Chip for alkali cracking

Abstract

The utility model belongs to the field of biological medicine, and particularly relates to a chip for alkaline lysis, which comprises a substrate and a channel positioned in the substrate, the channel comprises a main channel, an alkali liquid sample inlet and a bacteria liquid sample inlet which are positioned at one end of the main channel, a mixed sample outlet positioned at the other end of the main channel, and an acid liquid sample inlet positioned in the middle of the main channel; and the alkali liquor sample inlet, the bacteria liquid sample inlet and the acid liquid sample inlet are communicated with the main channel through sample inlet channels. According to the chip for alkali lysis disclosed by the utility model, the plurality of S-shaped section small units are connected in series to carry out mixed lysis on bacterial liquid and alkali liquor, so that the mixing uniformity can be quickly realized, and the defect that plasmid DNA (Deoxyribose Nucleic Acid) is degraded due to over-thick local alkali liquor is effectively reduced; compared with alkali cracking in the prior art, the method is beneficial to accurate regulation and control of alkali cracking operation, improves the plasmid yield, is beneficial to linear amplification, and has excellent industrial practicability.

Description

TECHNICAL FIELD

[0001] The utility model belongs to the field of biological medicine, and relates to a chip for alkali lysis. BACKGROUND

[0002] The mRNA vaccine is a nucleic acid vaccine, which produces antigens by injecting part of mRNA fragments of viruses into human cells, and then excites specific immune responses to achieve the effect of forming immune memory. The plasmid production is the first step of the mRNA vaccine, and usually adopts recombinant E. coli containing a nucleotide sequence to ferment and culture, so that the plasmid is amplified in the bacterial body. The bacterial body is collected, then resuspended by using a resuspension solution, and the bacterial body is lysed by using a strong alkali, so that the mRNA is obtained after subsequent enzyme cutting, transcription and further purification.

[0003] The alkali lysis method is a commonly used method for extracting plasmids, and has been widely used in the field of gene therapy for extracting and purifying DNA. The principle is that the bacterial suspension is exposed to a strong anion detergent with high pH value, so that the cell wall is broken, the chromosomal DNA and the protein are denatured, and the plasmid DNA is released into the supernatant. During the lysis process, the bacterial protein, the broken cell wall and the denatured chromosomal DNA will be intertwined into a large complex, the latter is covered and precipitated by dodecyl sulfate, and the supernatant containing the plasmid DNA is obtained by a certain clarification process for subsequent purification of the plasmid DNA

[0004] In the prior art, under the laboratory scale, the alkali lysis generally adopts a beaker or a container with a volume of less than 50L, and the alkali solution and the acid solution are prepared respectively, then the alkali solution is directly poured into the bacterial suspension by using a manual method, and the alkali lysis time can be accurately controlled. This operation cannot be applied to high-intensity batch operation in scale-up, and cannot meet the needs of large-scale industrial production. In large-scale industrial production, how to keep the alkali solution uniform and avoid local over-strong alkalinity to cause degradation of the plasmid DNA is a major technical difficulty at present; how to accurately control the alkali lysis time and add the acid solution in time for neutralization is also another major difficulty of the current technology, which also limits the large-scale batch production of the corresponding mRNA vaccine.

[0005] Therefore, how to quickly and efficiently implement the process steps of alkali lysis to meet the needs of large-scale batch production is an important technical problem to be solved by the person skilled in the art at present. UTILITY MODEL CONTENT

[0006] Based on the defects of the existing alkali lysis operation that is not suitable for scale-up production and the local over-high solubility of the alkali solution, which easily leads to the breakage and loss of the plasmid DNA.

[0007] The utility model discloses a first aspect provides a kind of chip for alkali lysis;Including substrate and the channel located in substrate interior;Wherein, the channel includes main channel and located alkali inlet and bacteria liquid inlet in one end of main channel, mixed sample outlet in the other end of main channel, acid liquid inlet in the middle of main channel;The alkali inlet, bacteria liquid inlet, acid liquid inlet are all connected with main channel by sample inlet channel and communicate with each other;

[0008] Further, the main channel includes S-shaped section;

[0009] Further, the main channel includes a plurality of S-shaped sections connected in series;

[0010] Further, the length of the S-shaped section is 0.5-50mm;

[0011] Further, the length of the S-shaped section is 0.5-30mm;

[0012] Further, the main channel includes 2-300 S-shaped sections connected in series;

[0013] Further, the main channel, the S-shaped section between the acid liquid inlet and the alkali inlet or the bacteria liquid inlet is 1-200; the S-shaped section between the acid liquid inlet and the mixed sample outlet is 1-300;

