, the volume of the single qPCR reaction solution preparation chamber is 201-300 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 301-500 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 501-700 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 701-1000 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 1001-1200 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 1201-1500 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 1501-2000 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 2001-3000 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 3001-3500 microliters. In certain embodiments, the volume of a single qPCR reaction solution preparation chamber is 3501-3999 μl.

[0064] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the volume of a single qPCR detection chamber provided on the substrate of the detection chip is 1 microliter-4000 microliters. In one embodiment, the volume of the single qPCR detection chamber is 2 microliters. In certain embodiments, the volumes of the single qPCR detection chambers are 3 microliters, 4 microliters, 5 microliters, 6 microliters, 7 microliters, 8 microliters, 9 microliters, and 10 microliters, respectively. In certain embodiments, the volume of a single qPCR detection chamber is 11-50 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 51-100 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 101-150 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 151-200 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 201-300 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 301-500 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 501-700 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 701-1000 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 1001-1200 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 1201-1500 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 1501-2000 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 2001-3000 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 3001-3500 microliters. In certain embodiments, the volume of a single qPCR detection chamber is 3501-3999 microliters.

[0065] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the microfluidic channel between the plasmid standard sample solution mother solution preparation tank and the plasmid standard sample solution mother solution finished product tank is provided with an atomizing nozzle with a check function to form microdroplets with a diameter of 0.05-50 μm.

[0066] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the microfluidic channel between the plasmid standard sample solution gradient solution preparation tank and the plasmid standard sample solution gradient solution finished tank is provided with an atomizing nozzle with a check function to form microdroplets with a diameter of 0.05-50 μm.

[0067] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the diameter of microdroplets formed by the atomizing nozzle with a check function arranged in the microfluidic channel between the spiked recovery solution preparation tank and the spiked recovery solution finished product tank is 0.05-50 μm.

[0068] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the diameter of microdroplets formed by the atomizing nozzle with a check function arranged in the microfluidic channel between the MIX reaction solution preparation tank and the MIX reaction solution finished product tank is 0.05-50 μm.

[0069] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the diameter of microdroplets formed by the atomizing nozzle with a check function provided in the microfluidic channel between the qPCR reaction liquid preparation chamber and the qPCR detection chamber is 0.05-50 μm.

[0070] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the quantitative volume of a single quantitative sampling valve with a check function provided in the test solution injection pipeline connected to the qPCR reaction solution preparation chamber is 0.1 microliters-50 microliters. In one embodiment, the quantitative volume of the single quantitative sampling valve is 0.2 microliters. In certain embodiments, the quantitative volumes of the single quantitative sampling valve are 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1.0 microliters, respectively. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 1.1-5 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 5.1-10 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 10.1-15 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 15.1-20 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 20.1-25 μl. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 25.1-49.9 μl.

[0071] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the quantitative volume of a single quantitative sampling valve with a check function provided in the standard solution injection pipeline connected to the qPCR reaction solution preparation chamber is 0.1 microliters-375 microliters. In one embodiment, the quantitative volume of the single quantitative sampling valve is 0.2 microliters. In certain embodiments, the quantitative volumes of the single quantitative sampling valve are 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 2-10 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 11-20 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 21-30 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 31-40 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 41-50 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 51-60 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 61-70 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 71-80 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 81-90 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 91-100 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 101-120 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 121-150 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 151-200 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 201-250 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 251-300 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 301-374 microliters.

[0072] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the qPCRMIX reaction solution injection pipeline connected to the qPCR reaction solution preparation chamber is provided with a single quantitative sampling valve with a check function, and the quantitative volume is 0.2 microliters-400 microliters. In one embodiment, the quantitative volume of the single quantitative sampling valve is 0.3 microliters. In certain embodiments, the quantitative volumes of the single quantitative sampling valve are 0.4 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 2-10 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 11-20 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 21-30 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 31-40 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 41-50 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 51-60 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 61-70 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 71-80 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 81-90 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 91-100 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 101-120 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 121-150 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 151-200 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 201-250 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 251-300 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 301-350 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 351-399 microliters.

[0073] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the quantitative volume of a single quantitative sampling valve with a check function provided in the DNA dilution liquid output pipeline connected to the DNA dilution liquid chamber is 0.9 microliters-500 microliters. In one embodiment, the quantitative volume of the single quantitative sampling valve is 1.35 microliters. In certain embodiments, the quantitative volumes of the single quantitative sampling valve are 1.8 microliters, 2.25 microliters, 2.7 microliters, 3.15 microliters, 3.6 microliters, 5.4 microliters, 6 microliters, and 6.6 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 5-12.5 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 12.6-25 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 25.1-37.5 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 37.6-50 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 50.1-75 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 75.1-112.5 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 112.6-150 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 150.1-225 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 225.1-300 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 300.1-374.9 microliters.

[0074] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the quantitative volume of a single quantitative sampling valve with a check function provided in the test solution injection pipeline connected to the spiked recovery solution preparation chamber is 0.05 microliters-25 microliters. In one embodiment, the quantitative volume of the single quantitative sampling valve is 0.1 microliter. In certain embodiments, the quantitative volumes of the single quantitative sampling valve are 0.2 microliters, 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1.0 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 1.1-2.5 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 2.6-5 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 5.1-10 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 10.1-15 microliters. In some embodiments, the quantitative volume of a single quantitative sampling valve is 15.1-20 microliters. In some embodiments, the quantitative volume of a single quantitative sampling valve is 20.1-24.9 microliters.

[0075] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the plasmid standard sample solution gradient solution injection pipeline connected to the spiked recovery solution preparation chamber is provided with a single quantitative sampling valve with a check function of 0.05 microliters-20 microliters. In one embodiment, the quantitative volume of the single quantitative sampling valve is 0.1 microliter. In certain embodiments, the quantitative volumes of the single quantitative sampling valve are 0.2 microliters, 0.3 microliters, 0.4 microliters, 0.5 microliters, 0.6 microliters, 0.7 microliters, 0.8 microliters, 0.9 microliters, and 1 microliter. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 1.1-2 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 2.1-5 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 5.1-10 microliters. In certain embodiments, the quantitative volume of the single quantitative sampling valve is 10.1-15 microliters. In certain embodiments, the quantitative volume of a single quantitative sampling valve is 15-19.9 μl.

[0076] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the number of qPCR detection cavities provided on the substrate of the detection chip is 1-3. In one embodiment, the number of the qPCR detection cavities is 2.

[0077] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA is provided, wherein a substrate and a cover plate of the detection chip are bonded to form a two-layer structure.

[0078] In one preferred embodiment, a detection chip for residual plasmids in in vitro synthesized circular RNA, the thickness of the cover plate of the detection chip is 0.1-0.5 mm. In one embodiment, the thickness of the cover plate is 0.11 mm. In certain embodiments, the thickness of the cover plate is 0.12 mm, 0.13 mm, 0.14 mm, 0.15 mm, 0.16 mm, 0.17 mm, 0.18 mm, 0.19 mm, 0.2 mm. In certain embodiments, the thickness of the cover plate is 0.21 mm-0.3 mm. In certain embodiments, the thickness of the cover plate is 0.31 mm-0.4 mm. In certain embodiments, the thickness of the cover plate is 0.41 mm-0.5 mm.

[0079] A method for preparing a detection chip for residual plasmids in in vitro synthesized circular RNA comprises the following steps:

[0080] The first step is to use photolithography technology to make a silicon wafer mold, and etch out the test solution cavity, DNA dilution liquid cavity, plasmid standard sample solution mother solution preparation tank, plasmid standard sample solution mother solution finished tank, plasmid standard sample solution gradient solution preparation tank, plasmid standard sample solution gradient solution finished tank, spike recovery solution preparation tank, spike recovery solution finished tank, qPCRMIX reaction solution preparation tank, qPCR MIX reaction solution finished tank, qPCR reaction solution preparation cavity and qPCR detection cavity edge lines and pipelines or microfluidic channels connecting each cavity or tank in sequence; the second step is to cast on the silicon wafer mold, and form a chip substrate after inverting the mold, and in the test solution cavity, DNA dilution liquid cavity, plasmid standard sample solution mother solution preparation tank, plasmid standard sample solution mother solution finished tank, plasmid standard sample solution gradient solution preparation tank, plasmid standard sample solution gradient solution finished tank, spike recovery solution preparation tank, spike recovery solution finished tank, qPCR MIX reaction solution preparation tank, qPCR The MIX reaction solution finished product tank, qPCR reaction solution preparation chamber and qPCR detection chamber are connected with pipelines or microfluidic channels and functionalized; the third step is to use bonding packaging technology to bond the functionalized chip substrate to one side of the cover plate which is not marked with the words "test solution chamber", "DNA dilution chamber", "plasmid standard sample solution mother solution preparation tank", "plasmid standard sample solution mother solution finished product tank", "plasmid standard sample solution gradient solution preparation tank", "plasmid standard sample solution gradient solution finished product tank", "spiked recovery solution preparation tank", "spiked recovery solution finished product tank", "qPCR MIX reaction solution preparation tank", "qPCR MIX reaction solution finished tank", "qPCR reaction solution preparation chamber" and "qPCR detection chamber" to complete the chip production.

[0081] A method for using a detection chip for residual plasmids in in vitro synthesized circular RNA comprises the following steps:

