Hepatocarcinoma diagnosis kit combined with DNA logic gate and aptamer sequencing

Abstract

The application discloses a liver cancer diagnosis kit combined with DNA logic gate and nucleic acid aptamer sequencing, belongs to the field of diagnosis kits, and particularly relates to a double-stranded "and gate" logic operation composed of DNA sequences of cT1, cT2, cT35 and I, and an amplification reagent containing S-II, W, F, F-Cy5, M1 and M1-Cy3 based on entropy-driven amplification reaction. After PBMC is extracted from whole blood, the kit can perform logic analysis on whether target proteins corresponding to nucleic acid aptamers Apt51, GR-30 and IBA are simultaneously present on the PBMC, so that early screening of liver cancer is realized. The specific target of liver cancer patients found by high-throughput sequencing of nucleic acid aptamers can be effectively distinguished from healthy people and cancer patients by the kit.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of diagnostic kits, and particularly relates to a liver cancer diagnostic kit combining DNA logic gate and aptamer sequencing. BACKGROUND

[0002] Liver cancer refers to liver malignant tumor, which can be divided into primary and secondary types. The primary liver malignant tumor originates from the epithelial or mesenchymal tissue of the liver, the former is called primary liver cancer, which is a high-incidence and extremely harmful malignant tumor, and the latter is called sarcoma, which is less common compared with the primary liver cancer. The secondary or metastatic liver cancer refers to the liver invasion of malignant tumors originating from multiple organs in the body. It is generally found in liver metastasis of malignant tumors of organs such as stomach, biliary tract, pancreas, colorectum, ovary, uterus, lung and breast.

[0003] The prior art has the following defects: As the liver cancer ranks the top three in the global mortality rate, the early screening and diagnosis of the liver cancer is crucial to improve the survival rate of patients. However, the current clinical routine diagnosis methods have significant limitations: first, the serum markers such as alpha-fetal protein AFP have a low detection sensitivity of about 40%-60%, and are easily interfered by benign lesions such as hepatitis and cirrhosis; second, the imaging such as ultrasound, CT / MRI examination has insufficient resolution for early lesions smaller than 1 cm, and cannot provide molecular pathological information; third, the tissue biopsy is invasive, and it is difficult to realize dynamic monitoring and heterogeneity analysis.

[0004] Therefore, it is urgent to develop a non-invasive liquid biopsy technology based on peripheral blood to realize the early discovery and accurate molecular typing of the liver cancer. SUMMARY

[0005] The application aims to provide a liver cancer early screening kit based on aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operation, which is convenient and flexible, has high sensitivity, good specificity and is non-invasive.

[0006] The technical scheme adopted by the application to achieve the above-mentioned purpose is as follows:

[0007] The liver cancer early screening kit based on aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operation comprises a probe reagent based on DNA logic operation and a nuclease-free signal amplification reagent, and the probe reagent comprises nucleic acid sequences as shown in SEQ ID NO. 1-SEQ ID NO. 4.

[0008] In the probe reagent, the SEQ ID NO. 1 sequence, the SEQ ID NO. 2 sequence, the SEQ ID NO. 3 sequence and the SEQ ID NO. 4 sequence are used in a molar ratio of 1:0.5-2:0.5-2:0.5-2.

[0009] The present application can simultaneously find multiple specific proteins with high expression quantity related to liver cancer in PBMC through high-throughput sequencing of nucleic acid aptamers, and can effectively distinguish healthy people from cancer patients through comprehensive analysis by DNA logic operation. Compared with the traditional detection method based on a single nucleic acid aptamer, the multiplex detection method of the present application significantly improves the recognition accuracy and anti-interference ability of the PMBC surface protein. Since the method has good sensitivity, specificity and expandability, it provides a new means for accurately identifying and analyzing the PMBC surface protein of liver cancer patients. In addition, this multi-parameter input logic diagnosis method is not limited to the early screening of liver cancer, but can also be extended to the early screening of other malignant tumors and diseases, which makes the method have a wide application prospect in early clinical diagnosis. The present application is based on the programmability of DNA, and a logic gate is designed to directly output signals through logic calculation, thereby simplifying the data processing process and making the signals more intuitive.

[0010] Preferably, the nuclease-free signal amplification reagent comprises any one or more of the nucleic acid sequences shown as SEQ ID NO. 5~ SEQ ID NO. 8.

[0011] More preferably, the nuclease-free signal amplification reagent further comprises F-Cy5 and M1-Cy3, wherein the sequence of F is shown as SEQ ID NO. 7, F-Cy5 is a Cy5 group connected to the 3' end of the F sequence; the sequence of M1 is shown as SEQ ID NO. 8, and M1-Cy3 is a Cy3 group connected to the 5' end of the M1 sequence.

[0012] More preferably, in the nuclease-free signal amplification reagent, the SEQ ID NO. 5 sequence, the SEQ ID NO. 6 sequence, the SEQ ID NO. 7 sequence and the SEQ ID NO. 8 sequence are used in a molar ratio of 1:0.5-2:0.5-2:0.5-2.

[0013] More preferably, in the nuclease-free signal amplification reagent, the SEQ ID NO. 5 sequence, the F-Cy5 and the M1-Cy3 are used in a molar ratio of 1:0.5-2:0.5-2.

[0014] Preferably, the probe reagent is formed by base complementary pairing of the nucleic acid sequences shown as SEQ ID NO. 1~ SEQ ID NO. 4.

[0015] Preferably, the probe reagent further comprises a buffer; or, the nuclease-free signal amplification reagent comprises a buffer.

[0016] More preferably, the buffer is a TE buffer.

[0017] Preferably, the kit further comprises a PBMC extraction agent, and the PBMC extraction agent is a DPBS solution. The PBMC is peripheral blood mononuclear cell.

