A cas13a-microdroplet chip system and detection method for ionizing radiation quantification

Abstract

The application belongs to the technical field of molecular biological analysis, and particularly relates to a Cas13a-microdroplet chip system and a detection method for ionizing radiation quantification. The system comprises a microdroplet chip, magnetic beads and a CRISPR-Cas13a mixed system. The microdroplet chip is composed of a sample inlet, a Cas13a mixed system inlet, an oil phase inlet, a droplet generation area, a droplet collection cavity and microchannels, and is connected through the microchannels in the droplet generation area, and further connected with the droplet collection cavity through the microchannels. The magnetic beads are anti-m6A antibody modified magnetic beads. The CRISPR-Cas13a mixed system comprises Cas13a protein, primer crRNA and fluorescent reporter molecules. The microdroplet chip system can realize in-situ detection of NCOA4-m6A level within 1 hour, and can realize highly sensitive quantification for low target concentration (5 copies / μL).

Description

Technical Field

[0001] This invention belongs to the field of molecular biological analysis technology, specifically relating to a Cas13a-microdroplet chip system and detection method for quantitative analysis of ionizing radiation. Background Technology

[0002] Humans are constantly exposed to ionizing radiation in their daily lives, especially in specific environments such as medical CT scans, radioactive isotope reagents, and radiotherapy. Ionizing radiation can cause irreversible damage to human health, particularly for patients undergoing radiotherapy, where excessive exposure to high-energy particles (such as gamma rays) can damage healthy organs and cells. According to the World Health Organization's 2020 Global Cancer Report, ionizing radiation has been listed as a major carcinogenic factor for many cancers. Studies on the quantitative effects of radiation on tumorigenesis have confirmed that an increase of 1 Gy in cumulative radiation can increase the risk of leukemia by 2.96 times. Therefore, the ability to sensitively detect low radiation doses to quantify their damage is crucial. Furthermore, human metabolism can cause dynamic changes in the actual radiation dose applied to the body, placing higher demands on rapid, real-time detection strategies.

[0003] Traditional methods for detecting ionizing radiation are divided into physical and biochemical methods. Physical methods for detecting ionizing radiation are based on portable dosimeters with infrared sensors. This is a device that calculates high-energy particles through avalanche discharges induced by ionizing gases and is commonly used to assess radiation levels on the skin surface. However, due to its limited detection range, this device is not easily applicable for measuring whole-body radiation. Another widely used method is to identify radiation-sensitive biomarkers in body fluids, which is more accurate than detecting ionizing radiation only on the body surface. For example, enzyme-linked immunosorbent assays (ELISA) or immunofluorescence staining are used to detect the levels of γ-H2AX (a key protein involved in DNA damage repair) in peripheral blood mononuclear cells (PBMCs) to provide feedback on ionizing radiation damage. However, this method relies on the immune recognition of proteins by antibodies and cannot amplify the signal. Due to its limited sensitivity and long workflow, it is not suitable for detecting low-dose radiation. Therefore, these technical limitations pose a significant obstacle to the clinical need for effective screening and protection against ionizing radiation damage.

[0004] Existing literature has confirmed that the level (or modification site) of NCOA4 RNA m6A modification can be used to determine whether gamma-ray radiation has occurred. Patent application CN202210031406.0, entitled "An Application of NCOA4 mRNA Modification as a Marker of Gamma-Ray Radiation," discloses a method using MeRIP-qPCR to detect the level or relative modification of NCOA4 RNA m6A modification to determine whether gamma-ray radiation has occurred. This method involves capturing the RNA of the sample with magnetic beads, followed by elution, purification, and reverse transcription of the m6A-modified RNA, making the process complex. Furthermore, the results of the MeRIP-qPCR method require relative quantification using an internal control, and relative quantification methods are subject to systematic errors.

[0005] In summary, sensitive and rapid quantitative detection of low-dose ionizing radiation remains a challenge, thus necessitating the development of new methods and strategies. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a Cas13a-microdroplet chip system and detection method for quantitative ionizing radiation, so as to achieve high sensitivity and rapid detection of the actual ionizing radiation dose received by a human or animal. The specific technical solution is as follows.