[0082] Step 1: Open the injection port protective plugs of the plasmid standard sample solution mother solution preparation tank and the DNA dilution liquid chamber in turn, inject the plasmid standard sample and the DNA dilution liquid respectively through a pipette, and then reset the injection port protective plug; Step 2: Open the quantitative sampling valve and the check valve of the DNA dilution injection microfluidic channel connected to the plasmid standard sample solution mother solution preparation tank, wait for the quantitative sampling of the DNA dilution liquid to be completed, and then reset the quantitative sampling valve and the check valve; Step 3: Open the check valve and the spray nozzle of the microfluidic channel connected between the plasmid standard sample solution mother solution preparation tank and the plasmid standard sample solution mother solution finished product tank. When the mixed liquid in the plasmid standard sample solution mother solution preparation tank flows through the edge of the atomizing nozzle hole with a check function, it expands into a liquid layer. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tubular pore-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid is impacted and rebounded by the vortex cavity of the quartz blade to form a mixed solution and enter the plasmid standard sample solution mother liquor finished product tank. After the solution in the plasmid standard sample mother liquor preparation tank is atomized and mixed, the check valve and the spray nozzle are reset; Step 4: Open the plasmid standard sample solution mother liquor output microfluidic channel connected between the plasmid standard sample solution mother liquor finished product tank and the plasmid standard sample solution first gradient solution preparation tank After the quantitative sampling of the plasmid standard sample solution mother solution is completed, the quantitative sampling valve and the check valve are reset; Step five: open the quantitative sampling valve and the check valve of the DNA dilution solution output microfluidic channel connected to the first gradient solution preparation tank of the plasmid standard sample solution, after the quantitative sampling of the DNA dilution solution is completed, the quantitative sampling valve and the check valve are reset; Step six: open the microfluidic channel connected between the first gradient solution preparation tank of the plasmid standard sample solution and the first gradient solution finished tank of the plasmid standard sample solution The check valve and the spray nozzle are reset after the solution in the first gradient solution preparation tank of the plasmid standard sample solution is atomized and mixed; Step seven: open the quantitative sampling valve and the check valve of the first gradient solution output microfluidic channel of the plasmid standard sample solution connected between the first gradient solution finished tank of the plasmid standard sample solution and the second gradient solution preparation tank of the plasmid standard sample solution, and reset the quantitative sampling valve and the check valve after the quantitative sampling of the first gradient solution of the plasmid standard sample solution is completed; Step eight: open the plasmid standard sample solution. The quantitative sampling valve and the check valve of the DNA dilution output microfluidic channel connected to the second gradient solution preparation tank of the plasmid standard sample solution are opened, and after the quantitative sampling of the DNA dilution is completed, the quantitative sampling valve and the check valve are reset; Step nine: open the check valve and the spray nozzle of the microfluidic channel connected between the second gradient solution preparation tank of the plasmid standard sample solution and the second gradient solution finished tank of the plasmid standard sample solution, and after the atomization and mixing of the solution in the second gradient solution preparation tank of the plasmid standard sample solution is completed, the check valve and the spray nozzle are reset;Step 10: Open the quantitative sampling valve and check valve of the second gradient solution output pipeline of the plasmid standard sample solution of the microfluidic channel connected between the second gradient solution finished tank of the plasmid standard sample solution and the third gradient solution preparation tank of the plasmid standard sample solution, and reset the quantitative sampling valve and check valve after the quantitative sampling of the second gradient solution of the plasmid standard sample solution is completed; Step 11: Open the quantitative sampling valve and check valve of the DNA dilution solution output microfluidic channel connected to the third gradient solution preparation tank of the plasmid standard sample solution, and reset the quantitative sampling valve and check valve after the quantitative sampling of the DNA dilution solution is completed; Step 12: Open the third gradient solution preparation tank of the plasmid standard sample solution The check valve and the spray nozzle of the microfluidic channel connected to the finished product tank of the third gradient solution of the plasmid standard sample solution are reset after the atomization and mixing of the solution in the preparation tank of the third gradient solution of the plasmid standard sample solution is completed; Step thirteen: open the quantitative sampling valve and the check valve of the third gradient solution output microfluidic channel of the plasmid standard sample solution connected between the finished product tank of the third gradient solution of the plasmid standard sample solution and the preparation tank of the fourth gradient solution of the plasmid standard sample solution, and reset the quantitative sampling valve and the check valve after the quantitative sampling of the third gradient solution of the plasmid standard sample solution is completed; Step fourteen: open the quantitative sampling valve and the check valve of the microfluidic channel of the third gradient solution output microfluidic channel of the plasmid standard sample solution connected between the finished product tank of the third gradient solution of the plasmid standard sample solution and the preparation tank of the fourth gradient solution of the plasmid standard sample solution The quantitative sampling valve and the check valve of the DNA dilution injection pipeline are connected, and after the DNA dilution sampling is completed, the quantitative sampling valve and the check valve are reset; Step 15: Open the check valve and the spray nozzle connected between the 4th gradient solution preparation tank of the plasmid standard sample solution and the 4th gradient solution finished tank of the plasmid standard sample solution, and after the atomization and mixing of the solution in the 4th gradient solution preparation tank of the plasmid standard sample solution is completed, reset the check valve and the spray nozzle; Step 16: Open the quantitative sampling valve of the 4th gradient solution output microfluidic channel of the plasmid standard sample solution connected between the 4th gradient solution finished tank of the plasmid standard sample solution and the 5th gradient solution preparation tank of the plasmid standard sample solution and check valve, after the quantitative sampling of the 4th gradient solution of the plasmid standard sample solution is completed, the quantitative sampling valve and the check valve are reset; step 17: open the quantitative sampling valve and the check valve of the DNA dilution output microfluidic channel connected to the 5th gradient solution preparation tank of the plasmid standard sample solution, after the DNA dilution sampling is completed, the quantitative sampling valve and the check valve are reset; step 18: open the check valve and the spray nozzle of the microfluidic channel connected between the 5th gradient solution preparation tank of the plasmid standard sample solution and the 5th gradient solution finished tank of the plasmid standard sample solution, after the atomization and mixing of the solution in the 5th gradient solution preparation tank of the plasmid standard sample solution is completed, the check valve and the spray nozzle are reset;For a detection chip whose substrate is provided with N or more (where N is greater than 5) plasmid standard sample solution gradient solution preparation slots, the plasmid standard sample solution gradient solution injection microfluidic channel of the Nth gradient concentration solution preparation slot of the plasmid standard sample solution is the plasmid standard sample solution gradient solution output microfluidic channel of the N-1th plasmid standard sample solution gradient solution finished slot; Step 19: open the injection port protective plug of the test solution, inject the test solution, and then reset the injection port protective plug; Step 20: open the quantitative sampling valve and check valve of the test solution output microfluidic channel connected to the test solution cavity and the spiked recovery solution preparation slot, and reset the quantitative sampling valve and check valve after the quantitative sampling of the test solution is completed; Step 21: open the spiked recovery solution preparation slot and the plasmid standard solution 4th gradient After the quantitative sampling of the fourth gradient solution of the plasmid standard sample solution is completed, the quantitative sampling valve and the check valve are reset; for a detection chip whose substrate is provided with more than N (where N is greater than 5) plasmid standard sample solution gradient solution preparation grooves, the spiked recovery solution preparation groove is connected to the (N+1) / 2th gradient solution finished groove of the plasmid standard sample solution; Step 22: open the check valve and the spray nozzle of the microfluidic channel connected between the spiked recovery solution preparation groove and the spiked recovery solution finished groove, and the mixed solution in the spiked recovery solution preparation groove expands into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid is impacted and rebounded by the vortex cavity of the quartz blade to form a mixed solution that enters the finished product tank of the spiked recovery solution. After the atomization and mixing of the solution in the spiked recovery solution preparation tank is completed, the check valve and the spray nozzle are reset; Step 23, the injection port protective plugs of the qPCR reaction buffer, plasmid primer and / or probe mixture are opened in sequence, and the qPCR reaction buffer, plasmid primer and / or probe mixture are respectively injected through a pipette, and then the injection port protective plug is reset; Step 24, the check valves of the qPCR reaction buffer injection pipeline and the plasmid primer and / or probe mixture injection pipeline are opened in sequence, and the qPCR reaction buffer and the plasmid primer and / or probe mixture are mixed in the qPCR MIX reaction solution preparation tank, and then the check valve is reset;Step 25: Open the check valve and the spray nozzle connected between the qPCR MIX reaction solution preparation tank and the qPCR MIX reaction solution finished product tank. When the mixed liquid in the qPCR MIX reaction solution preparation tank flows through the edge of the atomizing nozzle hole with a check function, it expands into a liquid layer. Due to the instability of aerodynamic force, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes droplets. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a mixed solution that enters the qPCR MIX reaction solution finished tank. After the solution in the qPCR MIX reaction solution preparation tank is atomized and mixed, the check valve and the spray nozzle are reset; step 26, open the injection port protective plug of the template-free control solution, inject the template-free control solution, and then reset the injection port protective plug; step 27, open the check valve of the template-free control solution injection pipeline, let the template-free control solution enter the qPCR reaction solution preparation tank, and then reset the check valve; step 28, open the check valve of the template-free control solution injection pipeline, let the template-free control solution enter the qPCR reaction solution preparation tank, and then reset the check valve; step 29, open the quantitative sampling valve and the check valve of the microfluidic channel connecting the plasmid standard sample solution gradient solution finished tank, the qPCR MIX reaction solution finished tank and the qPCR reaction solution preparation chamber in turn, and wait for the plasmid standard sample gradient solution, qPCR After the MIX reaction solution and quantitative sampling are completed, the quantitative sampling valve and the check valve are reset in sequence; step 30, the check valve and the spray nozzle connected between the qPCR reaction solution preparation chamber and the qPCR detection chamber are opened, and the mixed liquid in the qPCR reaction solution preparation chamber flows through the edge of the atomizing nozzle hole with a check function and expands into a liquid layer. Due to the instability of aerodynamics, the liquid layer is broken into an elongated tubular-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a mixed solution that enters the qPCR detection cavity. After the atomization and mixing of the solution in the qPCR reaction liquid preparation cavity is completed, the check valve and the spray nozzle are reset; step 31, the chip is placed in the qPCR instrument for thermal cycling and fluorescence qPCR quantification; step 32, after the detection is completed, the safety plug and the safety valve are opened, the liquid in the PCR detection cavity is discharged through the safety pipeline and the safety outlet, and the safety valve and the safety plug are reset; step 33, steps 26, 27 and 32 are repeated as needed to clean the PCR reaction liquid preparation cavity and the PCR detection cavity until no plasmid is detected;Step 34, sequentially open the quantitative sampling valve and the check valve of the microfluidic channel connecting the test solution chamber, the qPCR MIX reaction solution finished tank and the qPCR reaction solution preparation chamber, and reset the quantitative sampling valve and the check valve after the test solution, qPCR MIX reaction solution and quantitative sampling are completed; Step 35, repeat steps 30 to 33; Step 36, sequentially open the quantitative sampling valve and the check valve of the microfluidic channel connecting the spike recovery solution finished tank, the qPCR MIX reaction solution finished tank and the qPCR reaction solution preparation chamber, and reset the quantitative sampling valve and the check valve after the spike recovery solution, qPCR MIX reaction solution and quantitative sampling are completed; Step 37, repeat steps 30 to 31; Step 38, place the chip in the qPCR instrument for thermal cycling, perform fluorescence qPCR quantification, and use the software to calculate the Ct value of the plasmid standard sample. The value is plotted against the logarithmic value of its concentration for linear regression analysis. The linear correlation coefficient R2 of the standard curve is greater than 0.99, no detection is found in the template-free control, and the recovery rate of the spiked recovery solution is greater than 50% and less than 150%. Then the Ct value of the test solution obtained is combined with the plasmid length in bp to convert the plasmid DNA residue value. The unit of the plasmid DNA residue value is ng / dose. If a safety failure occurs, the safety outlet plug and safety valve can be opened, and the safety failure can be eliminated through the safety outlet and safety pipeline to ensure safe operation; Step 39, for samples that need to be retested, repeat steps 32 and 33, and then follow the above test steps; Step 40, after the test, discharge the liquid in all cavities or grooves of the chip through the safety pipeline, and then clean, dry or rinse it for reuse. ;

[0083] Compared with the prior art, the beneficial effects of the utility model are:

[0084] (1) Filled the gap in detection chips for residual plasmid detection in in vitro synthesized circular RNA at home and abroad;

[0085] (2) The chip for detecting residual plasmids in in vitro synthesized circular RNA integrates the functions of preparing test solution, plasmid standard sample solution, spike recovery solution, qPCR MIX reaction solution, qPCR reaction solution, and qPCR detection, providing a strong guarantee for the vast number of in vitro synthesized circular RNA drug research and development institutions to efficiently, accurately and reliably implement the detection of residual plasmids in in vitro synthesized circular RNA, and can realize the entire process of residual plasmid detection "sample in, result out";

[0086] (3) The detection chip for residual plasmids in in vitro synthesized circular RNA uses a microfluidic channel quantitative sampling valve and a check valve with a check function between different cavities or grooves to realize the different functions of multiple cavities or grooves connected in series, and the liquids between the cavities or grooves can be fully isolated without cross contamination; in addition, the atomizing nozzle can fully mix the various parts of the mixed solution;

[0087] (4) The detection chip for residual plasmids in in vitro synthesized circular RNA is designed with "non-stick" materials, which avoids the loss or residue caused by the adsorption of various solutions on the surface of different chip cavities or grooves or even on the surface of channels or pipes during the residual plasmid detection process. The chip can be reused repeatedly through appropriate design. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 It is a schematic diagram of the substrate structure of a detection chip for residual plasmids in in vitro synthesized circular RNA according to an embodiment of the utility model.