[0018] More preferably, the DPBS solution contains FBS and / or nicotinoyl dimethylaminopropionyl hydrazide; the nicotinoyl dimethylaminopropionyl hydrazide is prepared by reacting L-proline methyl ester hydrochloride, 6-methoxynicotinoyl chloride, hydrazine hydrate and (dimethylamino)acetyl chloride. The probe reagent based on DNA logic operation of the present application can release signal chain I only when T1-Apt51, T2-GR-30 and T3-IBA are simultaneously present on PBMC, and further form fluorescence under the action of nuclease-free signal amplification reagent, which can effectively distinguish healthy people from cancer patients. The cell viability of PBMC can ensure more accurate detection results. In the present application, the PBMC extraction agent is a DPBS solution, and FBS and / or nicotinoyl dimethylaminopropionyl hydrazide can be added to the DPBS solution. When only a small amount of FBS or nicotinoyl dimethylaminopropionyl hydrazide is added, the cell survival rate of PBMC is low, which affects the detection effect. However, by adding an appropriate amount of FBS or nicotinoyl dimethylaminopropionyl hydrazide to the DPBS solution, the cell survival rate of PBMC can be improved.

[0019] Preferably, the DPBS solution contains 0.03-0.5wt% of nicotinoyl dimethylaminopropionyl hydrazide; or, the DPBS solution contains 0.1-10wt% of FBS.

[0020] More preferably, in the preparation of nicotinoyl dimethylaminopropionyl hydrazide, nicotinoyl proline ester is first prepared by reacting L-proline methyl ester hydrochloride and 6-methoxynicotinoyl chloride, then reacted with hydrazine hydrate to generate nicotinoyl proline hydrazide, and finally reacted with (dimethylamino)acetyl chloride to generate nicotinoyl dimethylaminopropionyl hydrazide.

[0021] More preferably, in the preparation of nicotinoyl proline ester, L-proline methyl ester hydrochloride is added to a saturated sodium bicarbonate solution, then 6-methoxynicotinoyl chloride solution is added, and the reaction is carried out at 0-10℃ for 1-4h, then at 20-40℃ for 12-48h. After the reaction is completed, ethyl acetate is extracted, the organic layer is washed with saturated sodium bicarbonate solution, dried, and the organic solvent is removed by rotary evaporation to obtain nicotinoyl proline ester.

[0022] Further preferably, in the preparation of the nicotinoyl proline hydrazide, the nicotinoyl proline ester is added into methanol, then hydrazine hydrate is added, and the reaction is carried out at 20-40 DEG C for 8-24 h; the solvent methanol is removed by rotary evaporation, and the product is obtained after washing and drying.

[0023] More preferably, in the preparation of the nicotinoyl proline hydrazide, the nicotinoyl proline ester is added into methanol, then hydrazine hydrate is added, and the reaction is carried out at 20-40 DEG C for 8-24 h; the solvent methanol is removed by rotary evaporation, and the product is obtained after washing and drying.

[0024] Further preferably, in the preparation of the nicotinoyl proline hydrazide, the nicotinoyl proline ester is added into methanol, then hydrazine hydrate is added, and the reaction is carried out at 20-40 DEG C for 8-24 h; the solvent methanol is removed by rotary evaporation, and the product is obtained after washing and drying.

[0025] More preferably, in the preparation of the nicotinoyl proline hydrazide, the nicotinoyl proline ester is added into methanol, then hydrazine hydrate is added, and the reaction is carried out at 20-40 DEG C for 8-24 h; the solvent methanol is removed by rotary evaporation, and the product is obtained after washing and drying.

[0026] Further preferably, in the preparation of the nicotinoyl proline hydrazide, the nicotinoyl proline ester is added into methanol, then hydrazine hydrate is added, and the reaction is carried out at 20-40 DEG C for 8-24 h; the solvent methanol is removed by rotary evaporation, and the product is obtained after washing and drying.

[0027] The application discloses a liver cancer early screening model based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA "and gate" logic operation, and comprises the following steps:

[0028] The PBMC is mixed with a probe reagent based on DNA logic operation and a nuclease-free signal amplification reagent, fluorescence signals are detected, and any one of results (1) and (2) is output according to the detection result:

[0029] Result (1): the fluorescence signal exists, and the liver cancer is positive;

[0030] Result (2): the fluorescence signal does not exist, and the liver cancer is negative.

[0031] Preferably, the PBMC is extracted by a PBMC extraction agent, and the PBMC extraction agent is a DPBS solution.

[0032] The application discloses nucleic acid aptamer Apt51, GR-30 and IBA and the use of corresponding target proteins in early screening of liver cancer.

[0033] The nucleic acid aptamer used in the application is a short-chain oligonucleotide screened by SELEX technology, and can specifically bind to target molecules such as GPC3, ASGPR1 and other liver cancer-related membrane proteins. Compared with traditional antibodies, the nucleic acid aptamer has the following advantages: high affinity and flexible chemical modification, supporting multiple target parallel labeling, wherein the Kd can reach the nM level; programmability: the end of the aptamer can be connected to a functional DNA sequence, realizing the integrated design of "recognition-signal conversion".

[0034] The nucleic acid aptamer is an oligonucleotide sequence with a length of about 15-60 nt, has a series of advantages such as wide target range, high affinity, strong specificity and easy synthesis, and can accurately recognize metal ions, small molecules, amino acids, polypeptides, proteins and cells, and is praised as "chemists' antibodies". As a kind of recognition molecule, the nucleic acid aptamer can recognize disease markers and identify diseased cells when applied to molecular medicine, and is suitable for molecular probes and therapeutic drugs for clinical application.

[0035] One key advantage of nucleic acid aptamers over other molecular probes is that they are essentially nucleic acids, which allows them to perform complex DNA computations. Through DNA strand displacement reactions, nucleic acid aptamers can implement a variety of Boolean logic gates for biomolecular computation, processing multiple input signals into a single output. This allows nucleic acid aptamers to accurately detect multiple biomarkers in a single cell, thereby improving the sensitivity and specificity of cancer diagnosis. In vitro screening, also known as SELEX, is a ligand that is exponentially enriched by system evolution. Nucleic acid aptamers are DNA / RNA oligonucleotide sequences generated by in vitro screening, and have high affinity and specificity, and can bind to a variety of targets such as proteins and small molecules. As a powerful molecular recognition tool, nucleic acid aptamers have wide application prospects in the fields of molecular diagnosis, biological imaging, targeted drug delivery and biomarker discovery. The computing power of this DNA provides new ways for disease diagnosis and treatment, such as pattern recognition, DNA robots and DNA molecular networks.