[0014] Further, the inner diameter of the main channel is 0.1-100mm;

[0015] Further, the diameter of the acid liquid inlet, the alkali inlet and the bacteria liquid inlet is 0.5-20mm;

[0016] Further, the main channel, the inner bending radius of the S-shaped section is 0.3-30mm; the outer bending radius is 0.4-40mm;

[0017] Further, the included angle between the alkali inlet and the bacteria liquid inlet is 10-180 degrees;

[0018] Further, the included angle between the acid liquid inlet and the S-shaped section is 10-170 degrees;

[0019] Further, the chip for alkali lysis, the substrate includes 1-20 channels located in the substrate;

[0020] Further, the substrate is one of circular, oval, square, trapezoidal, hexagonal, pentagonal, triangular and star-shaped;

[0021] In the specific embodiment of the utility model, the inner diameter of the main channel is 5mm;

[0022] In another specific implementation of the utility model, the inner diameter of the main channel is 10mm;

[0023] In another specific implementation of the utility model, the inner diameter of the main channel is 30mm;

[0024] In another specific implementation of the utility model, the inner diameter of the main channel is 45mm;

[0025] In another specific implementation of the utility model, the inner diameter of the main channel is 50mm;

[0026] In another specific implementation of the utility model, the inner diameter of the main channel is 80mm;

[0027] In the specific implementation of the utility model, the inner bending radius of the S-shaped section is 2mm; the outer bending radius is 4mm;

[0028] In another specific implementation of the utility model, the inner bending radius of the S-shaped section is 10mm; the outer bending radius is 20mm;

[0029] In another specific implementation of the utility model, the inner bending radius of the S-shaped section is 15mm; the outer bending radius is 30mm;

[0030] In the specific implementation of the utility model, the length of the S-shaped section is 2mm;

[0031] In another specific implementation of the utility model, the length of the S-shaped section is 10mm;

[0032] In another specific implementation of the utility model, the length of the S-shaped section is 15mm;

[0033] In another specific implementation of the utility model, the length of the S-shaped section is 20mm;

[0034] In another specific implementation of the utility model, the length of the S-shaped section is 30mm;

[0035] In the specific implementation of the utility model, the included angle between the lye inlet and the bacteria liquid inlet is 30 degrees;

[0036] In another specific implementation of the utility model, the included angle between the lye inlet and the bacteria liquid inlet is 60 degrees;

[0037] In another specific implementation of the utility model, the included angle between the lye inlet and the bacteria liquid inlet is 90 degrees;

[0038] In another specific implementation of the utility model, the included angle between the lye inlet and the bacteria liquid inlet is 120 degrees;

[0039] In the embodiment of the utility model, the included angle between the acid liquid sample inlet and the S-shaped section is 30 degrees.

[0040] In another embodiment of the utility model, the included angle between the acid liquid sample inlet and the S-shaped section is 60 degrees.

[0041] In another embodiment of the utility model, the included angle between the acid liquid sample inlet and the S-shaped section is 90 degrees.

[0042] In another embodiment of the utility model, the included angle between the acid liquid sample inlet and the S-shaped section is 130 degrees.

[0043] In the embodiment of the utility model, the chip for alkali lysis, the base plate includes 1 passageway in the base plate;

[0044] In another embodiment of the utility model, the chip for alkali lysis, the base plate includes 2 passageways in the base plate;

[0045] In another embodiment of the utility model, the chip for alkali lysis, the base plate includes 3 passageways in the base plate;

[0046] In another embodiment of the utility model, the chip for alkali lysis, the base plate includes 5 passageways in the base plate;

[0047] In another embodiment of the utility model, the chip for alkali lysis, the base plate includes 8 passageways in the base plate;

[0048] In another embodiment of the utility model, the chip for alkali lysis, the base plate includes 12 passageways in the base plate;

[0049] In the embodiment of the utility model, the base plate is square;

[0050] In the embodiment of the utility model, the multiple passageways in the base plate can be connected in parallel to realize higher flux alkali lysis.

[0051] In the embodiment of the utility model, the chip for alkali lysis, the bacteria liquid to be lysed flows from the bacteria liquid sample inlet to the main passageway through the sample passageway, the lye flows from the lye sample inlet to the main passageway through the sample passageway, and the acid liquid flows from the acid liquid sample inlet to the middle part of the main passageway through the sample passageway, mixes with the mixed liquid flowing in the S-shaped section, neutralizes the alkalinity of the mixed liquid, and finally the mixed liquid flows out from the mixed sample outlet through the S-shaped section of the latter section.