[0089] Figure 2 It is a schematic diagram of the cover structure of a detection chip for residual plasmids in in vitro synthesized circular RNA according to an embodiment of the utility model.

[0090] Figure 3 yes Figure 1 Schematic diagram of the structure of the atomizing nozzle 40.

[0091] Figure 4 yes Figure 1 Representative example photos of contact angle test results of the chip substrate for 1-test solution, 2-plasmid standard sample solution gradient solution, 3-qPCRMIX reaction solution, and 4-no template control solution.

[0092] Figure 5 yes Figure 1 Representative example photos of the contact angle test results of the middle chip cover for 1-test solution, 2-plasmid standard sample solution gradient solution, 3-qPCRMIX reaction solution, and 4-no template control solution. DETAILED DESCRIPTION

[0093] The utility model will be described in detail below with reference to the accompanying drawings and in combination with the embodiments. It should be noted that, in the absence of conflict, the embodiments of the utility model and the features in the embodiments can be combined with each other. For the convenience of description, if the words "upper", "lower", "left" and "right" appear in the following text, it only means that they are consistent with the upper, lower, left and right directions of the accompanying drawings themselves, and do not limit the structure. The embodiments of the utility model are only for explaining the utility model, not for limiting the utility model, and the embodiments of the utility model are not limited to the embodiments given in the specification. The specific experimental conditions or operating conditions not indicated in the embodiments are made under conventional conditions, or made under the conditions recommended by the material supplier. In addition, it should be understood that one or more method steps mentioned in the utility model do not exclude that there may be other method steps before and after the combination steps or other method steps may be inserted between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combined connection relationship mentioned in the utility model does not exclude that there may be other connections before and after the combination or other connections may be inserted between these two explicitly mentioned connections, unless otherwise specified. Furthermore, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the utility model. The change or adjustment of the relative relationship, without substantial change in the technical content, should also be regarded as the scope of the utility model. In the following examples, the reagents, materials and instruments used can be commercially available unless otherwise specified.

[0094] The utility model comprises a substrate 1, a cover plate 2, a template-free control solution injection port 3, a template-free control solution injection port plug 4, a qPCR reaction buffer injection port 5, a qPCR reaction buffer injection port plug 6, a plasmid primer or probe mixture injection port 7, a plasmid primer or probe mixture injection port plug 8, a test solution injection port 9, a test solution injection port plug 10, a plasmid standard sample injection port 11, a plasmid standard sample injection port plug 12, a DNA diluent injection port 13, a DNA diluent injection port plug 14, a template-free control solution injection pipeline 15 and a check valve 16, a qPCR reaction buffer injection pipeline 17 and a check valve 18, a plasmid primer or probe mixture injection pipeline 19 and a check valve 20, a qPCR MIX reaction solution preparation tank 21, a microfluidic channel 22 between the qPCR MIX reaction solution preparation tank 21 and the qPCR MIX reaction solution finished product tank 25, a check valve 23 and an atomizing nozzle 24, and a qPCR MIX reaction solution finished product tank 25, microfluidic channel 26, quantitative sampling valve 27 and check valve 28 between qPCR MIX reaction solution finished product tank 25 and qPCR reaction solution preparation chamber 131, test solution injection pipeline 29 and check valve 30, test solution chamber 31, microfluidic channel 32, quantitative sampling valve 33 and check valve 34 between test solution chamber 31 and qPCR reaction solution preparation chamber 131, plasmid standard sample injection pipeline 35 and check valve 36, plasmid standard sample solution mother solution preparation tank 37, plasmid standard sample solution The microfluidic channel 38, the check valve 39 and the atomizing nozzle 40 between the plasmid standard sample solution mother solution preparation tank 37 and the plasmid standard sample solution mother solution finished tank 41, the plasmid standard sample solution mother solution finished tank 41, the DNA dilution liquid injection pipeline 42 and the check valve 43, the DNA dilution liquid chamber 44, the microfluidic channel 45, the check valve 46 between the plasmid standard sample solution mother solution preparation tank 37 and the DNA dilution liquid chamber 44, the plasmid standard sample solution mother solution finished tank 41 and the first gradient solution of the plasmid standard sample solution The microfluidic channel 47, the quantitative sampling valve 48 and the check valve 49 between the DNA dilution liquid chamber 44 and the first gradient solution preparation tank 53 of the plasmid standard sample solution, the microfluidic channel 50, the quantitative sampling valve 51 and the check valve 52 between the first gradient solution preparation tank 53 of the plasmid standard sample solution, the microfluidic channel 54, the check valve 55 and the mist control valve 56 between the first gradient solution preparation tank 53 of the plasmid standard sample solution and the first gradient solution finished tank 57 of the plasmid standard sample solution. a microfluidic channel 58, a quantitative sampling valve 59 and a check valve 60 between the first gradient solution finished tank 57 of the plasmid standard sample solution and the second gradient solution preparation tank 64 of the plasmid standard sample solution; a microfluidic channel 61, a quantitative sampling valve 62 and a check valve 63 between the DNA dilution liquid chamber 44 and the second gradient solution preparation tank 64 of the plasmid standard sample solution; and a second gradient solution preparation tank 64 of the plasmid standard sample solution.The microfluidic channel 65, the check valve 66 and the atomizing nozzle 67 between the second gradient solution preparation tank 64 of the plasmid standard sample solution and the second gradient solution finished tank 68 of the plasmid standard sample solution, the microfluidic channel 69, the quantitative sampling valve 70 and the check valve 71 between the second gradient solution finished tank 68 of the plasmid standard sample solution and the third gradient solution preparation tank 75 of the plasmid standard sample solution, the microfluidic channel 72, the quantitative sampling valve 73 and the check valve 74 between the DNA dilution liquid chamber 44 and the third gradient solution preparation tank 75 of the plasmid standard sample solution, the third gradient solution preparation tank 75 of the plasmid standard sample solution, the third gradient solution preparation tank 75 of the plasmid standard sample solution and the plasmid standard sample solution. the microfluidic channel 76, the check valve 77 and the atomizing nozzle 78 between the third gradient solution finished tank 79 of the plasmid standard sample solution, the third gradient solution finished tank 79 of the plasmid standard sample solution, the microfluidic channel 80, the quantitative sampling valve 81 and the check valve 82 between the third gradient solution finished tank 79 of the plasmid standard sample solution and the fourth gradient solution preparation tank 86 of the plasmid standard sample solution, the microfluidic channel 83, the quantitative sampling valve 84 and the check valve 85 between the DNA dilution liquid chamber 44 and the fourth gradient solution preparation tank 86 of the plasmid standard sample solution, the fourth gradient solution preparation tank 86 of the plasmid standard sample solution, the microfluidic channel 87, the check valve 88 between the fourth gradient solution preparation tank 86 of the plasmid standard sample solution and the fourth gradient solution finished tank 90 of the plasmid standard sample solution and atomizing nozzle 89, plasmid standard sample solution fourth gradient solution finished tank 90, microfluidic channel 91, quantitative sampling valve 92 and check valve 93 between plasmid standard sample solution fourth gradient solution finished tank 90 and plasmid standard sample solution fifth gradient solution preparation tank 97, microfluidic channel 94, quantitative sampling valve 95 and check valve 96 between DNA dilution liquid chamber 44 and plasmid standard sample solution fifth gradient solution preparation tank 97, plasmid standard sample solution fifth gradient solution preparation tank 97, microfluidic channel 98, check valve 99 and atomizing nozzle 100 between plasmid standard sample solution fifth gradient solution preparation tank 97 and plasmid standard sample solution fifth gradient solution finished tank 101, plasmid standard sample solution fifth gradient solution finished tank 101, the microfluidic channel 102, the quantitative sampling valve 103 and the check valve 104 between the fifth gradient solution finished tank 101 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 105, the quantitative sampling valve 106 and the check valve 107 between the fourth gradient solution finished tank 90 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 108, the quantitative sampling valve 109 and the check valve 110 between the third gradient solution finished tank 79 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 111, the quantitative sampling valve 112 and the check valve 113 between the second gradient solution finished tank 68 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131,The microfluidic channel 114, the quantitative sampling valve 115 and the check valve 116 between the first gradient solution finished tank 57 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 117, the quantitative sampling valve 118 and the check valve 119 between the third gradient solution finished tank 79 of the plasmid standard sample solution and the spiked recovery preparation tank 120, the microfluidic channel 121, the check valve 122 and the atomizing nozzle 123 between the spiked recovery liquid preparation tank 120 and the spiked recovery liquid finished tank 124, the spiked recovery liquid finished tank 124, the microfluidic channel 125, the quantitative sampling valve 118 and the check valve 119 between the spiked recovery liquid finished tank 124 and the qPCR reaction solution preparation chamber 131. The quantitative sampling valve 126 and the check valve 127, the microfluidic channel 128, the check valve 129 and the quantitative sampling valve 130 between the spike recovery liquid preparation tank 120 and the test solution chamber 31, the qPCR reaction liquid preparation chamber 131, the microfluidic channel 132, the check valve 133 and the atomizing nozzle 134 between the qPCR reaction liquid preparation chamber 131 and the qPCR detection chamber 135, the qPCR detection chamber 135, the safety pipeline 136, the safety valve 137, the safety outlet 138, and the safety outlet plug 139 are all universal standard parts or parts known to those skilled in the art, and their structures and principles can be known to those skilled in the art through technical manuals or conventional experimental methods.