[0036] Compared to antibodies, nucleic acid aptamers have the following characteristics: (1) Simple preparation: Nucleic acid aptamers can be screened and prepared in vitro using SELEX technology, without relying on immunized animals like antibodies, thus avoiding individual differences that may occur during immunization. The process is relatively simple. (2) Short screening cycle: Generally speaking, the screening cycle for nucleic acid aptamers is shorter than that for antibodies, usually only a few weeks, while it often takes several months from immunizing animals to obtaining specific antibodies. (3) Easy to modify: The sequences of nucleic acid aptamers are known, and they can be easily chemically modified and altered in vitro as needed, such as by adding different functional groups and markers to meet various application requirements, while the structural modification of antibodies is relatively complex. (4) Good stability: Nucleic acid aptamers have good stability under certain conditions and can withstand certain temperature and pH changes, making them easier to preserve during storage and use. In contrast, antibodies, as proteins, are more sensitive to conditions such as temperature and pH, and are prone to denaturation and inactivation under extreme conditions. (5) Low immunogenicity: Nucleic acid aptamers are generally very low in immunogenicity, and have a low risk of triggering an immune response when used in vivo. They can be administered multiple times without easily causing immune rejection, which is very beneficial for their application in clinical treatment. Antibodies, as proteins, especially heterologous antibodies, may trigger an immune response in vivo, affecting their efficacy and safety. (6) Adjustable affinity and specificity: Through SELEX technology, different target molecules can be screened to obtain nucleic acid aptamers with different affinities and specificities. Their affinity can reach a level comparable to or even higher than that of antibodies, and they can more accurately target specific structural domains or epitopes of target molecules to achieve highly specific binding. (7) Strong tissue penetration: Nucleic acid aptamers have small molecular weights, generally between 10 kDa and 50 kDa, which is easier to penetrate tissues and blood vessel walls than antibodies of about 150 kDa. They can reach the target site more quickly and have advantages in in vivo imaging and tumor targeted therapy. (8) Cost advantage: The production cost of nucleic acid aptamers is relatively low. Especially when produced on a large scale, the cost of chemical synthesis can be further reduced. Moreover, the production process is relatively simple and easy to standardize, which can reduce application costs.

[0037] This invention discloses the potential and integration needs of DNA molecular computing:

[0038] DNA molecular computing, by designing logic-gated reactions between nucleic acid molecules, namely AND / OR / NOT, can simulate the signal processing functions of electronic circuits, demonstrating unique advantages in the field of biological detection.

[0039] 1. Multiple logic judgments: Through the "AND gate" design, the signal is triggered only when ≥2 markers are identified simultaneously, significantly improving specificity;

[0040] 2. Signal amplification capability: Cascade hybridization chain reaction (HCR) or catalytic hairpin assembly (CHA) can convert single molecule events into detectable macro signals;

[0041] 3. Microfluidic integration: DNA computing units can be combined with microfluidic chips to achieve automated single-cell manipulation and parallel analysis.

[0042] In the present application, nucleic acid aptamer specific labeling technology is adopted, a liver cancer targeting aptamer library is constructed, high-affinity aptamers against liver cancer cell surface markers such as GPC3, ASGPR1, CD44v6 and intracellular metabolites such as ATP are screened through SELEX technology, and the Kd is less than or equal to 2 nM, and phosphorothioate modification is carried out to enhance the nuclease resistance. The present application also adopts aptamer-sequence probe coupling, introduces sequence Read2 and unique molecular identifier UMI at the 3' end of the aptamer, and realizes direct library sequencing of aptamer binding signals at the single cell level.

[0043] The present application combines the high-specificity target recognition ability of nucleic acid aptamer with the intelligent signal processing ability of DNA molecular logic operation, realizes the integrated operation of "recognition-coding-operation" on the surface of single cells, breaks through the limitation of traditional step-by-step detection, and significantly improves the detection efficiency.

[0044] The DPBS solution used to extract PBMC in the present application can also add cocamide propyl hydroxy sultaine, and the content of cocamide propyl hydroxy sultaine in the DPBS solution is 0.01-0.1wt%. The DPBS solution contains an appropriate amount of FBS and nicotinoyl dimethylaminopropyl hydrazine, and cocamide propyl hydroxy sultaine cooperates with them to further improve the cell viability of PBMC.

[0045] The present application adopts a probe reagent based on DNA logic operation composed of cT1, cT2, cT35 and I, and then prepares a nuclease-free signal amplification reagent containing S-II, W, F, F-Cy5, M1 and M1-Cy3 based on entropy-driven amplification EDA, and after extracting PBMC from whole blood, whether T1-Apt51, T2-GR-30 and T3-IBA are present on PBMC can be detected. The probe reagent based on DNA logic operation of the present application will only release signal chain I when T1-Apt51, T2-GR-30 and T3-IBA are present on PBMC, and further form fluorescence under the action of the nuclease-free signal amplification reagent, which can effectively distinguish healthy people from cancer patients. The present application has the following beneficial effects: in vitro detection, high sensitivity, good detection effect, signal amplification and non-invasiveness.

[0046] Therefore, this invention is a convenient, flexible, highly sensitive, specific, and non-invasive early liver cancer screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operations. Attached Figure Description

[0047] Figure 1 This is a graph showing the total number of nucleic acid aptamers.

[0048] Figure 2 This is a dimensionality-reduced clustering group diagram.

[0049] Figure 3 This is a distribution map of nucleic acid aptamers.

[0050] Figure 4 This diagram shows the expression of cell subpopulations and various classic genes.

[0051] Figure 5 This figure shows the aptamer binding status in single-cell high-throughput sequencing of PMBC in liver cancer patients.

[0052] Figure 6 The final fluorescence output signal result of the liver cancer early screening kit based on DNA logic gates combined with nucleic acid aptamer high-throughput sequencing is shown in the figure.

[0053] Figure 7 This is a schematic diagram of logic diagnosis based on DNA AND gate logic operation.

[0054] Figure 8 This is a schematic diagram of the AND gate logic operation section and signal amplification in the kit.

[0055] Figure 9 This is an infrared spectrum.