[0007] A Cas13a-microdroplet chip system for quantitative detection of ionizing radiation, the system comprising a microdroplet chip 100, magnetic beads, and a CRISPR-Cas13a hybrid system; the microdroplet chip 100 consists of a sample inlet 110, a Cas13a hybrid system inlet 120, an oil phase inlet 130, a droplet generation region 140, a droplet collection cavity 150, and various microchannels; the sample inlet 110, the Cas13a hybrid system inlet 120, and the oil phase inlet 130 are respectively connected to the microchannels, which converge at the droplet generation region 140, and are further connected to the droplet collection cavity 150 through the microchannels; the magnetic beads are anti-m6 A. Antibody-modified magnetic beads; the CRISPR-Cas13a mixture system includes Cas13a protein, primer crRNA, and fluorescent reporter molecule; when using the system for detection, the components in the CRISPR-Cas13a mixture system are mixed and injected from the Cas13a mixture system inlet 120; the magnetic beads that have captured the gene to be detected are injected from the sample inlet 110; at the same time, the oil phase is injected from the oil phase inlet 130; the oil phase reacts with the CRISPR-Cas13a mixture system and the magnetic beads to form multiple microdroplets, which enter the droplet collection chamber 150 for in-situ fluorescence detection.

[0008] Furthermore, the sample inlet 110 is connected to the sample flow channel 113, and the Cas13a mixing system inlet 120 is connected to the mixing system flow channel 123. The sample flow channel 113 and the mixing system flow channel 123 merge in the microdroplet chip 100 to form an aqueous phase channel, which is connected to the droplet generation region 140 through the droplet generation cavity flow channel 151. The microchannel connected to the oil phase inlet 130 is divided into two to form a first oil phase flow channel 132 and a second oil phase flow channel 134, which merge with the aqueous phase channel at the droplet generation region 140.

[0009] Furthermore, the droplet collection cavity 150 is connected to the droplet generation area 140 through the droplet generation cavity flow channel 151; the droplet collection cavity 150 is provided with a plurality of wall-like structures 153 arranged in parallel with vertical spacing and a plurality of regularly arranged column-like structures 154, the wall-like structures 153 and the column-like structures 154 dividing the interior of the droplet collection cavity 150 into a plurality of interconnected small areas.

[0010] Preferably, the wall-like structure 153 and the column-like structure 154 divide the interior of the droplet collection cavity 150 into 64 interconnected small regions to facilitate observation and counting.

[0011] Furthermore, a diamond-shaped filter structure may be optionally provided at the sample inlet 110, the Cas13a mixing system inlet 120, and the oil phase inlet 130.

[0012] Furthermore, a curved flow resistance structure may be optionally provided on one side of the sample flow channel 113 and the mixing system flow channel 123 to connect with the droplet generation cavity flow channel 151; and a curved flow resistance structure may be optionally provided on one side of the first oil phase flow channel 132 and the second oil phase flow channel 134 to connect with the droplet generation region 140.

[0013] Furthermore, a curved flow resistance structure may be optionally provided on one side of the droplet generation cavity flow channel 151 and connected to one end of the droplet collection cavity 150; an outlet 155 is provided on the other end of the droplet collection cavity 150.

[0014] Furthermore, the CRISPR-Cas13a hybrid system comprises two primer crRNAs, the sequences of which are shown in SEQ ID NO.1 and NO.2, or the sequences of which are shown in SEQ ID NO.3 and NO.4; and the fluorescent reporter molecule is an RNA molecule with a fluorescent group attached to one end and a quenching group attached to the other end.