[0095] Example 1

[0096] A detection chip for residual plasmid in in vitro synthesized circular RNA, as shown in the attached Figure 1 and attached Figure 2As shown, it mainly consists of a two-layer structure of a substrate 1 and a cover plate 2, wherein the substrate 1 is provided with a template-free control solution injection port 3, a template-free control solution injection port plug 4, a qPCR reaction buffer injection port 5, a qPCR reaction buffer injection port plug 6, a plasmid primer or probe mixture injection port 7, a plasmid primer or probe mixture injection port plug 8, a test solution injection port 9, a test solution injection port plug 10, a plasmid standard sample injection port 11, a plasmid standard sample injection port plug 12, a DNA diluent injection port 13, a DNA diluent injection port plug 14, a template-free control solution injection line 15 and a check valve 16, a qPCR reaction buffer injection line 17 and a check valve 18, a plasmid primer or probe mixture injection line 19 and a check valve 20, a qPCR MIX reaction solution preparation tank 21, a qPCR MIX reaction solution preparation tank 21 and a qPCR The microfluidic channel 22, the check valve 23 and the atomizing nozzle 24 between the MIX reaction solution finished product tank 25, the qPCR MIX reaction solution finished product tank 25, the microfluidic channel 26, the quantitative sampling valve 27 and the check valve 28 between the qPCR MIX reaction solution finished product tank 25 and the qPCR reaction solution preparation chamber 131, the test solution injection pipeline 29 and the check valve 30, the test solution chamber 31, the microfluidic channel 32, the quantitative sampling valve 33 and the check valve 34 between the test solution chamber 31 and the qPCR reaction solution preparation chamber 131, the plasmid standard sample injection pipeline 35 and the check valve 36, the plasmid standard sample solution mother solution preparation tank 37, the plasmid The microfluidic channel 38, the check valve 39 and the atomizing nozzle 40 between the standard sample solution mother solution preparation tank 37 and the plasmid standard sample solution mother solution finished product tank 41, the plasmid standard sample solution mother solution finished product tank 41, the DNA dilution liquid injection pipeline 42 and the check valve 43, the DNA dilution liquid chamber 44, the microfluidic channel 45, the check valve 46 between the plasmid standard sample solution mother solution preparation tank 37 and the DNA dilution liquid chamber 44, the plasmid standard sample solution mother solution finished product tank 41 and the plasmid standard a microfluidic channel 47, a quantitative sampling valve 48 and a check valve 49 between the first gradient solution preparation tank 53 of the sample solution, a microfluidic channel 50, a quantitative sampling valve 51 and a check valve 52 between the DNA dilution liquid chamber 44 and the first gradient solution preparation tank 53 of the plasmid standard sample solution, a microfluidic channel 54, a check valve 55 and an atomizing nozzle 56 between the first gradient solution preparation tank 53 of the plasmid standard sample solution and the first gradient solution finished tank 57 of the plasmid standard sample solution, a microfluidic channel 58, a quantitative sampling valve 59 and a check valve 60 between the first gradient solution finished tank 57 of the plasmid standard sample solution and the second gradient solution preparation tank 64 of the plasmid standard sample solution, a microfluidic channel 61, a quantitative sampling valve 62 and a check valve 63 between the DNA dilution liquid chamber 44 and the second gradient solution preparation tank 64 of the plasmid standard sample solution,Plasmid standard sample solution second gradient solution preparation tank 64, microfluidic channel 65, check valve 66 and atomizing nozzle 67 between plasmid standard sample solution second gradient solution preparation tank 64 and plasmid standard sample solution second gradient solution finished tank 68, plasmid standard sample solution second gradient solution finished tank 68, microfluidic channel 69, quantitative sampling valve 70 and check valve 71 between plasmid standard sample solution second gradient solution finished tank 68 and plasmid standard sample solution third gradient solution preparation tank 75, microfluidic channel 72, quantitative sampling valve 73 and check valve 74 between DNA dilution liquid chamber 44 and plasmid standard sample solution third gradient solution preparation tank 75, plasmid standard sample solution third gradient solution preparation tank 75, microfluidic channel 76, check valve 77 and atomizing nozzle 78 between plasmid standard sample solution third gradient solution preparation tank 75 and plasmid standard sample solution third gradient solution finished tank 79, plasmid standard sample solution third gradient solution finished tank 79, microfluidic channel 80, quantitative sampling valve 81 and check valve 82 between plasmid standard sample solution third gradient solution finished tank 79 and plasmid standard sample solution fourth gradient solution preparation tank 86, microfluidic channel 83, quantitative sampling valve 84 and check valve 85 between DNA dilution liquid chamber 44 and plasmid standard sample solution fourth gradient solution preparation tank 86, plasmid standard sample solution fourth gradient solution preparation tank 86, plasmid standard sample solution fourth gradient solution The microfluidic channel 87, the check valve 88 and the atomizing nozzle 89 between the preparation tank 86 and the fourth gradient solution finished tank 90 of the plasmid standard sample solution, the fourth gradient solution finished tank 90 of the plasmid standard sample solution, the microfluidic channel 91, the quantitative sampling valve 92 and the check valve 93 between the fourth gradient solution finished tank 90 of the plasmid standard sample solution and the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the microfluidic channel 94, the quantitative sampling valve 95 and the check valve 96 between the DNA dilution liquid chamber 44 and the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the fifth gradient solution preparation tank 97 of the plasmid standard sample solution and the fifth gradient solution preparation tank 97 of the plasmid standard sample solution. the microfluidic channel 98, the check valve 99 and the atomizing nozzle 100 between the finished solution tank 101 of the plasmid standard sample solution, the fifth gradient solution finished solution tank 101, the microfluidic channel 102, the quantitative sampling valve 103 and the check valve 104 between the fifth gradient solution finished solution tank 101 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 105, the quantitative sampling valve 106 and the check valve 107 between the fourth gradient solution finished solution tank 90 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 108, the quantitative sampling valve 109 and the check valve 110 between the third gradient solution finished solution tank 79 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131,The microfluidic channel 111, the quantitative sampling valve 112 and the check valve 113 between the second gradient solution finished tank 68 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 114, the quantitative sampling valve 115 and the check valve 116 between the first gradient solution finished tank 57 of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 117, the quantitative sampling valve 118 and the check valve 119 between the third gradient solution finished tank 79 of the plasmid standard sample solution and the spiked recovery preparation tank 120, the microfluidic channel 121, the check valve 122 between the spiked recovery solution preparation tank 120 and the spiked recovery solution finished tank 124 and an atomizing nozzle 123, a finished product tank for spiked recovery liquid 124, a microfluidic channel 125, a quantitative sampling valve 126 and a check valve 127 between the finished product tank for spiked recovery liquid 124 and the qPCR reaction solution preparation chamber 131, a microfluidic channel 128, a check valve 129 and a quantitative sampling valve 130 between the preparation tank for spiked recovery liquid 120 and the test solution chamber 31, a qPCR reaction solution preparation chamber 131, a microfluidic channel 132, a check valve 133 and an atomizing nozzle 134 between the qPCR reaction solution preparation chamber 131 and the qPCR detection chamber 135, a qPCR detection chamber 135, a safety pipeline 136, a safety valve 137, a safety outlet 138, and a safety outlet plug 139;

[0097] In Example 1, the atomizing nozzle 24, the atomizing nozzle 40, the atomizing nozzle 56, the atomizing nozzle 67, the atomizing nozzle 78, the atomizing nozzle 89, the atomizing nozzle 100, the atomizing nozzle 123 and the atomizing nozzle 134 are all equipped with filter membranes and quartz blades, and are as shown in the attached Figure 3 The middle atomizing nozzle 40 is shown to be equipped with a filter membrane 140 and a quartz blade 141;

[0098] In Example 1, the template-free control solution is injected into the pipeline 15, the qPCR reaction buffer is injected into the pipeline 17, the plasmid primer or the probe mixture is injected into the pipeline 19, the microfluidic channel 22, the microfluidic channel 26, the test solution is injected into the pipeline 29, the microfluidic channel 32, the plasmid standard sample is injected into the pipeline 35, the microfluidic channel 38, The diameters of the DNA diluent injection pipeline 42, microfluidic channel 45, microfluidic channel 50, microfluidic channel 54, microfluidic channel 61, microfluidic channel 65, microfluidic channel 72, microfluidic channel 76, microfluidic channel 83, microfluidic channel 87, microfluidic channel 94, microfluidic channel 98, microfluidic channel 102, microfluidic channel 105, microfluidic channel 108, microfluidic channel 111, microfluidic channel 114, microfluidic channel 121, microfluidic channel 125, microfluidic channel 132 and safety pipeline 136 are all 0.5 mm;

[0099] In Example 1, the diameters of microfluidic channel 47, microfluidic channel 58, microfluidic channel 69, microfluidic channel 80, microfluidic channel 91, microfluidic channel 117, and microfluidic channel 128 are all 0.2 mm;

[0100] In Example 1, the quantitative sampling volume of the quantitative sampling valve 27 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 33 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 46 is 90 microliters, the quantitative sampling volume of the quantitative sampling valve 48 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 51 is 90 microliters, the quantitative sampling volume of the quantitative sampling valve 59 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 62 is 90 microliters, the quantitative sampling volume of the quantitative sampling valve 70 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 73 is 90 microliters, the quantitative sampling volume of the quantitative sampling valve 81 is 10 microliters, and the quantitative sampling volume of the quantitative sampling valve 84 is 10 microliters. The sampling volume is 90 microliters, the quantitative sampling volume of the quantitative sampling valve 92 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 95 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 103 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 106 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 109 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 112 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 115 is 10 microliters, the quantitative sampling volume of the quantitative sampling valve 118 is 5 microliters, and the quantitative sampling volume of the quantitative sampling valve 126 is 10 microliters; the quantitative sampling volume of the quantitative sampling valve 126 is 45 microliters;

[0101] In Example 1, the number of detection chambers is 1, and the volume of the single detection chamber is 125 μl;

[0102] In Example 1, the substrate 1 is made of a silicon dioxide composite material, wherein the silicon dioxide mass content of the silicon dioxide composite material is 78%, and the mass content of PDMS is 22%; the substrate 1 is non-sticky to liquid samples encountered in the detection application of the utility model, such as the attached Figure 4 As shown;

[0103] In Example 1, the cover plate 2 is made of a PC composite material, wherein the PC mass content of the PC composite material is 93.2%, and the content of boron silicon is 6.8%; the cover plate 2 is non-sticky to the liquid samples encountered in the detection application of the utility model, such as the attached Figure 5 As shown;

[0104] In Example 1, the size of the cover plate is 0.25×45×75 mm;

[0105] In Example 1, the volume of the qPCR MIX reaction solution preparation tank 21 is 50 μl, the volume of the qPCR MIX reaction solution finished tank 25 is 50 μl, the volume of the test solution chamber 31 is 100 μl, the volume of the plasmid standard sample solution mother solution preparation tank 37 is 100 μl, the volume of the plasmid standard sample solution mother solution finished tank 41 is 100 μl, the volume of the DNA dilution liquid chamber 44 is 540 μl, the volume of the plasmid standard sample solution first gradient solution preparation tank 53 is 100 μl, the volume of the plasmid standard sample solution first gradient solution finished tank 57 is 100 μl, the volume of the plasmid standard sample solution second gradient solution preparation tank 64 is 100 μl, the volume of the plasmid standard sample solution second gradient solution finished tank 68 is 100 μl, and the volume of the plasmid standard sample solution third gradient solution preparation tank 54 is 100 μl. The volume of slot 75 is 100 microliters, the volume of plasmid standard sample solution third gradient solution finished slot 79 is 100 microliters, the volume of plasmid standard sample solution fourth gradient solution preparation slot 86 is 100 microliters, the volume of plasmid standard sample solution fourth gradient solution finished slot 90 is 100 microliters, the volume of plasmid standard sample solution fifth gradient solution preparation slot 97 is 100 microliters, the volume of plasmid standard sample solution fifth gradient solution finished slot 101 is 100 microliters, the volume of spike recovery preparation slot 120 is 50 microliters, the volume of spike recovery solution finished slot 124 is 50 microliters, the volume of qPCR reaction solution preparation chamber 131 is 100 microliters, and the volume of qPCR detection chamber 135 is 100 microliters;

[0106] In Example 1, the diameter of the micro droplets formed by the atomizing nozzle 24, the atomizing nozzle 40, the atomizing nozzle 56, the atomizing nozzle 67, the atomizing nozzle 78, the atomizing nozzle 89, the atomizing nozzle 100, the atomizing nozzle 123 and the atomizing nozzle 134 in Example 1 is 0.05-50 μm.