[0056] Figure 10 This is a cell viability graph. Detailed Implementation

[0057] 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 embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0059] The research basis of the present application is as follows:

[0060] 1. PBMC separation in blood samples

[0061] Fresh whole blood in an anticoagulant tube was centrifuged at 3000 rpm / min for 10 min, the upper plasma was discarded, 2% FBS DPBS was added to 20 mL, and the diluted sample was slowly added to a SepMate™ tube with a density gradient centrifugation liquid (Ficoll) passing through the central small hole of the SepMate™ insert, the density gradient centrifugation liquid should be just submerged in the insert, and centrifugation was carried out at room temperature at 1200 g for 10 min, for samples stored for more than 24 h, the recommended centrifugation time is 20 min, then the white membrane layer rich in PBMCs was quickly transferred to a new tube, the PBMCs were washed with 10 mL of 2% FBS DPBS, centrifuged at 300 g for 8 min at room temperature, the supernatant was discarded, if the above-mentioned precipitate is light pink, it can not be red, if the color is deep red, red blood cell lysis treatment is needed: add 1:3 1x DPBS and red blood cell lysis solution (Solebao), mix gently with a gun head, stand on ice for 15 min, and mix gently up and down at intervals, centrifuge at 500 g at 4°C for 5 min, remove the supernatant; resuspend according to the cell density, and count the dead and live cells with a cell counting plate to determine the cell viability and cell concentration.

[0062] 2. Nucleic acid aptamer incubation and machine operation

[0063] According to the sample concentration determined in the above step, 1 million cells were taken in 400 μL of binding buffer solution, 200 nM of nucleic acid aptamer was added, and incubation was carried out on ice for 30 min, and then the cells were centrifuged at 800 g at room temperature for 3 min after gentle mixing up and down at intervals, the supernatant was discarded, and the cells were washed once with washing buffer, resuspended with an appropriate amount of washing buffer, and counted the dead and live cells with a cell counting plate to determine the cell viability and cell concentration, and 22,000 cells were taken for the next step of machine operation test. The nucleic acid aptamer used in this step is the nucleic acid aptamer of the nucleotide sequence disclosed in CN119506289A.

[0064] 3. Nucleic acid aptamer single cell high-throughput sequencing library construction and sequencing

[0065] The library construction and sequencing were carried out according to the DNBelab C series high-throughput single cell RNA library preparation kit provided by Huada and the Huada gene sequencer instruction manual.

[0066] Bioinformatics analysis shows that the total number of nucleic acid aptamers is as follows: Figure 1As shown, in liver cancer patient samples, the number of nucleic acid aptamers detected by this invention ranged from 500 to 1500. The number of nucleic acid aptamers identified varied among different patients, indicating significant heterogeneity in PBMC cell samples from different patients. Dimensionality reduction clustering is shown below. Figure 2 As shown, by integrating Unsupervised Dimensionality Reduction (UMAP) technology with unsupervised clustering algorithms to achieve data visualization and cell subpopulation identification, 17 immune cell subpopulations with significant transcriptomic differences were successfully identified, including CD14+ Monocytes, CD16+ Monocytes, Intermediate Cells, Dendritic Cells, Erythrocytes and Platelets, Naïve CD4+ T Cells, NKT Cells, Mucosa-Associated Invariant T Cells (MAIT T), Circulating T Cells, Plasma-1 Cells, Plasma-2 Cells, Plasma-3 Cells, and Circulating B Cells. This invention quantitatively analyzed the nucleic acid aptamers in each of the 17 cell subpopulations at the single-cell level. The distribution of nucleic acid aptamers is shown in the figure. Figure 3 As shown. The results of cell subsets and the expression of various classical genes are as follows. Figure 4 As shown, based on the expression of 27 classic cell markers, PMBC cells can be divided into 17 subpopulations, such as... Figure 4 As shown in Figure a, the 27 classic cellular markers are H MGB2, IGLC3, JCHAIN, MZB1, IGHG1, XBP1, MKI67, STMN1, MAGI2, NKG7, GZMB, GZMA, CD8A, CD3D, TCF7, LTB, PF4, PPBP, HBA1, HBD, CD1C, CLEC10A, CEBPD, FCN1, MS4A7, FCGR3A, CDKN1C, LYZ, S100A8, S100A12, and CD14; the expression of multiple classic genes in 17 cell subpopulations is as follows: Figure 4 As shown in Figure b, over 2000 aptamers were identified in a single cell at an incubation concentration of 200 nM. Based on the optimal incubation concentration, multiple aptamers that specifically bind to biliary tract cancer cell lines were identified, with the top three targets being CD49c, PTPRF, and alkaline phosphatase heterodimers. Figure 5In the nucleic acid aptamer binding condition in the single cell high-throughput sequencing of the PMBC of the liver cancer patient, the top three nucleic acid aptamers in 293 different nucleic acid aptamers are: Apt51, GR-30 and IBA. The protein target corresponding to Apt51 (the sequence is shown as SEQ ID NO. 9) is CD49c; the protein target corresponding to GR-30 (the sequence is shown as SEQ ID NO. 10) is PTPRF; and the protein target corresponding to IBA (the sequence is shown as SEQ ID NO. 11) is alkaline phosphatase heterodimer. First, the present application extends a domain at the end of the nucleic acid aptamer Apt51, GR-30 and IBA, respectively, and the probes are named as T1-Apt51, T2-GR-30 and T3-IBA. Then, for the above three domains, the present application designs the DNA sequences of cT1, cT2, cT3 and I according to the principle of base complementary pairing, and the four sequences are used to construct the "and" logic gate. The sequence of cT1 is shown as SEQ ID NO. 1, the sequence of cT2 is shown as SEQ ID NO. 2, the sequence of cT3 is shown as SEQ ID NO. 3, and the sequence of I is shown as SEQ ID NO. 4. The present application adopts a nuclease-free isothermal amplification technology, i.e. entropy-driven signal amplification, and designs the DNA sequences of S-II, W, F, F-Cy5, M1 and M1-Cy3. The sequence of S-II is shown as SEQ ID NO. 5, the sequence of W is shown as SEQ ID NO. 6, the sequence of F is shown as SEQ ID NO. 7, the sequence of F-Cy5 is TCCCGAGTGAGACTCGGTGG-GGTC-AACATTTCTCCAACTAACTTACGT-Cy5, the sequence of M1 is shown as SEQ ID NO. 8, and the sequence of M1-Cy3 is Cy3-ACGTAAGTTAGTTGGAGAAATGTT-GACCCCACCGAGTCTCACTCGGGAGTCAG. The specific sequences are shown in Table 1.