[0015] The method for quantitative detection of ionizing radiation using the Cas13a-microdroplet chip system described above quantifies the radiation dose by detecting the m6A content in RNA (on the NCOA4 protein) in situ using in-situ fluorescence. The specific steps are as follows: Step 1: Mix the magnetic beads in the system with the RNA of the sample to be tested to capture the gene containing m6A, and then separate the magnetic beads under the action of a magnetic field; Step 2: Inject the magnetic beads that captured the m6A gene in Step 1 into the sample inlet of the microdroplet chip. Mix the components of the CRISPR-Cas13a mixture in a buffer solution and then inject them into the Cas13a mixture inlet of the chip. The crRNA primers in the CRISPR-Cas13a mixture pair complementaryly with specific sequences in the RNA of the sample to be tested, and then initiate Cas13a enzyme side-branch cleavage, non-specifically cleaving the fluorescent reporter molecules in the system, exposing their fluorescent groups to display fluorescence. Step 3: Fluorinated oil is injected through the oil phase inlet of the chip. The fluorinated oil reacts with the product (aqueous phase) in Step 2 in the droplet generation area of ​​the chip to generate several microdroplets. The size of the microdroplets is 44-56μm. The microdroplets enter the droplet collection cavity of the chip through the droplet generation cavity channel. Step 4: Heat the microdroplets in situ at 30-40°C in the droplet collection chamber of the chip, and detect and count the fluorescence of the microdroplets.

[0016] Furthermore, the concentration of NCOA4-m6A is obtained by dividing the number of fluorescent microdroplets in step 4 by the total number of droplets (m6A concentration = number of fluorescent droplets / total number of droplets).

[0017] In the droplet collection chamber, droplets containing zero targets will not fluoresce, while fluorescent droplets typically contain one or more targets. When the total target copy number is less than the total number of droplets, the targets can be considered uniformly distributed within the droplets. However, when the total target copy number exceeds the total number of droplets, all droplets will glow, exceeding the detection limit. Therefore, this method is suitable for detecting low-concentration samples (i.e., low-radiation-dose samples).

[0018] The method for fabricating the microdroplet chip in the Cas13a-microdroplet chip system of the present invention includes the following steps: Step 1: Place the silicon wafer in the center of the spin coater, apply room temperature photoresist SU8-2050 to the center of the silicon wafer, spin coat to a thickness of 50μm and heat to cure; Step 2: Place the mask with the flow channel pattern into the photolithography machine, expose it with ultraviolet light, heat and cure it. Then immerse and shake the cured silicon wafer in SU8 developer to obtain a template with microstructure. Step 3: Place the template obtained in Step 2 and trichlorosilane together in a vacuum dryer for silanization treatment to obtain a hydrophobic surface; Step 4: Mix the polydimethylsiloxane elastomer and curing agent at a mass ratio of 10:1 and pour the mixture into the mold. Place the mold in a vacuum pump for degassing. Then, heat the mold containing the polydimethylsiloxane at 80°C for 30 minutes. After curing, peel, cut, and punch holes to obtain the polydimethylsiloxane chip. Step 5: Place the polydimethylsiloxane chip from Step 4 with the structured side facing up, and put it together with the glass slide into a plasma cleaner for oxygen bonding. After the process is completed, press the polydimethylsiloxane chip and the glass slide with the oxygen-treated side together to form a closed flow channel.

[0019] Beneficial technical effects This invention proposes a method for in-situ direct quantitative detection of NCOA4-m6A levels using a Cas13a-droplet microfluidic chip platform system to assess ionizing radiation exposure in organisms. First, magnetic beads modified with anti-m6A antibodies are used to capture samples containing m6A, a process that takes approximately 30 minutes, achieving enrichment of the m6A gene. Second, to specifically identify the NCOA4 gene sequence, a CRISPR / Cas13a detection system is used to distinguish the target gene sequence, simultaneously generating an amplified fluorescent signal; this process takes approximately 20 minutes. Finally, a microdroplet chip is used as the reactor. Due to the chip's design with oil and water phase channels, the reactants can be broken down into approximately 200,000 water-oil mixed microdroplets within approximately 10 minutes, reducing the reaction volume from microliters to picoliters within 10 minutes, thus achieving highly sensitive quantification at a lower target concentration (5 copies / μL). Compared to large-volume reactions (microliter systems within PCR tubes), the target and detection solution are more likely to meet within the droplet (picoliter system). At low concentrations, the target concentration is concentrated within the positive droplets, effectively increasing the target concentration, resulting in less background noise and greater accuracy in the detection method. Furthermore, this invention provides absolute quantification; the number of bright spots within the droplet collection chamber of the chip directly reflects the target copy number, and the m6A content is directly obtained by dividing the number of bright spots by the total number of droplets. Overall, the microdroplet chip system method provided by this invention can achieve in-situ detection of NCOA4-m6A levels within one hour, offering simple operation and shorter processing time. Compared to traditional qPCR, it significantly improves both the detection limit and ease of operation (this invention's system method can complete detection under 0.5 Gy of radiation).