[0107] A method for preparing a detection chip for residual plasmids in in vitro synthesized circular RNA comprises the following steps:

[0108] The first step is to use AutoCAD software to draw the designed detection chip for residual plasmids in in vitro synthesized circular RNA, and make it into a film mask for photolithography; use a four-inch single crystal silicon wafer as a substrate, use photolithography technology to make a silicon wafer mold, and use photoresist to etch the basic chip: the qPCR MIX reaction solution preparation tank 21, the qPCR MIX reaction solution finished tank 25, the test solution cavity 31, the plasmid standard sample solution mother solution preparation tank 37, the plasmid standard sample solution mother solution finished tank 41, the DNA dilution liquid cavity 44, the plasmid standard sample solution first gradient solution preparation tank 53, the plasmid standard sample solution first gradient solution finished tank 57, the plasmid standard sample solution second gradient solution preparation tank 64, the plasmid standard sample solution second gradient solution finished tank 68, the plasmid standard sample solution third gradient solution preparation tank 75, the plasmid standard sample solution third gradient solution finished tank 79, the plasmid standard sample solution The fourth gradient solution preparation tank 86 of the plasmid standard sample solution, the fourth gradient solution finished tank 90 of the plasmid standard sample solution, the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the fifth gradient solution finished tank 101 of the plasmid standard sample solution, the spike recovery preparation tank 120, the spike recovery finished tank 124, the qPCR reaction solution preparation chamber 131, the edge line of the qPCR detection chamber 135 and the template-free control solution injection pipeline 15 connected to each chamber or tank, the qPCR reaction buffer injection pipeline 17, the plasmid primer or probe mixture injection pipeline 19, the microfluidic channel 22 between the qPCR MIX reaction solution preparation tank 21 and the qPCR MIX reaction solution finished tank 25, and the qPCR The microfluidic channel 26 between the MIX reaction solution finished tank 25 and the qPCR reaction solution preparation chamber 131, the test solution injection pipeline 29, the microfluidic channel 32 between the test solution chamber 31 and the qPCR reaction solution preparation chamber 131, the plasmid standard sample injection pipeline 35, the microfluidic channel 38 between the plasmid standard sample solution mother solution preparation tank 37 and the plasmid standard sample solution mother solution finished tank 41, the DNA diluent injection pipeline 42, the microfluidic channel 45 between the plasmid standard sample solution mother solution preparation tank 37 and the DNA diluent chamber 44, the plasmid standard sample solution mother solution finished tank 41 and the plasmid standard sample solution the microfluidic channel 47 between the first gradient solution preparation tank 53 of the DNA dilution liquid chamber, the microfluidic channel 50 between the first gradient solution preparation tank 53 of the plasmid standard sample solution, the microfluidic channel 54 between the first gradient solution preparation tank 53 of the plasmid standard sample solution and the first gradient solution finished tank 57 of the plasmid standard sample solution, the microfluidic channel 58 between the first gradient solution finished tank 57 of the plasmid standard sample solution and the second gradient solution preparation tank 64 of the plasmid standard sample solution, the microfluidic channel 61 between the DNA dilution liquid chamber 44 and the second gradient solution preparation tank 64 of the plasmid standard sample solution,The microfluidic channel 65 between the plasmid standard sample solution second gradient solution preparation tank 64 and the plasmid standard sample solution second gradient solution finished tank 68, the microfluidic channel 69 between the plasmid standard sample solution second gradient solution finished tank 68 and the plasmid standard sample solution third gradient solution preparation tank 75, the microfluidic channel 72 between the DNA dilution liquid chamber 44 and the plasmid standard sample solution third gradient solution preparation tank 75, the microfluidic channel 76 between the plasmid standard sample solution third gradient solution preparation tank 75 and the plasmid standard sample solution third gradient solution finished tank 79, the microfluidic channel 77 between the plasmid standard sample solution third gradient solution finished tank 79 and the plasmid standard sample solution third gradient solution preparation tank 78, and the microfluidic channel 78 between the plasmid standard sample solution third gradient solution finished tank 79 and the plasmid standard sample solution third gradient solution preparation tank 79. the microfluidic channel 80 between the fourth gradient solution preparation tank 86, the microfluidic channel 83 between the DNA diluent chamber 44 and the fourth gradient solution preparation tank 86 of the plasmid standard sample solution, the microfluidic channel 87 between the fourth gradient solution preparation tank 86 of the plasmid standard sample solution and the fourth gradient solution finished tank 90 of the plasmid standard sample solution, the microfluidic channel 91 between the fourth gradient solution finished tank 90 of the plasmid standard sample solution and the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the microfluidic channel 94 between the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the microfluidic channel 95 between the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the microfluidic channel 96 between the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, and the microfluidic channel 97 between the fifth gradient solution preparation tank 97 of the plasmid standard sample solution and the microfluidic channel 98 between the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, and the microfluidic channel 99 between the fifth gradient solution preparation tank 97 of the plasmid standard sample solution and the microfluidic channel 91 between the fourth gradient solution finished tank 90 of the plasmid standard sample solution and the fifth gradient solution preparation tank 97 of the plasmid standard sample solution. The microfluidic channel 98 between the finished trough 101 of the fifth gradient solution of the plasmid standard sample solution, the microfluidic channel 102 between the finished trough 101 of the fifth gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 105 between the finished trough 90 of the fourth gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 108 between the finished trough 79 of the third gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 111 between the finished trough 68 of the second gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 112 between the finished trough 68 of the second gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 113 between the finished trough 68 of the second gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 114 between the finished trough 68 of the second gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 115 between the finished trough 79 of the third gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 116 between the finished trough 68 of the second gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 117 between the finished trough 68 of the second gradient solution of the plasmid standard sample solution and the qPCR reaction solution preparation chamber 131, the microfluidic channel 118 between the finished trough The microfluidic channel 114 between the tank 57 and the qPCR reaction solution preparation chamber 131, the microfluidic channel 117 between the plasmid standard sample solution third gradient solution finished tank 79 and the spiked recovery preparation tank 120, the microfluidic channel 121 between the spiked recovery solution preparation tank 120 and the spiked recovery solution finished tank 124, the microfluidic channel 125 between the spiked recovery solution finished tank 124 and the qPCR reaction solution preparation chamber 131, the microfluidic channel 128 between the spiked recovery solution preparation tank 120 and the test solution chamber 31, the microfluidic channel 132 between the qPCR reaction solution preparation chamber 131 and the qPCR detection chamber 135, and the safety pipeline 136;

[0109] The second step is to cast on the mold, and after the mold is reversed, the qPCR MIX reaction solution preparation tank 21, the qPCR MIX reaction solution finished tank 25, the test solution chamber 31, the plasmid standard sample solution mother solution preparation tank 37, the plasmid standard sample solution mother solution finished tank 41, the DNA dilution liquid chamber 44, the plasmid standard sample solution first gradient solution preparation tank 53, the plasmid standard sample solution first gradient solution finished tank 57, the plasmid standard sample solution second gradient solution preparation tank 64, the plasmid standard sample solution second gradient solution finished tank 68, the plasmid standard sample solution third gradient solution preparation tank 75, the plasmid standard sample solution third gradient solution finished tank 79, the plasmid standard sample solution fourth gradient solution preparation tank 86, the plasmid standard sample solution fourth gradient solution finished tank 90, the plasmid standard sample solution fifth gradient solution preparation tank 91, the plasmid standard sample solution fifth gradient solution finished tank 92, the plasmid standard sample solution fifth gradient solution preparation tank 93, the plasmid standard sample solution fifth gradient solution finished tank 94, the plasmid standard sample solution fifth gradient solution preparation tank 95, the plasmid standard sample solution fifth gradient solution finished tank 96, the plasmid standard sample solution fifth gradient solution preparation tank 97, the plasmid standard sample solution fifth gradient solution finished tank 98, the plasmid standard sample solution fifth gradient solution preparation tank 99, the plasmid standard sample solution fifth gradient solution finished tank 99, the plasmid standard sample solution fifth gradient solution preparation tank 98, the plasmid standard sample solution fifth gradient solution finished tank 99, the plasmid standard sample solution fifth gradient solution preparation tank 94, the plasmid standard sample solution fifth gradient solution finished tank 97, the plasmid standard sample solution fifth gradient solution preparation tank 98, the plasmid standard sample solution fifth gradient solution preparation tank 99, the plasmid standard sample solution fifth gradient solution finished tank 99, the plasmid standard sample solution fifth gradient solution preparation tank 93, The preparation tank 97 of the concentration solution, the fifth gradient solution product tank 101 of the plasmid standard sample solution, the preparation tank 120 of the spiked recovery solution, the finished tank 124 of the spiked recovery solution, the qPCR reaction solution preparation chamber 131, and the qPCR detection chamber 135 are functionalized, and the check valve 16 of the template-free control solution injection line 15, the check valve 18 of the qPCR reaction buffer injection line 17, the check valve 20 of the plasmid primer or probe mixture injection line 19, the check valve 23 and the atomizing nozzle 24 of the microfluidic channel 22, the quantitative sampling valve 27 and the check valve 28 of the microfluidic channel 26, the check valve 30 of the test solution injection line 29, the quantitative sampling valve 33 and the check valve 34 of the microfluidic channel 32, the plasmid standard sample solution preparation tank 97, the fifth gradient solution product tank 101 of the plasmid standard sample solution, the spiked recovery solution preparation tank 120, the spiked recovery solution finished tank 124, the qPCR reaction solution preparation chamber 131, and the qPCR detection chamber 135 are assembled. The check valve 36 of the quasi-sample injection pipeline 35, the check valve 39 and the atomizing nozzle 40 of the microfluidic channel 38, the check valve 43 of the DNA diluent injection pipeline 42, the check valve 46 of the microfluidic channel 45, the quantitative sampling valve 48 and the check valve 49 of the microfluidic channel 47, the quantitative sampling valve 51 and the check valve 52 of the microfluidic channel 50, the check valve 55 and the atomizing nozzle 56 of the microfluidic channel 54, the quantitative sampling valve 59 and the check valve 60 of the microfluidic channel 58, the quantitative sampling valve 62 and the check valve 63 of the microfluidic channel 61, the check valve 66 and the atomizing nozzle 67 of the microfluidic channel 65, the quantitative sampling valve 70 and the check valve 71 of the microfluidic channel 69, the quantitative sampling valve 72 of the microfluidic channel 73 and check valve 74, check valve 77 and atomizing nozzle 78 of microfluidic channel 76, quantitative sampling valve 81 and check valve 82 of microfluidic channel 80, quantitative sampling valve 84 and check valve 85 of microfluidic channel 83, check valve 88 and atomizing nozzle 89 of microfluidic channel 87, quantitative sampling valve 92 and check valve 93 of microfluidic channel 91, quantitative sampling valve 95 and check valve 96 of microfluidic channel 94, check valve 99 and atomizing nozzle 100 of microfluidic channel 98, quantitative sampling valve 103 and check valve 104 of microfluidic channel 102, quantitative sampling valve 106 and check valve 107 of microfluidic channel 105, quantitative sampling valve 109 and check valve 110 of microfluidic channel 108,The quantitative sampling valve 112 and the check valve 113 of the microfluidic channel 111, the quantitative sampling valve 115 and the check valve 116 of the microfluidic channel 114, the quantitative sampling valve 118 and the check valve 119 of the microfluidic channel 117, the check valve 122 and the atomizing nozzle 123 of the microfluidic channel 121, the quantitative sampling valve 126 and the check valve 127 of the microfluidic channel 125, the check valve 129 and the quantitative sampling valve 130 of the microfluidic channel 128, the check valve 133 and the atomizing nozzle 134 of the microfluidic channel 132, and the safety valve 137 of the safety pipeline 136;

[0110] The third step is to prepare a cover plate 2 corresponding to the substrate 1, and the cover plate 2 not facing the substrate 1 has markings corresponding to the cavities or grooves and channels of the substrate 1;

[0111] The fourth step is to bond the functionalized substrate and the cover plate by using hot pressing packaging technology, and equip the chip with a template-free control solution injection port plug 4, a qPCR reaction buffer injection port plug 6, a plasmid primer or probe mixture injection port plug 8, a test solution injection port plug 10, a plasmid standard sample injection port plug 12, a DNA diluent injection port 13, a DNA diluent injection port plug 14, and a safety outlet plug 139.