[0067] Table 1 DNA sequence table

[0068]

[0069] 4. Construction of DNA logic gate

[0070] The DNA sequences cT1, cT2, cT3 and I are annealed in 1×TE / Mg 2+ buffer to self-assemble to form double-stranded DNA logic gate. The 1×TE / Mg 2+ buffer contains 40mM Tris, 1mM EDTA and 5mM Mg 2+ . 2+The usage amount of the buffer is 1 μM: 1 mL, the molar ratio of cT2 to cT1 is 1:1, the molar ratio of cT3 to cT1 is 1:1, and the molar ratio of I to cT1 is 1:1

[0071] 5. Preparation of clinical samples

[0072] Human serum samples were collected at Zhejiang Cancer Hospital. The study has been approved by the Ethics Committee of Zhejiang Cancer Hospital, with the ethics number IRB-2024-26 (IIT), and in accordance with all relevant ethical guidelines. All samples (n = 20) were anonymous samples, and only gender, age, and pathological diagnosis information were recorded. In the validation experiment, the selected cohort patients (n = 10) were diagnosed with liver cancer, while the healthy donors (n = 10) had no history of cancer before sample collection.

[0073] 6. DNA logic computation on clinical samples

[0074] The extracted PBMCs were incubated with T1-Apt51, T2-GR-30, and T3-IBA at equal concentrations in 200 μL of 1x TE buffer at 37°C for 1 h. Subsequently, the excess aptamers were removed by washing with washing buffer solution for 3 times. Then, the treated solution was incubated with 1 μM double-stranded AND, 1 μM M strand, and 1.2 μM F strand in 200 μL of 1x TE / Mg 2+ buffer at 4°C for 30 min. Finally, the fluorescence signal was measured using a fluorescence spectrophotometer.

[0075] The present application conducted an in-depth analysis of serum samples of 10 liver cancer patients and 10 healthy donors, and the results are shown in FIG. 2a. The results showed that there was a significant statistical difference between the PMBCs of liver cancer patients and healthy donors (P < 0.05), as shown in FIG. 2b. When further analyzed using the receiver operating characteristic curve (ROC), the present application observed that the area under the curve (AUC) for distinguishing liver cancer patients and healthy individuals reached 0.82, as shown in FIG. 2c. By using a three-parameter input AND gate logic, the present application can effectively distinguish liver cancer patients and healthy donors. The research results of the present application have shown that the AND gate logic detection has great potential in the field of cancer diagnosis. Figure 6 Figure 6 Figure 6

[0076] Based on the above research results, the present application proposes the following design:

[0077] ​​​This invention, based on single-cell high-throughput sequencing results of nucleic acid aptamers in peripheral blood cells (PMBCs) from hepatocellular carcinoma (HCC) patients, employs a strategy of encoding membrane proteins specifically expressed on the surface of PMBCs using nucleic acid aptamers. This leads to the development of a logical diagnostic technique targeting multiple proteins on the surface of PMBCs from HCC patients. Nucleic acid aptamers specifically bind to the cell membrane surface to label the target proteins on the PMBCs, which are then used as input for an AND gate logical operation. Normal human PMBCs do not bear these labeled nucleic acid aptamers. More specifically, in the probe reagents of this invention, three selected nucleic acid aptamers target three specific membrane proteins on PMBCs from HCC patients, and each aptamer extends a specific fragment (T) from its terminal. These probes are named T1-Apt51, T2-GR-30, and T3-IBA, respectively. To achieve comprehensive analysis of specific membrane proteins on PMBCs from HCC patients, this invention designs and constructs an AND gate logical operation. Figure 7 As shown, its operation includes the following parts: When the target PMBC is present, T1-SYL3C targets and binds to EpCAM. The T1 domain at the probe tip hybridizes with the cT1 chain using an AND gate logic, activating the second-stage logic operation. Similarly, the second probe extension domain T2, bound to the cell membrane surface, hybridizes with cT2, activating the third-stage logic operation. The third probe extension domain T3, bound to the cell membrane surface, hybridizes with cT3, ultimately releasing the signal trigger chain I. Therefore, signal output is only triggered when all three target proteins are present simultaneously; otherwise, no signal output is achieved. To amplify the weak trigger signal in the reaction system, such as... Figure 8 As shown, this invention employs a nuclease-free isothermal amplification technique, namely entropy-driven signal amplification, to amplify nucleic acids from a trigger signal. This is a simple, low-cost, and efficient signal amplification method. Therefore, after the above three-input logic operations and signal amplification, the detection system outputs a fluorescence signal based on the disease state of PMBC in the patient's serum.

[0078] Example 1: A liver cancer diagnostic kit based on DNA logic gates combined with nucleic acid aptamer sequencing

[0079] The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extractants.

[0080] In this embodiment, the PBMC extractant includes a DPBS solution. The DPBS solution contains 2 wt% FBS.

[0081] DNA logic gate-based probe reagents: cT1, cT2, cT3, and I are mixed in TE / Mg 2+ Annealing in buffer solution yields double-stranded DNA based on DNA logic gates. TE / Mg 2+The buffer contains 40 mM Tris, TE / Mg 2+ The buffer contains 1 mM EDTA, TE / Mg 2+ The buffer contains 10 mM Mg 2+ cT1 and TE / Mg 2+ The use amount of the buffer is 1 μM: 1 mL, the molar ratio of cT2 to cT1 is 1:1, the molar ratio of cT3 to cT1 is 1:1, and the molar ratio of I to cT1 is 1:1.