[0052] The utility model has the advantages that:

[0053] The alkali lysis chip of the utility model, adopt multiple S shape section small unit series connection, carry out bacteria liquid and lye mixed lysis, can realize the uniformity of mixing fast, effectively reduce the defect that plasmid DNA happens degradation caused by local lye over concentration;And introduce acid liquid in the suitable position of main channel composed of S shape section small unit to neutralize the alkaline in mixed liquid after alkali lysis;By adjusting the position of acid liquid sample inlet, cooperate with the speed of fluid in each sample inlet channel, the time of alkali lysis is accurately controlled, the alkali lysis is neither caused by acid liquid addition too early, nor because acid liquid addition too late causes the excessive loss of plasmid;Compared with prior art alkali lysis, it is favorable to accurately control alkali lysis operation, improve plasmid yield, and is favorable to linear amplification to have the excellent effect of industrial practicability. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 It is alkali lysis chip structure schematic view of the utility model;

[0055] Figure 2 It is alkali lysis chip partial schematic view of the utility model;

[0056] Figure 3 It is alkali lysis chip structure schematic view of another embodiment of the utility model;

[0057] In the drawing, 1, base plate;2, main channel;3, lye sample inlet;4, bacteria liquid sample inlet;5, S shape section;6, acid liquid sample inlet;7, mixed sample outlet;8, sample inlet channel. DETAILED DESCRIPTION

[0058] The utility model will be further explained in detail in combination with the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the related content, and not limited to the utility model. In addition, it needs to be explained that only the part related to the utility model is shown in the drawing for the convenience of description.

[0059] It needs to be explained that the embodiments in the utility model and the features in the embodiments can be combined with each other without conflict. The technical scheme of the utility model will be explained in detail below with reference to the drawings and in combination with the embodiments.

[0060] Unless otherwise specified, the exemplary embodiments shown will be understood to provide exemplary features of various details that can implement the technical concept of the utility model in practice. Therefore, unless otherwise specified, the features of various embodiments can be additionally combined, separated, interchanged and / or rearranged without departing from the technical concept of the utility model.

[0061] The use of cross-hatching and / or shading in the drawings is generally used to illustrate the boundaries and / or transitions from one portion of a part to another portion of a part. As such, unless specified, the presence of cross-hatching or shading in no way supercedes or otherwise clarifies any aspect of the parts depicted in the drawings. Moreover, in the drawings, the size and relative sizes of parts can be exaggerated for clarity. When exemplary embodiments can be carried out in different ways, the specific sequences of processes can be performed in different sequences from described unless otherwise specified. For example, two sequentially described processes can be performed at about the same time or in the reverse order unless otherwise specified. Also, like reference numerals denote like parts throughout.

[0062] When a part is referred to as being "on" or "on" another part, "connected to" or "coupled to" another part, it can be directly on, directly connected to, or directly coupled to the other part, or intervening parts can be present. In contrast, when a part is referred to as being "directly on," "directly connected to," or "directly coupled to" another part, there are no intervening parts. In this regard, the term "connected" can refer to physical or electrical connection, with or without intervening parts.

[0063] For descriptive purposes, the present application can use spatial or relative terms, such as "below," "lower," "lower," "down," "down," "up," "up," "above," "higher," and "side" (e.g., as in "side wall") to describe the relationship between one part and another part as shown in the drawings. In addition to the orientation depicted in the drawings, spatially relative terms can also encompass different orientations of the device in use, operation, and / or manufacture. For example, if the device in the drawings is turned over, the part described as "below" or "under" other parts or features would then be oriented "above" the other parts or features. Therefore, the exemplary term "below" can encompass both an "above" and "below" orientation. In addition, the device can be otherwise oriented (e.g., rotated 90 degrees or at other orientations), and accordingly, spatially relative descriptions used herein are interpreted accordingly.

[0064] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "comprising," "including," "containing," and / or "having" and variations thereof are used herein, such terms are intended to be inclusive, in an aspect, it is noted that the terms "substantial," "approximately," and other similar terms are used as terms of approximation and not as terms of degree, unless otherwise indicated herein, as such, they are used to account for inherent deviations in measurements, calculations, and / or provided values that would be recognized by those of ordinary skill in the art.

[0065] Figures 1-2 It is the structure schematic diagram of the chip for alkali lysis of the utility model.