[0112] A method for using a detection chip for residual plasmids in in vitro synthesized circular RNA comprises the following steps:

[0113] Step 1: Open the protective plug 12 of the plasmid standard sample injection port and the protective plug 14 of the DNA diluent cavity injection port in sequence, and inject the plasmid standard sample and the DNA diluent into the plasmid standard sample solution mother solution preparation tank 37 and the DNA diluent cavity 44 respectively through a pipette, and then reset the injection port protective plugs 12 and 14; Step 2: Open the quantitative sampling valve 46 with a non-return function of the DNA diluent output pipeline 45, and after the quantitative sampling of the DNA diluent is completed and injected into the plasmid standard sample solution mother solution preparation tank 37, reset the quantitative sampling valve 46; Step 3: Open the non-return valve 39 and the spray nozzle 40 of the microfluidic channel 38, and let the mixed solution in the plasmid standard sample solution mother solution preparation tank 37 flow through the edge of the atomizing nozzle hole with a non-return function to expand into a liquid layer. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid is impacted and rebounded by the vortex cavity of the quartz blade to form a mixed solution and enter the plasmid standard sample solution mother solution finished product tank 41. After the solution in the plasmid standard sample mother solution preparation tank 37 is atomized and mixed, the check valve 39 and the spraying mist nozzle 40 are reset; Step 4: Open the quantitative sampling valve 48 and the check valve 49 of the microfluidic channel 47, and wait for the quantitative sampling of the plasmid standard sample solution mother solution to be completed and injected into the plasmid standard sample solution mother solution. After the plasmid standard sample solution is prepared in the first gradient solution preparation tank 53, the quantitative sampling valve 48 and the check valve 49 are reset; Step five: open the quantitative sampling valve 51 and the check valve 52 of the microfluidic channel 50, and after the quantitative sampling of the DNA diluent is completed and injected into the plasmid standard sample solution first gradient solution preparation tank 53, reset the quantitative sampling valve 51 and the check valve 52; Step six: open the check valve 55 and the spray nozzle 56 of the microfluidic channel 54, and after the solution in the plasmid standard sample solution first gradient solution preparation tank 53 is atomized and mixed, After that, reset the check valve 55 and the spray nozzle 56; Step 7: Open the quantitative sampling valve 59 and the check valve 60 of the microfluidic channel 58, wait for the quantitative sampling of the first gradient solution of the plasmid standard sample solution to be completed and injected into the second gradient solution preparation tank 64 of the plasmid standard sample solution, and then reset the quantitative sampling valve 59 and the check valve 60; Step 8: Open the quantitative sampling valve 62 and the check valve 63 of the microfluidic channel 61, wait for the quantitative sampling of the DNA dilution solution to be completed, and then reset the quantitative sampling valve 59 and the check valve 60. The quantitative sampling valve 62 and the check valve 63 are opened; Step nine: the check valve 66 and the spray nozzle 67 of the microfluidic channel 65 are opened, and after the atomization and mixing of the solution in the second plasmid standard sample solution gradient solution preparation tank is completed, the check valve 66 and the spray nozzle 67 are reset; Step ten: the quantitative sampling valve 70 and the check valve 71 of the microfluidic channel 69 are opened, and after the quantitative sampling of the second plasmid standard sample solution gradient solution is completed and injected into the third plasmid standard sample solution gradient solution preparation tank 75, the quantitative sampling valve 70 and the check valve 71 are reset;Step 11: Open the quantitative sampling valve 73 and the check valve 74 of the microfluidic channel 72, and reset the quantitative sampling valve 73 and the check valve 74 after the quantitative sampling of the DNA diluent is completed and injected into the third gradient solution preparation tank 75 of the plasmid standard sample solution; Step 12: Open the check valve 77 and the spray mist nozzle 78 of the microfluidic channel 76, and reset the check valve 77 and the spray mist nozzle 78 after the atomization and mixing of the solution in the third gradient solution preparation tank 75 of the plasmid standard sample solution is completed; Step 13: Open the quantitative sampling valve 81 and the check valve 82 of the microfluidic channel 80, and reset the quantitative sampling valve after the quantitative sampling of the third gradient solution of the plasmid standard sample solution is completed and injected into the fourth gradient solution preparation tank 86 of the plasmid standard sample solution. 81 and check valve 82; Step 14: Open the quantitative sampling valve 84 and check valve 85 of the microfluidic channel 83, wait for the DNA dilution solution to be sampled and injected into the fourth gradient solution preparation tank 86 of the plasmid standard sample solution, and then reset the quantitative sampling valve 84 and check valve 85; Step 15: Open the check valve 88 and spray mist nozzle 89 of the microfluidic channel 87, wait for the atomization and mixing of the solution in the fourth plasmid standard sample solution gradient solution preparation tank to be completed, and then reset the check valve 88 and spray mist nozzle 89; Step 16: Open the quantitative sampling valve 92 and check valve 93 of the microfluidic channel 91, wait for the fourth plasmid standard sample solution gradient solution to be sampled and injected into the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, and then reset the quantitative sampling valve 84 and check valve 85. Quantitative sampling valve 92 and check valve 93; Step 17: Open the quantitative sampling valve 95 and check valve 96 of the microfluidic channel 94, and after the DNA dilution solution is sampled and injected into the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, reset the quantitative sampling valve 95 and check valve 96; Step 18: Open the check valve 99 and the spray mist nozzle 100 of the microfluidic channel 98, and after the atomization and mixing of the solution in the fifth plasmid standard sample solution gradient solution preparation tank 97 is completed, reset the check valve and the spray mist nozzle; For a detection chip with more than N (where N is greater than 5) plasmid standard sample solution gradient solution preparation tanks provided on the substrate, the plasmid standard sample solution gradient solution of the Nth gradient concentration solution preparation tank of the plasmid standard sample solution is injected. The inlet pipeline is connected to the N-1th plasmid standard sample solution gradient solution finished tank; step 19: open the test solution injection port protective plug 10, inject the test solution through the pipette, and then reset the injection port protective plug 10; step 20: open the quantitative sampling valve 118 and the check valve 119 of the microfluidic channel 117, wait for the fourth plasmid standard sample solution to be quantitatively sampled and injected into the spiked recovery solution preparation tank 120, and then reset the quantitative sampling valve 118 and the check valve 119. For a detection chip with more than N (where N is greater than 5) plasmid standard sample solution gradient solution preparation tanks provided on the substrate, the spiked recovery solution preparation tank is connected to the (N+1) / 2th plasmid standard sample solution gradient solution finished tank;Step 21: Open the quantitative sampling valve 130 and the check valve 129 of the microfluidic channel 128, and after the quantitative sampling of the test solution is completed and injected into the spiked recovery solution preparation tank 120, reset the quantitative sampling valve 130 and the check valve 129; Step 22: Open the check valve 122 and the spray nozzle 123 of the microfluidic channel 121, and the mixed liquid in the spiked recovery solution preparation tank 120 will expand into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a mixed solution that enters the finished product tank of the spiked recovery solution. After the atomization and mixing of the solution in the spiked recovery solution preparation tank is completed, the check valve 122 and the spray mist nozzle 123 are reset; step 23, open the injection port protective plug 6 of the qPCR reaction buffer and the injection port protective plug 8 of the plasmid primer and / or probe mixture in turn, inject the qPCR reaction buffer, the plasmid primer and / or probe mixture through a pipette, and then reset the injection port protective plugs 6 and 8; step 24, open the check valve 18 of the qPCR reaction buffer injection pipeline 17 and the check valve 20 of the plasmid primer and / or probe mixture injection pipeline 19 in turn, and let the qPCR reaction buffer and the plasmid primer and / or probe mixture in the qPCR The MIX reaction solution preparation tank 21 is mixed, and then the check valves 18 and 20 are reset; Step 25: The check valve 23 and the spray nozzle 24 of the microfluidic channel 22 are opened, and the mixed solution in the qPCR MIX reaction solution preparation tank 21 is expanded into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of aerodynamics, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid is impacted and rebounded by the vortex cavity of the quartz blade to form a mixed solution that enters the qPCR MIX reaction solution finished tank 25. After the solution in the qPCR MIX reaction solution preparation tank 21 is atomized and mixed, the check valve 23 and the spraying nozzle 24 are reset; step 26, open the injection port protective plug 4 of the template-free control solution, inject the template-free control solution through the pipette, and then reset the injection port protective plug 4; step 27, open the check valve 16 of the template-free control solution injection pipeline 15, let the template-free control solution enter the qPCR reaction solution preparation tank 131, and then reset the check valve 16;Step 28, open the quantitative sampling valve 27 and the check valve 28 of the microfluidic channel 26 in sequence, and after the quantitative sampling of the qPCR MIX reaction solution is completed and injected into the qPCR reaction solution preparation chamber 131, reset the quantitative sampling valve 27 and the check valve 28 in sequence; Step 29, open the quantitative sampling valve 103 and the check valve 104 of the microfluidic channel 102 in sequence, and after the quantitative sampling of the fifth gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction solution preparation chamber 131, reset the quantitative sampling valve 103 and the check valve 104 in sequence; Step 30, open the check valve 133 and the spray nozzle 134 of the microfluidic channel 132, and the mixed solution in the qPCR reaction solution preparation chamber 131 will expand into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid is impacted and rebounded by the vortex cavity of the quartz blade to form a mixed solution that enters the qPCR detection cavity 135. After the atomization and mixing of the solution in the qPCR reaction liquid preparation cavity 131 is completed, the check valve 133 and the spraying mist nozzle 134 are reset; Step 30, the chip is placed in the qPCR instrument for thermal cycling and fluorescence qPCR quantification; Step 31, after the detection is completed, the safety outlet plug 139 and the safety valve 137 are opened, and the solution in the qPCR detection cavity 135 is discharged through the safety outlet 138 and the safety pipeline 136; Step Step 32, open the injection port protective plug 4 of the template-free control solution, inject the template-free control solution through a pipette, and then reset the injection port protective plug 4; Step 33, open the check valve 16 of the template-free control solution injection pipeline 15, allow the template-free control solution to enter the qPCR reaction solution preparation tank 131, and then reset the check valve 16; Step 34, open the check valve 133 and the spray nozzle 134 of the microfluidic channel 132, and the template-free control solution in the qPCR reaction solution preparation chamber 131 will expand into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of aerodynamics, the liquid layer is broken into an elongated tubular pore-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a cleaning solution that enters the qPCR detection cavity 135. After the atomization cleaning of the solution in the qPCR reaction liquid preparation cavity 131 is completed, the check valve 133 and the spraying mist nozzle 134 are reset; step 35, after cleaning, open the safety outlet plug 139 and the safety valve 137, and open the safety outlet 138 and the safety pipeline 136 to drain the template-free control solution of the qPCR detection cavity 135; step 36, repeat steps 32 to 35, generally 3 times, or more times, until no plasmid is detected; step 37, repeat steps 27 and 28;Step 38, sequentially open the quantitative sampling valve 106 and the check valve 107 of the microfluidic channel 105, and after the quantitative sampling of the fourth gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction solution preparation chamber 131, reset the quantitative sampling valve 106 and the check valve 107 in sequence; Step 39, repeat steps 30 to 37; Step 40, sequentially open the quantitative sampling valve 109 and the check valve 110 of the microfluidic channel 108, and after the quantitative sampling of the gradient solution of the plasmid standard sample solution 3 is completed and injected into the qPCR reaction solution preparation chamber 131, respectively. After the liquid preparation chamber 131 is filled, the quantitative sampling valve 109 and the check valve 110 are reset in sequence; step 41, repeat steps 30 to 37; step 42, the quantitative sampling valve 112 and the check valve 113 of the microfluidic channel 111 are opened in sequence, and after the quantitative sampling of the second gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction liquid preparation chamber 131, the quantitative sampling valve 112 and the check valve 113 are reset in sequence; step 43, repeat steps 30 to 37; step 44, the microfluidic channel is opened in sequence The quantitative sampling valve 115 and the check valve 116 of 114 are reset in sequence after the quantitative sampling of the first gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction solution preparation chamber 131; step 45, repeating steps 30 to 37; step 46, opening the quantitative sampling valve 33 and the check valve 34 of the microfluidic channel 32 in sequence, and after the quantitative sampling of 10 microliters of the test solution is completed and injected into the qPCR reaction solution preparation chamber 131, the quantitative sampling valve 33 and the check valve 34 are reset in sequence. and check valve 34; step 47, repeating steps 30 to 37; step 48, opening the quantitative sampling valve 126 and the check valve 127 of the microfluidic channel 125 in sequence, and after the quantitative sampling of the spiked recovery solution is completed and injected into the qPCR reaction solution preparation chamber 131, resetting the quantitative sampling valve 126 and the check valve 127 in sequence; step 49, repeating step 30; step 50, using the software to plot the Ct value of the plasmid standard sample against the logarithmic value of its concentration for linear regression analysis, the linear correlation coefficient of the standard curve is R; 2 Greater than 0.99, no detection in the template-free control, the recovery rate of the spiked recovery solution is greater than 50% and less than 150%, and then the obtained Ct value of the test solution is combined with the plasmid length in bp to convert the plasmid DNA residual value. The unit of the plasmid DNA residual value is ng / dose. If a safety failure occurs, the safety outlet plug 139 and the safety valve 137 can be opened in turn, and the safety failure can be eliminated through the safety outlet 138 and the safety pipeline 136 to ensure safe operation.