[0082] Nuclease-free signal amplification reagent: S-II, W, F, F-Cy5, M1 and M1-Cy3 are mixed in a TE buffer to obtain a nuclease-free signal amplification reagent. The TE buffer contains 40 mM Tris, and the TE buffer contains 1 mM EDTA. The use amount of S-II to the TE buffer is 1 μM: 1 mL, the molar ratio of W to S-II is 1:1, the molar ratio of F to S-II is 1:1, the molar ratio of F-Cy5 to S-II is 1:1, the molar ratio of M1 to S-II is 1:1, and the molar ratio of M1-Cy3 to S-II is 1:1.

[0083] A diagnostic method based on aptamer single-cell sequencing combined with a DNA logic gate, comprising:

[0084] S1, centrifuge 20 mL of fresh whole blood, discard the upper plasma, then use DPBS solution to make up to 20 mL, mix well, then add the density gradient centrifugation liquid (Ficoll) through the central small hole of the SepMate™ insert into the SepMate™ test tube, the density gradient centrifugation liquid should be just submerged in the insert, centrifuge, take the white membrane layer, wash with 10 mL of DPBS solution, centrifuge to remove the supernatant, and obtain PBMC. In subsequent use, according to the cell density, add an appropriate amount of binding buffer solution to resuspend to obtain a PBMC liquid. The DPBS solution contains 2wt% FBS.

[0085] S2, add the probe reagent based on the DNA logic gate and the nuclease-free signal amplification reagent to the PBMC liquid, and stand. The volume ratio of the probe reagent based on the DNA logic gate to the PBMC liquid is 1:1, and the volume ratio of the nuclease-free signal amplification reagent to the probe reagent based on the DNA logic gate is 1:1.

[0086] Result (1): There is a fluorescent signal, which is positive for liver cancer.

[0087] Result (2): There is no fluorescent signal, which is negative for liver cancer.

[0088] The diagnostic method in the application has high specificity, the probe reagent based on the DNA logic gate designed by the AND gate logic reduces the false positive rate to below 5%, while the false positive rate of the traditional method is above 20%; the diagnostic method in the application has high sensitivity, more than 2000 aptamers per cell can be captured at the single cell level; the diagnostic method in the application can be used for non-invasive dynamic monitoring, based on peripheral blood PBMC, and the invasiveness of tissue biopsy is avoided; the diagnostic method in the application has cost advantage, compared with PCR amplification, the EDA technology reduces the signal amplification cost by 50%.

[0089] Embodiment 2: A liver cancer diagnostic kit combining DNA logic gate and aptamer sequencing

[0090] The diagnostic kit comprises a probe reagent based on a DNA logic gate, a nuclease-free signal amplification reagent and a PBMC extraction agent. The probe reagent and the nuclease-free signal amplification reagent of the AND gate logic in the embodiment are described in Embodiment 1.

[0091] The PBMC extraction agent in the embodiment comprises a DPBS solution. The DPBS solution contains 0.2wt% of FBS and 0.05wt% of nicotinoyl dimethylaminopropionyl hydrazine. The preparation of nicotinoyl dimethylaminopropionyl hydrazine comprises the preparation of nicotinoyl proline ester, the preparation of nicotinoyl proline hydrazide and the preparation of nicotinoyl dimethylaminopropionyl hydrazine.

[0092] Preparation of nicotinoyl proline ester: L-proline methyl ester hydrochloride is added into a saturated sodium bicarbonate solution, then a 6-methoxynicotinoyl chloride solution is added, and the reaction is carried out at 5℃ for 2h and then at 25℃ for 24h. After the reaction is completed, ethyl acetate is used for extraction, the organic layer is washed with a saturated sodium bicarbonate solution, dried, and the organic solvent is removed by rotary evaporation to obtain nicotinoyl proline ester. The use amount of L-proline methyl ester hydrochloride is 8.28wt% of the saturated sodium bicarbonate solution. The 6-methoxynicotinoyl chloride solution is obtained by mixing 6-methoxynicotinoyl chloride and dioxane, and the 6-methoxynicotinoyl chloride solution contains 8.58wt% of 6-methoxynicotinoyl chloride. The use amount of the 6-methoxynicotinoyl chloride solution is calculated based on the use amount of 6-methoxynicotinoyl chloride, and the use molar amount of 6-methoxynicotinoyl chloride is 100% of the use molar amount of L-proline methyl ester hydrochloride.

[0093] Preparation of nicotinoyl proline hydrazide: nicotinoyl proline ester is added into methanol, then hydrazine hydrate is added, and the reaction is carried out at 25℃ for 12h. The solvent methanol is removed by rotary evaporation, washed, and dried to obtain nicotinoyl proline hydrazide. The use amount of nicotinoyl proline ester is 8wt% of methanol, and the use amount of hydrazine hydrate is 3.6wt% of nicotinoyl proline ester. In the washing, chloroform and saturated brine are mixed for washing, and the organic layer is taken. In the drying, anhydrous magnesium sulfate is used to dry the organic layer, and the solvent is removed by rotary evaporation to obtain the product.

[0094] Preparation of nicotinoyl dimethylaminopropyl hydrazide: nicotinoyl proline hydrazide was added into chloroform, then (dimethylamino) acetyl chloride was added, and the mixture was treated at 25°C for 5h, and the solvent was removed by vacuum drying to obtain nicotinoyl dimethylaminopropyl hydrazide. The amount of nicotinoyl proline hydrazide used was 10wt% of chloroform, and the amount of (dimethylamino) acetyl chloride used was 32wt% of nicotinoyl proline hydrazide.

[0095] Example 3: A liver cancer diagnostic kit combined with DNA logic gate and aptamer sequencing

[0096] The diagnostic kit comprises probe reagents based on DNA logic gates, nuclease-free signal amplification reagents, and PBMC extraction reagents. The probe reagents and nuclease-free signal amplification reagents of the "and gate" logic in this embodiment are as described in Example 1.

[0097] The PBMC extraction reagent in this embodiment comprises a DPBS solution. The DPBS solution contains 0.2wt% FBS and 0.2wt% nicotinoyl dimethylaminopropyl hydrazide.

[0098] Example 4: A liver cancer diagnostic kit combined with DNA logic gate and aptamer sequencing

[0099] The diagnostic kit comprises probe reagents based on DNA logic gates, nuclease-free signal amplification reagents, and PBMC extraction reagents. The probe reagents and nuclease-free signal amplification reagents of the "and gate" logic in this embodiment are as described in Example 1.