[0020] Furthermore, the CRISPR-Cas13a hybrid system of the present invention uses crRNA primers with two target recognition sites in combination. The combination of two target recognition sites increases the probability of binding to the target RNA, which further increases the probability of activating the Cas13a cleavage enzyme function, thereby generating a stronger fluorescence signal.

[0021] Finally, to prevent impurities from flowing into the chip, a filter structure is added at each inlet. To stabilize the liquid flow rate inside the chip, a flow resistance structure is added at the end of each microchannel before droplet formation. This makes the formed microdroplets more uniform in size, thereby making the method more sensitive. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0023] Summary of attached labeling and identification: Microdroplet chip 100, sample inlet 110, first filter structure 111, first flow resistance structure 112, sample flow channel 113, Cas13a mixing system inlet 120, second filter structure 121, second flow resistance structure 122, mixing system flow channel 123, oil phase inlet 130, third filter structure 131, first oil phase flow channel 132, third flow resistance structure 133, second oil phase flow channel 134, fourth flow resistance structure 135, droplet generation zone 140, droplet collection chamber 150, first droplet generation chamber flow channel 151, fifth flow resistance structure 152, wall structure 153, column structure 154, outlet 155.

[0024] Figure 1 This is a schematic diagram of the detection process of the Cas13a-microdroplet chip system in an embodiment of the present invention; Figure 2 The following are experimental results of the Cas13a mixed system verification in the embodiments of the present invention (a is the result of fluorescence intensity change over time for Cas13a reaction with different concentrations of target, b is the linear fitting of the endpoint fluorescence intensity of different concentrations of target, and c is the experimental results of target specificity verification). Figure 3 This is a schematic diagram of the microdroplet chip structure in an embodiment of the present invention; Figure 4 The following are experimental diagrams of the Cas13a-microdroplet chip system functional verification in this embodiment of the invention (a is a statistical diagram of microdroplet particle size distribution, b is an experimental diagram of fluorescent bright spots of target at different concentrations, and c is a statistical diagram of the amount of target added and detected at different concentrations). Figure 5 Figure 2 shows the experimental results of the mouse radiation model in Invention Example 2 (a is a comparison of the effects of the Cas13a-microdroplet chip method and the traditional qPCR method, b is the result of different radiation doses and m6A concentrations for different days). Figure 6 Experimental diagrams of radiotherapy patient model detection in Invention Example 3 (a shows the detection results of the Cas13a-microdroplet chip method for increasing m6A by different radiation doses, b shows the detection results of the traditional qPCR method for increasing m6A by different radiation doses, and c shows a comparison of the Cas13a-microdroplet chip method and the traditional method for increasing m6A by different radiation doses). Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0027] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0028] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0029] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0030] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible subranges and independent numerical values ​​within those ranges. For example, range 1... The description of 6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0031] Example 1 This embodiment provides an example of the microdroplet chip structure described in this invention. The microdroplet chip 100 consists of a sample inlet 110, a Cas13a mixing system inlet 120, an oil phase inlet 130, a droplet generation region 140, a droplet collection chamber 150, and microchannels.

[0032] The sample inlet 110 connects to the sample channel 113 and the Cas13a mixing system inlet 120, which in turn connects to the mixing system channel 123. The sample channel 113 and the mixing system channel 123 merge in the microdroplet chip 100 to form an aqueous phase channel, which connects to the droplet generation region 140 via the droplet generation cavity channel 151. The microchannel connected to the oil phase inlet 130 splits into two to form a first oil phase channel 132 and a second oil phase channel 134, which merge with the aqueous phase channel at the droplet generation region 140. A first filter structure 111 can be installed at the sample inlet 110, a second filter structure 121 can be installed at the Cas13a mixing system inlet 120, and a third filter structure 131 can be installed at the oil phase inlet 130; all filter structures are arranged in a diamond pattern. Furthermore, a first flow resistance structure 112 arranged in a curved manner can be provided on one side of the sample flow channel 113 and connected to the droplet generation cavity flow channel 151; a second flow resistance structure 122 arranged in a curved manner can be provided on one side of the mixing system flow channel 123 and connected to the droplet generation cavity flow channel 151; and a third flow resistance structure 133 and a fourth flow resistance structure 135 are respectively provided on one side of the first oil phase flow channel 132 and the second oil phase flow channel 134 and connected to the droplet generation region 140.