[0114] Example 2

[0115] Step 1: Open the protective plug 12 of the plasmid standard sample injection port and the protective plug 14 of the DNA diluent cavity injection port in sequence, and inject 10 μl of the plasmid standard sample into the plasmid standard sample solution mother solution preparation tank 37 and 540 μl of the DNA diluent into the DNA diluent cavity 44 through a pipette, and then reset the injection port protective plugs 12 and 14; Step 2: Open the quantitative sampling valve 46 with a non-return function of the DNA diluent output pipeline 45, and after the quantitative sampling of 90 μl of the DNA diluent is completed and injected into the plasmid standard sample solution mother solution preparation tank 37, reset the quantitative sampling valve 46; Step 3: Open the non-return valve 39 and the spray nozzle 40 of the microfluidic channel 38, and let the mixed solution in the plasmid standard sample solution mother solution preparation tank 37 flow through the edge of the atomizing nozzle hole with a non-return function to expand into a liquid layer. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a mixed solution, which enters the plasmid standard sample solution mother solution finished tank 41. After the solution in the plasmid standard sample mother solution preparation tank 37 is atomized and mixed, the check valve 39 and the spraying mist nozzle 40 are reset; Step 4: Open the quantitative sampling valve 48 and the check valve 49 of the microfluidic channel 47, and after the quantitative sampling of 10 microliters of the plasmid standard sample solution mother solution is completed and injected into the plasmid standard sample solution first gradient solution preparation tank 53, the quantitative sampling valve 48 and the check valve 49 are reset; Step 5: Open the quantitative sampling valve 51 and the check valve 52 of the microfluidic channel 50, and after the quantitative sampling of 90 microliters of the DNA dilution solution is completed and injected into the plasmid standard sample solution first gradient solution preparation tank 53, the quantitative sampling valve 51 and the check valve 52 are reset; Step 6: Open the microfluidic channel 5 4, after the solution in the first gradient solution preparation tank 53 of the plasmid standard sample solution is atomized and mixed, the check valve 55 and the spray mist nozzle 56 are reset; Step 7: Open the quantitative sampling valve 59 and the check valve 60 of the microfluidic channel 58, after the quantitative sampling of 10 microliters of the first gradient solution of the plasmid standard sample solution is completed and injected into the second gradient solution preparation tank 64 of the plasmid standard sample solution, the quantitative sampling valve 59 and the check valve 60 are reset ; Step 8: Open the quantitative sampling valve 62 and the check valve 63 of the microfluidic channel 61, and reset the quantitative sampling valve 62 and the check valve 63 after the quantitative sampling of 90 μL of the DNA diluent is completed in the second gradient solution preparation tank 64 of the plasmid standard sample solution; Step 9: Open the check valve 66 and the spray mist nozzle 67 of the microfluidic channel 65, and reset the check valve 66 and the spray mist nozzle 67 after the atomization and mixing of the second plasmid standard sample solution gradient solution preparation tank solution is completed;Step 10: Open the quantitative sampling valve 70 and the check valve 71 of the microfluidic channel 69, and after the quantitative sampling of 10 μl of the second gradient solution of the plasmid standard sample solution is completed and injected into the third gradient solution preparation tank 75 of the plasmid standard sample solution, reset the quantitative sampling valve 70 and the check valve 71; Step 11: Open the quantitative sampling valve 73 and the check valve 74 of the microfluidic channel 72, and after the quantitative sampling of 90 μl of the DNA diluent is completed and injected into the third gradient solution preparation tank 75 of the plasmid standard sample solution, reset the quantitative sampling valve 73 and the check valve 74; Step 12: Open the check valve 77 and the spray nozzle 78 of the microfluidic channel 76, and after the quantitative sampling of 90 μl of the DNA diluent is completed and injected into the third gradient solution preparation tank 75 of the plasmid standard sample solution, reset the quantitative sampling valve 73 and the check valve 74; After the atomization and mixing of the solution in 75 is completed, the check valve 77 and the spray nozzle 78 are reset; Step 13: Open the quantitative sampling valve 81 and the check valve 82 of the microfluidic channel 80, and after the quantitative sampling of 10 microliters of the third gradient solution of the plasmid standard sample solution is completed and injected into the fourth gradient solution preparation tank 86 of the plasmid standard sample solution, reset the quantitative sampling valve 81 and the check valve 82; Step 14: Open the quantitative sampling valve 84 and the check valve 85 of the microfluidic channel 83, and after the quantitative sampling of 90 microliters of the DNA dilution solution is completed and injected into the fourth gradient solution preparation tank 86 of the plasmid standard sample solution, reset the quantitative sampling valve 84 and the check valve 85; Step 15: Open the check valve 86 of the microfluidic channel 87 The check valve 88 and the spray nozzle 89 are reset after the atomization and mixing of the solution in the gradient solution preparation tank of the fourth plasmid standard sample solution is completed; Step 16: Open the quantitative sampling valve 92 and the check valve 93 of the microfluidic channel 91, and after the quantitative sampling of 10 microliters of the gradient solution of the fourth plasmid standard sample solution is completed and injected into the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the quantitative sampling valve 92 and the check valve 93 are reset; Step 17: Open the quantitative sampling valve 95 and the check valve 96 of the microfluidic channel 94, and after the quantitative sampling of 90 microliters of the DNA dilution solution is completed and injected into the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the quantitative sampling valve 92 and the check valve 93 are reset; Step 17: Open the quantitative sampling valve 95 and the check valve 96 of the microfluidic channel 94, and after the quantitative sampling of 90 microliters of the DNA dilution solution is completed and injected into the fifth gradient solution preparation tank 97 of the plasmid standard sample solution, the quantitative sampling valve 92 and the check valve 93 are reset. Quantitative sampling valve 95 and check valve 96; Step 18: Open the check valve 99 and spray nozzle 100 of the microfluidic channel 98, and after the atomization and mixing of the solution in the gradient solution preparation tank 97 of the fifth plasmid standard sample solution is completed, reset the check valve and the spray nozzle; Step 19: Open the protective plug 10 of the injection port of the test solution, inject 95 microliters of the test solution through a pipette, and then reset the protective plug 10 of the injection port; Step 20: Open the quantitative sampling valve 118 and check valve 119 of the microfluidic channel 117, and after the quantitative sampling of 5 microliters of the fourth gradient solution of the plasmid standard sample solution is completed and injected into the spiked recovery solution preparation tank 120, reset the quantitative sampling valve 118 and check valve 119;Step 21: Open the quantitative sampling valve 130 and the check valve 129 of the microfluidic channel 128, and after the quantitative sampling of 45 μL of the test solution is completed and injected into the spiked recovery solution preparation tank 120, reset the quantitative sampling valve 130 and the check valve 129; Step 22: Open the check valve 122 and the spray nozzle 123 of the microfluidic channel 121, and the mixed liquid in the spiked recovery solution preparation tank 120 will expand into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tubular pore-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a mixed solution that enters the finished product tank of the spiked recovery solution. After the atomization and mixing of the solution in the spiked recovery solution preparation tank is completed, the check valve 122 and the spray mist nozzle 123 are reset; step 23, open the injection port protective plug 6 of the qPCR reaction buffer and the injection port protective plug 8 of the plasmid primer and / or probe mixture in turn, inject 85 microliters of qPCR reaction buffer and 15 microliters of plasmid primer and / or probe mixture through a pipette, and then reset the injection port protective plugs 6 and 8; step 24, open the check valve 18 of the qPCR reaction buffer injection pipeline 17 and the check valve 20 of the plasmid primer and / or probe mixture injection pipeline 19 in turn, and let the qPCR reaction buffer and the plasmid primer and / or probe mixture in the qPCR The MIX reaction solution preparation tank 21 is mixed, and then the check valves 18 and 20 are reset; Step 25: The check valve 23 and the spray nozzle 24 of the microfluidic channel 22 are opened, and the mixed solution in the qPCR MIX reaction solution preparation tank 21 is expanded into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of aerodynamics, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid is impacted and rebounded by the vortex cavity of the quartz blade to form a mixed solution that enters the qPCR MIX reaction solution finished tank 25. After the solution in the qPCR MIX reaction solution preparation tank 21 is atomized and mixed, the check valve 23 and the spraying nozzle 24 are reset; step 26, open the injection port protection plug 4 of the template-free control solution, inject 10 microliters of the template-free control solution through a pipette, and then reset the injection port protection plug 4; step 27, open the check valve 16 of the template-free control solution injection pipe 15, let the template-free control solution enter the qPCR reaction solution preparation tank 131, and then reset the check valve 16;Step 28, open the quantitative sampling valve 27 and the check valve 28 of the microfluidic channel 26 in sequence, and after the quantitative sampling of 10 microliters of the qPCR MIX reaction solution is completed and injected into the qPCR reaction solution preparation chamber 131, reset the quantitative sampling valve 27 and the check valve 28 in sequence; Step 29, open the quantitative sampling valve 103 and the check valve 104 of the microfluidic channel 102 in sequence, and after the quantitative sampling of 10 microliters of the fifth gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction solution preparation chamber 131, reset the quantitative sampling valve 103 and the check valve 104 in sequence; Step 30, open the check valve 133 and the spray nozzle 134 of the microfluidic channel 132, and the mixed solution in the qPCR reaction solution preparation chamber 131 will expand into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of the aerodynamic force, the liquid layer is broken into an elongated tube-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a mixed solution that enters the qPCR detection cavity 135. After the atomization and mixing of the solution in the qPCR reaction liquid preparation cavity 131 is completed, the check valve 133 and the spraying mist nozzle 134 are reset; Step 30, the chip is placed in the qPCR instrument for thermal cycling and fluorescence qPCR quantification; Step 31, after the detection, the safety outlet plug 139 and the safety valve 137 are opened, and the solution in the qPCR detection cavity 135 is discharged through the safety outlet 138 and the safety pipeline 136; Step 30 Second, open the injection port protective plug 4 of the template-free control solution, inject 125 μl of the template-free control solution through a pipette, and then reset the injection port protective plug 4; Step thirty-three, open the check valve 16 of the template-free control solution injection pipeline 15, allow the template-free control solution to enter the qPCR reaction solution preparation tank 131, and then reset the check valve 16; Step thirty-four, open the check valve 133 and the spray nozzle 134 of the microfluidic channel 132, and the template-free control solution in the qPCR reaction solution preparation chamber 131 will expand into a liquid layer when flowing through the edge of the atomizing nozzle hole with a check function. Due to the instability of aerodynamics, the liquid layer is broken into an elongated tubular-shaped thick and thin cylinder, and then becomes a droplet. The liquid is squeezed into the nozzle by the internal pressure. A quartz blade is placed inside the atomizing nozzle. The high-speed liquid passes through the vortex cavity of the quartz blade and rebounds to form a cleaning solution that enters the qPCR detection cavity 135. After the atomization cleaning of the solution in the qPCR reaction liquid preparation cavity 131 is completed, the check valve 133 and the spraying mist nozzle 134 are reset; step 35, after cleaning, open the safety outlet plug 139 and the safety valve 137, and open the safety outlet 138 and the safety pipeline 136 to drain the template-free control solution of the qPCR detection cavity 135; step 36, repeat steps 32 to 35, generally 3 times, or more times, until no plasmid is detected;Step 37, repeat steps 27 and 28; Step 38, open the quantitative sampling valve 106 and the check valve 107 of the microfluidic channel 105 in sequence, and after the quantitative sampling of 10 microliters of the fourth gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction solution preparation chamber 131, reset the quantitative sampling valve 106 and the check valve 107 in sequence; Step 39, repeat steps 30 to 37; Step 40, sequentially open the quantitative sampling valve 109 and the check valve 110 of the microfluidic channel 108 ...10 in sequence; After the quantitative sampling is completed and injected into the qPCR reaction solution preparation chamber 131, the quantitative sampling valve 109 and the check valve 110 are reset in sequence; step 41, repeat steps 30 to 37; step 42, open the quantitative sampling valve 112 and the check valve 113 of the microfluidic channel 111 in sequence, and after the quantitative sampling of 10 microliters of the second gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction solution preparation chamber 131, reset the quantitative sampling valve 112 and the check valve 113 in sequence; step 43, repeat steps 30 to 37; step 44, The quantitative sampling valve 115 and the check valve 116 of the microfluidic channel 114 are opened in sequence, and after the quantitative sampling of 10 μl of the first gradient solution of the plasmid standard sample solution is completed and injected into the qPCR reaction solution preparation chamber 131, the quantitative sampling valve 115 and the check valve 116 are reset in sequence; step 45, repeating steps 30 to 37; step 46, the quantitative sampling valve 33 and the check valve 34 of the microfluidic channel 32 are opened in sequence, and after the quantitative sampling of 10 μl of the test solution is completed and injected into the qPCR reaction solution preparation chamber 131, the quantitative sampling valve 115 and the check valve 116 are reset in sequence. The quantitative sampling valve 33 and the check valve 34 are opened; Step 47, repeating Steps 30 to 37; Step 48, opening the quantitative sampling valve 126 and the check valve 127 of the microfluidic channel 125 in sequence, and after the quantitative sampling of 10 microliters of the spiked recovery solution is completed and injected into the qPCR reaction solution preparation chamber 131, the quantitative sampling valve 126 and the check valve 127 are reset in sequence; Step 49, repeating Step 30; Step 50, using the software to plot the logarithmic value of the concentration of the plasmid standard sample with the Ct value of the plasmid standard sample for linear regression analysis, the linear correlation coefficient of the standard curve is R; 2 The Ct value of the test solution is greater than 0.99, no detection in the template-free control, and the recovery rate of the spiked recovery solution is greater than 50% and less than 150%. Then the Ct value of the test solution is combined with the plasmid length in bp to calculate the plasmid DNA residue value. The unit of the plasmid DNA residue value is ng / dose. If a safety failure occurs, the safety outlet plug 139 and the safety valve 137 can be opened in turn, and the safety failure can be eliminated through the safety outlet 138 and the safety pipeline 136 to ensure safe operation.