[0100] The PBMC extraction reagent in this embodiment comprises a DPBS solution. The DPBS solution contains 0.2wt% FBS, 0.2wt% nicotinoyl dimethylaminopropyl hydrazide, and 0.03% cocamidopropyl hydroxysultaine.

[0101] Example 5: A liver cancer diagnostic kit combined with DNA logic gate and aptamer sequencing

[0102] The diagnostic kit comprises probe reagents based on DNA logic gates, nuclease-free signal amplification reagents, and PBMC extraction reagents. The probe reagents and nuclease-free signal amplification reagents of the "and gate" logic in this embodiment are as described in Example 1.

[0103] The PBMC extraction reagent in this embodiment comprises a DPBS solution. The DPBS solution contains 0.2wt% FBS, 0.2wt% nicotinoyl dimethylaminopropyl hydrazide, and 0.05wt% cocamidopropyl hydroxysultaine.

[0104] Comparative Example 1: A liver cancer diagnostic kit combined with DNA logic gate and aptamer sequencing

[0105] The diagnostic kit comprises a probe reagent based on a DNA logic gate, a nuclease-free signal amplification reagent, and a PBMC extraction agent. The probe reagent and the nuclease-free signal amplification reagent of the "and gate" logic in this embodiment are described in Embodiment 1.

[0106] The PBMC extraction agent in the present comparative example comprises a DPBS solution. The DPBS solution contains 0.2wt% FBS.

[0107] Comparative Example 2:

[0108] The diagnostic kit comprises a probe reagent based on a DNA logic gate, a nuclease-free signal amplification reagent, and a PBMC extraction agent. The probe reagent and the nuclease-free signal amplification reagent of the "and gate" logic in this embodiment are described in Embodiment 1.

[0109] The PBMC extraction agent in the present comparative example comprises a DPBS solution. The DPBS solution contains 0.2wt% FBS and 0.001wt% nicotinoyl dimethylamino proline hydrazide. The preparation of nicotinoyl dimethylamino proline hydrazide is described in Embodiment 2.

[0110] Embodiment 6: An intelligent diagnostic model based on nucleic acid aptamer single cell sequencing combined with a DNA logic gate

[0111] The PBMC cells are mixed with a probe reagent based on a DNA logic gate and a nuclease-free signal amplification reagent, the fluorescence signal is detected, and the results are output:

[0112] Result (1): There is a fluorescence signal, which is positive for liver cancer.

[0113] Result (2): There is no fluorescence signal, which is negative for liver cancer.

[0114] Embodiment 7: An intelligent diagnostic model based on nucleic acid aptamer single cell sequencing combined with a DNA logic gate

[0115] PBMCs are extracted using a PBMC extraction agent, and the PBMCs are mixed with a probe reagent based on a DNA logic gate and a nuclease-free signal amplification reagent, the fluorescence signal is detected, and the results are output:

[0116] Result (1): There is a fluorescence signal, which is positive for liver cancer.

[0117] Result (2): There is no fluorescence signal, which is negative for liver cancer.

[0118] Test Example:

[0119] 1. Infrared characterization of nicotinoyl dimethylamino proline hydrazide

[0120] The nicotinoyl dimethylamino proline hydrazide prepared in Embodiment 2 was subjected to infrared characterization, and the results are as follows Figure 9shown, where the peak at 3228 cm -1 is the absorption peak of the hydrogen bonded to nitrogen, the peak at 3040 cm -1 is the absorption peak of the unsaturated carbon-hydrogen in the nicotinoyl group, the peak at 2932 cm -1 is the absorption peak of the carbon-hydrogen on the methyl group, the peak at 2848 cm -1 is the absorption peak of the carbon-hydrogen on the methylene group, the peak at 1702 cm -1 is the absorption peak of the carbon-oxygen double bond, the peak at 1042 cm -1 is the absorption peak of the carbon-oxygen-carbon.

[0121] 2. Cytotoxicity test

[0122] A test group was set up, nicotinoyl dimethylaminyl prolinyl hydrazide in Example 2 was added into DMEM culture medium, the DMEM culture medium contained 0.05wt% of nicotinoyl dimethylaminyl prolinyl hydrazide and 2wt% of FBS, then 100μL of the DMEM culture medium was added into a 96-well plate, 1×10 6 mouse fibroblasts L-929 were inoculated into the 96-well plate, and the plate was cultured in a 37℃, 5% CO2 incubator for 24h. A control group was set up, the DMEM culture medium in the control group did not contain nicotinoyl dimethylaminyl prolinyl hydrazide, and the other conditions were the same as those of the test group.

[0123] The survival rate of mouse fibroblasts L-929 was detected by the CCK-8 method, and the absorbance at 450nm was measured by an enzyme marker, and the cell survival rate was calculated.

[0124] The survival rate of the test group relative to the control group in the present application was 99.6%, indicating that the nicotinoyl dimethylaminyl prolinyl hydrazide in the present application had no cytotoxicity.

[0125] 3. Cell viability

[0126] The PBMCs obtained from S1 in Examples 1-5 and Comparative Examples 1-2 were subjected to cell survival rate test, the PBMCs obtained from S1 were resuspended in an appropriate amount of binding buffer solution, and dead and live cell counting was performed by using a cell counting plate to determine the cell viability.