[0033] The droplet collecting cavity 150 is connected to the droplet generating region 140 via a droplet generating cavity flow channel 151. The droplet collecting cavity 150 contains multiple parallel wall-like structures 153 arranged vertically at intervals and multiple regularly arranged columnar structures 154, which divide the interior of the droplet collecting cavity 150 into 64 interconnected small regions. A curved flow resistance structure can be provided on one side of the droplet generating cavity flow channel 151, connecting to one end of the droplet collecting cavity 150; an outlet 155 is provided at the other end of the droplet collecting cavity 150.

[0034] In this embodiment, the microdroplet chip 100 has a total length of 76 mm, a width of 26 mm, and each microchannel has a height of 50 μm. The droplet generation cavity channel 151 has a width of 50 μm. The droplet collection cavity 150 has a total length of 25.9 mm and a total width of 19.4 mm. The wall-like structure 153 is 200 μm wide, and the columnar structure 154 has a diameter of 200 μm.

[0035] Example 2 1. Validation of the Cas13a detection system.

[0036] Step 1: Antibody Modification: Prepare buffer (1×PBS pH=7.4, 0.05% Tween, 0.1% BSA). Disperse the original bottled magnetic beads on a vortex mixer. Transfer 100 μL (1 mg / μL) to a new centrifuge tube, magnetically separate, and discard the supernatant. Add 1 mL of buffer to wash the magnetic beads, magnetically separate, and discard the supernatant. Add 1 mL of buffer and 4 μL of biotin-modified antibody, vortex to resuspend, and incubate at room temperature for 1-2 hours. Magnetically separate, discard the supernatant, wash 1-3 times, add 1 mL of buffer and 2 μL of m6A antibody, and incubate at room temperature for 1-2 hours or overnight at 4°C. Magnetically separate, add 1 mL of buffer (magnetic bead concentration 1 mg / mL) to obtain anti-m6A antibody magnetic beads.

[0037] Step 2: Antibody capture: Add 5 μL of anti-m6A antibody magnetic beads to 40 μL of buffer, mix with 5 μL of standard m6A-RNA, and vortex thoroughly. Incubate at 37°C and 300 rpm for 30 min on a shaker. Magnetic separation removes the supernatant, wash once, and resuspend in 10 μL of buffer.

[0038] Step 3: Preparation of the Cas13a detection system: In a 10 μL system, there are 2 μM Cas13a, 2 μM crRNA, 2×Cas13a reaction buffer (10 mM Tris-HCl pH=8.3, 50 mM KCl, 1.5 mM MgCl2), and 10 μM fluorescent reporter molecule (sequence: FAM-UUUUUU-BHQ1, where BHQ1 is the quenching group and FAM is the fluorescent group).

[0039] Step 4: Mix 10 μL of m6A-RNA of different concentrations with 10 μL of Cas13a detection system in a centrifuge tube, heat at 40°C for 60 minutes, and record the changes in fluorescence intensity.

[0040] like Figure 2 As shown in a and b, for the target RNA, the linear range remains between 500 and 5 × 10⁻⁶. 7 The detection limit was 500 copies / μL, and the detection limit was 500 copies / μL. This result indicates that the detection system has high sensitivity.

[0041] Step 5: Specificity verification of the Cas13a detection system. RNA with correct sequence and m6A, RNA with correct sequence and no m6A, RNA with incorrect sequence and m6A, and RNA with incorrect sequence and no m6A were respectively tested at 5 × 10⁻⁶ ppm. 7 Copy / μL for steps 2, 3, and 4.

[0042] like Figure 2 As shown in c, a fluorescence intensity signal is observed only when the sequence is correct and contains m6A. RNA with a correct sequence but no m6A, RNA with an incorrect sequence but containing m6A, and RNA with an incorrect sequence but no m6A show no significant difference from the non-target group. This result indicates that the detection system has high specificity.