[0116] Example 3

[0117] The accuracy of the detection result of the detection chip in Example 2 of the present invention for the E. coli plasmid residue in the test solution is determined according to the following process: First, prepare a high concentration solution sample A: take the DNA dilution solution and the plasmid standard sample (3.00×10 5 femtograms per microliter) in a volume ratio of 1:1 and mix well. Secondly, prepare medium concentration solution sample B: take the DNA dilution solution and the plasmid standard sample (3.00×10 3 femtograms per microliter) in a volume ratio of 1:1 and mix well; Step 3: prepare low concentration solution sample C: take the DNA dilution solution and the plasmid standard sample (3.00×10 1 Femtograms per microliter) are mixed in a volume ratio of 1:1. Samples A, B, and C are prepared in 3 portions as test solutions, and then tested using the detection chip in Example 2 of the utility model. As shown in the results in Table 1, the recovery rate of each sample is between 50% and 150%. This shows that the detection accuracy of the detection chip in Example 2 of the utility model meets the accuracy requirements for E. Coli plasmid residue detection;

[0118] .

[0119] Example 4

[0120] The repeatability of the detection results of the detection chip in Example 2 of the present invention for the E. coli plasmid residue in the test solution was determined according to the following process: First, prepare the test solution sample: take the DNA dilution solution and the plasmid standard sample (3.00×10 3 femtogram per microliter) in a volume ratio of 1:1 and then mixed; secondly, the same experimenter performed 6 parallel tests on the prepared test solution samples. As shown in Table 2, the coefficient of variation of the 6 parallel test results was less than 15%, which met the repeatability requirements of E. coli plasmid residue detection;

[0121] .

[0122] Example 5

[0123] The intermediate precision of the detection result of the detection chip in Example 2 of the present invention for the E. coli plasmid residue in the test solution is determined according to the following process: First, prepare the test solution sample: take the DNA dilution solution and the plasmid standard sample (3.00×10 3 femtogram per microliter) and mixed at a volume ratio of 1:1; secondly, different inspectors took samples of the test solution at different times and made 6 parallels. As shown in Table 3 and Table 4, the coefficient of variation of the test results of different inspectors at different times was less than 15%, which met the intermediate precision requirements for E. Coli plasmid residue detection;

[0124] ;

[0125] .

[0126] Example 6

[0127] The linear range of the working curve of the plasmid standard sample gradient solution of the detection chip in Example 2 of the present utility model is 3.00×10 5 -3.00×10 1 Femtogram per microliter, R 2 =0.9999, the amplification efficiency is 99.89%; the accuracy of each plasmid standard sample gradient solution is between 50% and 150%, which meets the standard requirements for E.Coli plasmid residue detection. The results are shown in Table 5;

[0128] .

[0129] Example 7

[0130] The detection chip for residual plasmids in in vitro synthesized circular RNA in Example 2 of the utility model was used to compare the detection results of E.Coli plasmid residues in the test sample with the direct use of a commercial E.Coli plasmid residue detection kit (Shanghai, yeasen). The results showed that the detection results of the two were consistent, and the accuracy rate reached 100%. However, compared with the direct use of a commercial E.Coli plasmid residue detection kit (Shanghai, yeasen) to detect one test sample once, the utility model shortened the operation time required for 1 test sample by 45 minutes, and shortened the total operation time required for the test sample that needed to be repeated 5 times by 3 hours, greatly improving the efficiency of the detection process.

[0131] The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the embodiments by those skilled in the art fall within the scope defined by the claims attached to the present invention.

Claims

1. A detection chip for residual plasmids in in vitro synthesized circular RNA, characterized in that: The detection chip is composed of a substrate and a cover plate, and the substrate is provided with a test solution cavity, a DNA dilution cavity, a qPCR reaction solution preparation cavity, a qPCR detection cavity, a plasmid standard sample solution mother solution preparation tank, a plasmid standard sample solution mother solution finished product tank, a plasmid standard sample solution gradient solution preparation tank, a plasmid standard sample solution gradient solution finished product tank, a spiked recovery solution preparation tank, a spiked recovery solution finished product tank, a qPCR MIX reaction solution preparation tank, and a qPCR MIX reaction solution finished product tank.

2. The detection chip for residual plasmids in in vitro synthesized circular RNA according to claim 1, characterized in that: The test solution chamber, the standard sample solution mother solution preparation tank, the DNA dilution solution chamber, the qPCR MIX reaction solution preparation tank, and the qPCR reaction solution preparation chamber are respectively provided with injection pipelines.

3. The detection chip for residual plasmid in in vitro synthesized circular RNA according to claim 1, characterized in that: Microfluidic channels are provided between the plasmid standard sample solution mother solution preparation tank and the plasmid standard sample solution mother solution finished product tank, between the plasmid standard sample solution gradient solution preparation tank and the plasmid standard sample solution gradient solution finished product tank, between the spiked recovery solution preparation tank and the spiked recovery solution finished product tank, and between the qPCR MIX reaction solution preparation tank and the qPCR MIX reaction solution finished product tank. The microfluidic channels are provided with check valves and atomizing nozzles with check functions.

4. The detection chip for residual plasmid in in vitro synthesized circular RNA according to claim 1, characterized in that: The injection pipeline of the qPCR reaction solution preparation chamber includes an output plasmid standard product solution gradient solution injection pipeline of the plasmid standard product solution gradient solution finished tank, and the output plasmid standard product solution gradient solution injection pipeline of the plasmid standard product solution gradient solution finished tank is provided with a quantitative sampling valve and a check valve with a check function.

5. The detection chip for residual plasmid in in vitro synthesized circular RNA according to claim 1, characterized in that: The injection pipeline of the qPCR reaction solution preparation chamber includes an output qPCR MIX reaction solution injection pipeline of the qPCR MIX reaction solution finished product tank, and the output qPCR MIX reaction solution injection pipeline of the qPCR MIX reaction solution finished product tank is provided with a quantitative sampling valve and a check valve with a check function.

6. The detection chip for residual plasmid in in vitro synthesized circular RNA according to claim 1, characterized in that: The injection pipeline of the qPCR reaction solution preparation chamber includes an output spiked recovery solution injection pipeline of the spiked recovery solution finished product tank, and the output spiked recovery solution injection pipeline of the spiked recovery solution finished product tank is provided with a quantitative sampling valve and a check valve with a check function.

7. The detection chip for residual plasmids in in vitro synthesized circular RNA according to claim 1, characterized in that: The DNA diluent chamber is provided with a pipeline connected to a DNA diluent output, and the DNA diluent output pipeline is provided with a quantitative sampling valve with a non-return function.

8. The detection chip for residual plasmid in in vitro synthesized circular RNA according to claim 1, characterized in that: The side of the cover plate that is not in contact with the substrate is marked with words such as test solution chamber, DNA dilution chamber, qPCR reaction solution preparation chamber, qPCR detection chamber, plasmid standard sample solution mother solution preparation tank, plasmid standard sample solution mother solution finished product tank, plasmid standard sample solution gradient solution preparation tank, plasmid standard sample solution gradient solution finished product tank, spiked recovery solution preparation tank, spiked recovery solution finished product tank, qPCRMIX reaction solution preparation tank, and qPCR MIX reaction solution finished product tank. The substrate and the cover plate are bonded to form a two-layer structure.

9. The detection chip for residual plasmid in in vitro synthesized circular RNA according to claim 1, characterized in that: The substrate is provided with a safety pipeline.

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