[0066] As Figure 1 shown, the chip for alkali lysis of the utility model;Including square substrate 1 and the channel in the inside of substrate 1;Wherein, the channel includes main channel 2 and located in alkali sample inlet 3 and bacteria liquid sample inlet 4 in one end of main channel 2, mixed sample outlet 7 in the other end of main channel 2, acid liquid sample inlet 6 in the middle of main channel 2;Alkali sample inlet 3, bacteria liquid sample inlet 4, acid liquid sample inlet 6 are all communicated with main channel 2 through sample inlet channel 8;Main channel 2 includes 12 S shape sections 5 in series, and the S shape section between acid liquid sample inlet and alkali sample inlet or bacteria liquid sample inlet is 6, and the S shape section between acid liquid sample inlet and mixed sample outlet is 6;

[0067] As Figure 2 shown (numerical unit is μm), the inner diameter of main channel 2 is 450 μm;The inner bending radius of S shape section is 450 μm;The outer bending radius is 900 μm;The diameter of sample inlet is 600 μm;

[0068] In the embodiment, the length of S shape section is 5 mm;

[0069] The included angle between alkali sample inlet and bacteria liquid sample inlet is 60 degrees;

[0070] The included angle between acid liquid sample inlet and S shape section is 30 degrees;

[0071] As Figure 3 shown, in another embodiment of the utility model, the chip for alkali lysis, the substrate includes 2 channels in the inside of substrate;

[0072] In actual use, the bacteria solution to be lysed flows in through the bacteria solution inlet 4, the NaOH lye flows in through the lye inlet 3, the two liquids enter the main channel 2, and the Escherichia coli is lysed at a PH value of 12 in the S-shaped section inner cavity; the mixed liquid is mixed and neutralized with the acid liquid entering from the acid liquid inlet 6 after passing through the S-shaped section for 6 times, the alkalinity in the main channel 2 is neutralized, the mixed liquid flows through the S-shaped section for 6 times again, and then flows out from the mixed outlet 7; the lysed solution after alkaline lysis enters the next process.

[0073] By using the chip for alkaline lysis, the uniformity of mixing can be quickly realized, the defect that plasmid DNA is degraded due to excessive concentration of local lye is effectively reduced; the acid liquid is introduced at a suitable position of the main channel composed of S-shaped sections to neutralize the alkalinity in the mixed liquid; by adjusting the position of the acid liquid inlet and cooperating with the speed of the fluid in each inlet channel, the alkaline lysis time is accurately controlled, the alkaline lysis is neither caused by the alkaline lysis time being insufficient due to the alkaline lysis time being insufficient due to the acid liquid being added too early, nor caused by the plasmid being excessively lost due to the acid liquid being added too late; compared with the prior art alkaline lysis, the alkaline lysis is beneficial to accurately controlling the alkaline lysis operation, improving the plasmid quality and yield, and can realize the application of workshop industrialization amplification production.

Claims

1. A chip for alkaline lysis, characterized by: The chip comprises a substrate (1) and channels in the substrate (1); wherein the channels comprise a main channel (2), an alkali solution inlet (3) and a bacteria solution inlet (4) at one end of the main channel (2), a mixing outlet (7) at the other end of the main channel (2), and an acid solution inlet (6) in the middle of the main channel (2); the alkali solution inlet (3), the bacteria solution inlet (4) and the acid solution inlet (6) are connected to the main channel (2) through an inlet channel (8); the main channel (2) comprises 2-300 S-shaped sections (5) connected in series.

2. The chip for alkaline cleavage according to claim 1, characterized by: The length of the S-shaped section (5) is 0.5-50 mm.

3. The chip for alkaline cleavage according to claim 1, wherein: The S-shaped section (5) between the acid solution inlet (6) and the alkali solution inlet (3) or the bacteria solution inlet (4) is 1-200; the S-shaped section (5) between the acid solution inlet (6) and the mixing outlet (7) is 1-300.

4. The chip for alkaline cleavage according to claim 1, wherein: The inner diameter of the main channel (2) is 0.1-100 mm; the diameter of the acid solution inlet (6), the alkali solution inlet (3) and the bacteria solution inlet (4) is 0.5-20 mm.

5. The chip for alkaline cleavage according to claim 1, wherein: The inner bending radius of the S-shaped section (5) is 0.3-30 mm; the outer bending radius is 0.4-40 mm.

6. The chip for alkaline cleavage according to claim 1, wherein: The included angle between the alkali solution inlet (3) and the bacteria solution inlet (4) is 10-180 degrees; the included angle between the acid solution inlet (6) and the S-shaped section (5) is 10-170 degrees.

7. The chip for alkaline cleavage according to claim 1, wherein: The substrate (1) is one of a circle, an ellipse, a square, a trapezoid, a hexagon, a pentagon and a triangle.

8. The chip for alkaline cleavage according to claim 1, wherein: The chip for alkali lysis comprises 1-20 channels in the substrate (1).

9. The chip for alkaline cleavage according to claim 8, characterized in that: The multiple channels in the substrate (1) can be connected in parallel to realize higher throughput of alkali lysis.

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