[0043] 2. Cas13a droplet microfluidic chip detection.

[0044] Step 1: Setting up the detection platform. Inject 1 syringe containing 2% surfactant-rich fluorinated oil HFE7500, inject 10 μL of sample into syringe 2, and inject 10 μL of detection solution into syringe 3. Insert the syringes into the injection pump, connect the syringes to a Teflon tubing inserted into the chip inlet, and connect the steel needle to the chip outlet connected to air. Place the chip under a fluorescence microscope.

[0045] Step 2: Droplet Generation. Inject fluorinated oil into the chip until it completely wets the collection chamber, ensuring there are no air bubbles inside the chip. Set the flow rate of the fluorinated oil to 10 μL / min and the flow rate of the sample and detection solution to 1 μL / min, then start the injection pump. You should observe rapid droplet generation at the cross-shaped inlet and rapid droplet alignment in the collection chamber. Once the droplets are full, stop the injection pump and seal the inlet and outlet to ensure the system is closed.

[0046] Step 3: Heating. Place the hot water bag on the chip and heat in situ at 40°C for 20 minutes.

[0047] Step 4: Droplet analysis. Observe the droplet collection cavity under a fluorescence microscope and count the number of fluorescent bright spots and the particle size distribution.

[0048] like Figure 4 As shown in figure a, the generated microdroplets exhibit high uniformity in size, with a diameter of approximately 50 μm and a CV of 0.4%. Step 5: Mix 10 μL of m6A-RNA standard samples of different concentrations with 10 μL of the Cas13a detection system using microdroplet chip detection and analysis following steps 1, 2, 3, and 4. Figure 4 As shown in b and c, the number of detected fluorescent bright spots is highly consistent with the number of added target copies.

[0049] Example 3 Step 1: Establishment of the mouse radiation model. To establish the mouse radiation model, adult (6-8 weeks old) mice (C57BL / 6, Vital River Laboratories, China) were exposed to whole-body irradiation with a 60Co γ-ray source at a dose rate of 69 cGy / min. At least six mice were used for each radiation dose (0.5, 1, 2, 4, and 6.5 Gy) and time point (days 1, 7, and 14). Control mice were sham-exposed. Blood samples were collected from the mice via the retro-orbital plexus at each time point. PBMCs were then isolated by density gradient centrifugation using a PBMC isolation kit (TBD Sciences, China) according to the manufacturer's instructions. Total RNA from the PBMCs was extracted using TRIzol reagent (Invitrogen, USA) for subsequent experiments.

[0050] Table 1 Primer sequences for the mouse radiation model Step 2: Cas13a droplet microfluidic chip detection.

[0051] Step 3: Validation of the conventional qPCR method. After storing 500 ng of total RNA as input, the remaining RNA was incubated with anti-m6A-Ab MB (1 mg / mL) in PBS buffer (containing 0.05% Tween 20 and 0.1% BSA) at 37°C and 300 rpm for 30 min to obtain m6A-RNA. Then, the m6A RNA was eluted with elution buffer (5 mM Tris-HCl, 1 mM EDTA, pH 8.0, 0.05% SDS, 20 mg / mL proteinase K). The m6A RNA was then purified by Trizol and quantified using a Nano-300 (Allsheng, China). m6A RNA and 500 ng of input RNA were used as templates in the qRT-PCR assay. The enrichment rate of the m6A biomarker was calculated as the ratio of its amount in the IP to the amount in the input produced from the same amount of RNA. The relative levels of the m6A biomarker in each exposed group were normalized to their corresponding unirradiated control group.

[0052] Figure 5 The results showed that the Cas13a droplet microfluidic platform achieved high linearity in quantifying radiation dose, from 0.5 Gy to 6.5 Gy. Head-to-head comparisons also showed that the Cas13a droplet microfluidic chip detection platform achieved a lower limit of detection than traditional immunoqPCR methods, reaching 0.5 Gy of radiation that immunoqPCR could not detect. Figure 5 b shows that m6A-RNA can reflect differences in radiation dose as early as 1 day, and this difference can still be detected at 14 days.