[0127] The application first selects three terminal extension (T) nucleic acid aptamers, which are respectively targeted to three specific membrane proteins of liver cancer PMBC, and are respectively named T1-Apt51, T2-GR-30 and T3-IBA. Based on the coupling of T1-Apt51, T2-GR-30 and T3-IBA with nucleic acid aptamers, a double-stranded DNA based on a DNA logic gate composed of cT1, cT2, cT35 and I is prepared. Then, a nuclease-free signal amplification reagent containing S-II, W, F, F-Cy5, M1 and M1-Cy3 is prepared based on entropy-driven amplification (EDA). After PBMC is extracted from whole blood, whether T1-Apt51, T2-GR-30 and T3-IBA are present on PBMC can be detected. The double-stranded DNA based on the DNA logic gate of the application can only release the signal chain I when T1-Apt51, T2-GR-30 and T3-IBA are present on PBMC at the same time, and further form fluorescence under the action of the nuclease-free signal amplification reagent, so that healthy people and cancer patients can be effectively distinguished. The cell viability of PBMC can ensure more accurate detection results. In the application, the PBMC extraction agent is DPBS solution. In the DPBS solution of the application, FBS and / or nicotinoyl dimethylaminopropyl proline hydrazide can be added. Nicotinoyl dimethylaminopropyl proline hydrazide is prepared by reacting L-proline methyl ester hydrochloride and 6-methoxynicotinoyl chloride to form nicotinoyl proline ester, then reacting with hydrazine hydrate to form nicotinoyl proline hydrazide, and finally reacting nicotinoyl proline hydrazide with (dimethylamino) acetyl chloride to form nicotinoyl dimethylaminopropyl proline hydrazide. In the application, the cell viability is as shown in Figure 10 When the PBMC is extracted using the DPBS solution containing FBS, the obtained PBMC has good cell viability. If the content of FBS in the DPBS solution containing FBS is low, the cell viability of the obtained PBMC is poor. In the application, when the PBMC is extracted using the DPBS solution containing low usage amount of FBS and nicotinoyl dimethylaminopropyl proline hydrazide, the cell viability of the obtained PBMC is higher. The increase of the amount of nicotinoyl dimethylaminopropyl proline hydrazide in the suitable usage range can improve the cell viability of the obtained PBMC. If the usage amount of nicotinoyl dimethylaminopropyl proline hydrazide is too low, the cell viability of the obtained PBMC is poor. Further, in the application, the DPBS solution containing low usage amount of FBS, nicotinoyl dimethylaminopropyl proline hydrazide and cocamidopropyl hydroxysultaine can also be used for PBMC extraction. The use of cocamidopropyl hydroxysultaine, low usage amount of FBS and nicotinoyl dimethylaminopropyl proline hydrazide can further improve the cell viability of PBMC.

[0128] The above-described embodiments and / or implementations are merely intended to illustrate the preferred embodiments and / or implementations of the present technology, and are not intended to limit the embodiments of the present technology in any form, and any person skilled in the art can make some changes or modifications as other equivalent embodiments without departing from the scope of the technology disclosed in the present disclosure, but should be considered as the same technology or embodiments as the present disclosure.

[0129] The principles and implementations of the present application are described herein using specific examples. The above description of the embodiments is only intended to help understand the method and its core idea. The above description is only the preferred embodiments of the present application. It should be noted that due to the limited nature of the language, there are objectively infinite specific structures. For those skilled in the art, without departing from the principles of the present application, some improvements, refinements or changes can be made, and the above technical features can be combined in an appropriate manner. These improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, shall be considered as the protection scope of the present application.

Claims

1. A hepatocellular carcinoma early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA "and gate" logic operation, comprising: The probe reagent based on DNA logic operation and the nuclease-free signal amplification reagent and T1-Apt51, T2-GR-30 and T3-IBA, wherein the probe reagent comprises nucleic acid sequences shown as SEQ ID NO. 1-4; T1-Apt51, T2-GR-30 and T3-IBA are respectively formed by extending a domain from the end of aptamer Apt51, GR-30 and IBA, and the DNA sequences of cT1, cT2, cT3 and I are designed by using the principle of base complementary pairing, wherein the sequence of cT1 is shown as SEQ ID NO. 1, the sequence of cT2 is shown as SEQ ID NO. 2, the sequence of cT3 is shown as SEQ ID NO. 3, and the sequence of I is shown as SEQ ID NO. 4; the sequence of Apt51 is shown as SEQ ID NO. 9, the sequence of GR-30 is shown as SEQ ID NO. 10, and the sequence of IBA is shown as SEQ ID NO.

11. The nuclease-free signal amplification reagent comprises nucleic acid sequences shown as SEQ ID NO. 5-8, and further comprises F-Cy5 and M1-Cy3; the sequence of F-Cy5 is TCCCGAGTGAGACTCGGTGG-GGTC-AACATTTCTCCAACTAACTTACGT-Cy5, and the sequence of M1-Cy3 is Cy3-ACGTAAGTTAGTTGGAGAAATGTT-GACCCCACCGAGTCTCACTCGGGAGTCAG.

2. The early screening kit for liver cancer based on nucleic acid aptamer single cell high-throughput sequencing combined with DNA "and gate" logic operation according to claim 1, characterized in that: The probe reagent further comprises a buffer; or the nuclease-free signal amplification reagent comprises a buffer. 3.The early screening kit for liver cancer based on nucleic acid aptamer single cell high-throughput sequencing combined with DNA "and gate" logic operation according to claim 1, characterized in that: The kit further comprises a PBMC extraction agent, and the PBMC extraction agent is a DPBS solution.

4. The early screening kit for liver cancer based on nucleic acid aptamer single cell high-throughput sequencing combined with DNA "and gate" logic operation according to claim 3, characterized in that: The DPBS solution contains FBS and / or nicotinoyl dimethylaminopropionyl hydrazine; the nicotinoyl dimethylaminopropionyl hydrazine is prepared from L-proline methyl ester hydrochloride, 6-methoxynicotinoyl chloride, hydrazine hydrate and (dimethylamino) acetyl chloride.

5. The early screening kit for liver cancer based on nucleic acid aptamer single cell high-throughput sequencing combined with DNA "and gate" logic operation according to claim 4, characterized in that: The DPBS solution contains 0.03-0.5wt% of nicotinoyl dimethylaminopropionyl hydrazine; or the DPBS solution contains 0.1-10wt% of FBS.

6. The early screening kit for liver cancer based on nucleic acid aptamer single cell high-throughput sequencing combined with DNA "and gate" logic operation according to claim 5, characterized in that: In the preparation of the nicotinoyl dimethylaminopropionyl hydrazine, after the reaction of L-proline methyl ester hydrochloride and 6-methoxynicotinoyl chloride to prepare nicotinoyl proline ester, the reaction of the nicotinoyl proline ester with hydrazine hydrate to prepare nicotinoyl proline hydrazide, and finally the reaction of the nicotinoyl proline hydrazide with (dimethylamino) acetyl chloride to prepare the nicotinoyl dimethylaminopropionyl hydrazine.

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