[0053] Example 4 Step 1: PBMC samples were collected from 10 gynecological cancer patients who received radiation therapy. Samples were collected at different stages, such as before radiation therapy (0 Gy) and after each cycle with cumulative doses of 10 Gy, 20 Gy, 30 Gy, 40 Gy, and 50 Gy. PBMCs were then separated by density gradient centrifugation using a PBMC isolation kit (TBD Sciences, China) according to the manufacturer's instructions. Total RNA from the PBMCs was extracted using TRIzol reagent (Invitrogen, USA) for subsequent experiments.

[0054] Table 2 Primer sequences for radiotherapy patient models Step 2: Cas13a droplet microfluidic chip detection.

[0055] Step 3: Validation of the traditional qPCR method.

[0056] Quantitative analysis of the Cas13a droplet microfluidic platform showed that, although the initial levels of NCOA4-m6A varied among patients, all of these levels increased with increasing cumulative dose. Figure 6 a) further validated NCOA4-m6A as a biomarker for ionizing radiation dose. Next, we used the Cas13a droplet microfluidic platform and the traditional methylated RNA immunoprecipitation qPCR method (a) Figure 6 A head-to-head comparison was performed between b) and the two platforms. The continuously increasing trend in NCOA4-m6A levels detected by the CM platform and MeRIP-qPCR confirms the detectability of our platform. Furthermore, within the IR range of 10 Gy to 50 Gy, the CM platform exhibits nearly twice the sensitivity of MeRIP-qPCR in detecting NCOA4-m6A. Figure 6 c). In Figure 6 In diagram c, the slope represents sensitivity. Under the same radiation dose, the signal intensity (ordinate) of the method of this invention is approximately twice that of the MeRIP-qPCR method. The ordinate is a ratio of the signal from each radiation dose group to the control group, and has been normalized.

[0057] Example 5 A method for fabricating microdroplet chips is provided. Step 1: Master Fabrication: Clean and dry the silicon wafer. Place the wafer in the center of a spin coater and use a dropper to apply room temperature photoresist SU8-2050 to the center of the wafer, spreading it to a thickness of 50μm. Heat to cure. Place the mask with the flow channel pattern onto a photolithography machine for UV exposure and heat to cure. Immerse the post-baked silicon wafer in a dish containing SU8 developer, gently shaking until the visible flow channel pattern appears on the substrate. Rinse the wafer with isopropyl alcohol and dry it in an oven to obtain the master with microstructure.

[0058] Step 2: Silanization treatment: Place the master sample obtained in Step 1 and trichlorosilane together in a vacuum dryer for silanization treatment to obtain a hydrophobic surface.

[0059] Step 3: Chip Soft Lithography: Mix the polydimethylsiloxane elastomer and curing agent at a mass ratio of 10:1, pour the mixture into the master mold, and place it in a vacuum pump for degassing to completely eliminate air bubbles inside the PDMS. Place the master mold with the poured polydimethylsiloxane into an oven and heat it at 80°C for 30 minutes. After curing, peel, cut, and punch holes.

[0060] Step 4: Chip Bonding: With the polydimethylsiloxane chip structure side facing up, place it flat in a plasma cleaner along with a glass slide and initiate plasma oxygen bonding. After the program finishes, quickly press the plasma-treated side of the glass slide and the upward-facing flow channel structure side of the polydimethylsiloxane chip together to form a closed flow channel. Place the chip in an 80-degree oven and heat for 2 hours.

[0061] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A Cas13a-microdroplet chip system for quantitative detection of ionizing radiation, characterized in that, The system includes a microdroplet chip (100), magnetic beads, and a CRISPR-Cas13a hybrid system. The microdroplet chip (100) consists of a sample inlet (110), a Cas13a hybrid system inlet (120), an oil phase inlet (130), a droplet generation region (140), a droplet collection chamber (150), and various microchannels. The sample inlet (110) is connected to the sample channel (113), and the Cas13a hybrid system inlet (120) is connected to the hybrid system channel (123). The sample channel (113) and the hybrid system channel (123) are located within the microdroplet... The aqueous phase channel in the chip (100) is connected to the droplet generation region (140) through the first droplet generation cavity channel (151); the microchannel connected to the oil phase inlet (130) is divided into two to form the first oil phase channel (132) and the second oil phase channel (134), which merge with the aqueous phase channel at the droplet generation region (140); and further connected to the droplet collection cavity (150) through the microchannel; the magnetic beads are magnetic beads modified with anti-m6A antibody; the CRISPR-Cas13a hybrid system includes Cas13a protein, primer crRNA and fluorescent reporter molecule; When using the system for detection, the components in the CRISPR-Cas13a mixture are mixed and injected from the Cas13a mixture inlet (120); the magnetic beads that capture the RNA of the sample to be tested are injected from the sample inlet (110); at the same time, the oil phase is injected from the oil phase inlet (130); the oil phase reacts with the CRISPR-Cas13a mixture and the magnetic beads to form multiple microdroplets, which enter the droplet collection chamber (150) for in-situ fluorescence detection.

2. The Cas13a-microdroplet chip system as described in claim 1, characterized in that, The droplet collection cavity (150) is connected to the droplet generation area (140) through the flow channel of the second droplet generation cavity; the droplet collection cavity (150) is provided with a plurality of wall-like structures (153) and a plurality of column-like structures (154) arranged in parallel with vertical spacing, and the wall-like structures (153) and the column-like structures (154) divide the interior of the droplet collection cavity (150) into a plurality of interconnected small areas.

3. The Cas13a-microdroplet chip system as described in claim 2, characterized in that, A diamond-shaped filter structure is provided at the sample inlet (110), the Cas13a mixing system inlet (120), and the oil phase inlet (130).

4. The Cas13a-microdroplet chip system as described in claim 2, characterized in that, The sample flow channel (113) and the mixing system flow channel (123) are provided with a curved flow resistance structure on one side and connected to the first droplet generation cavity flow channel (151); and the first oil phase flow channel (132) and the second oil phase flow channel (134) are provided with a curved flow resistance structure on one side and connected to the droplet generation area (140).

5. The Cas13a-microdroplet chip system as described in claim 3, characterized in that, A curved flow resistance structure is provided on one side of the flow channel of the second droplet generating cavity and connected to one end of the droplet collecting cavity (150); an outlet (155) is provided on the other end of the droplet collecting cavity (150).

6. The Cas13a-microdroplet chip system as described in claim 1, characterized in that, The CRISPR-Cas13a hybrid system includes two primer crRNAs, the sequences of which are shown in SEQ ID NO.1 and NO.2, or the sequences of which are shown in SEQ ID NO.3 and NO.4; and the fluorescent reporter molecule is an RNA molecule with a fluorescent group attached to one end and a quenching group attached to the other end.

7. A method for quantitative detection of ionizing radiation using the Cas13a-microdroplet chip system according to any one of claims 2-6, characterized in that, The method quantifies radiation dose by detecting the m6A content in the RNA of the sample under test using in situ fluorescence. The specific steps are as follows: Step 1: Mix the magnetic beads in the system with the RNA of the sample to be tested to capture the gene containing m6A, and then separate the magnetic beads under the action of a magnetic field; Step 2: Inject the magnetic beads that captured the m6A gene in Step 1 into the sample inlet of the microdroplet chip. Mix the components of the CRISPR-Cas13a mixture in a buffer solution and then inject them into the Cas13a mixture inlet of the chip. The primer crRNA in the CRISPR-Cas13a mixture pairs complementaryly with a specific sequence in the RNA of the sample to be tested, and then initiates Cas13a enzyme side branch cleavage, non-specifically cleaving the fluorescent reporter molecules in the system, exposing their fluorescent groups to display fluorescence. Step 3: Fluorinated oil is injected through the oil phase inlet of the chip. The fluorinated oil reacts with the product in step 2 in the droplet generation area of ​​the chip to generate several microdroplets. The size of the microdroplets is 44-56μm. The microdroplets enter the droplet collection cavity of the chip through the flow channel of the second droplet generation cavity. Step 4: Heat the microdroplets in situ at 30-40°C in the droplet collection chamber of the chip, and detect and count the fluorescence of the microdroplets.

8. The method as described in claim 7, characterized in that, The concentration of NCOA4-m6A is obtained by dividing the number of fluorescent microdroplets in step 4 by the total number of droplets.

Images