Liver cancer diagnostic kit for sequencing by combining DNA logic gate with aptamer

Abstract

The invention discloses a liver cancer diagnostic kit for sequencing by combining a DNA logic gate with an aptamer, belongs to the field of diagnostic kits, and particularly relates to an AND gate logic operation double strand consisting 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 the PBMC is extracted from whole blood, the kit can be used for logically analyzing whether target proteins corresponding to the aptamers Apt51, GR-30 and IBA exist on the PBMC at the same time or not, so that early screening of the liver cancer is realized. A specific target of a liver cancer patient is found through aptamer single-cell high-throughput sequencing, and the kit can effectively distinguish healthy people from cancer patients.

Description

Technical Field

[0001] This invention belongs to the field of diagnostic reagent kits, specifically relating to a liver cancer diagnostic reagent kit based on DNA logic gates combined with nucleic acid aptamer sequencing. Background Technology

[0002] Liver cancer, or malignant tumor of the liver, can be divided into two main categories: primary and secondary. Primary malignant liver tumors originate from the epithelial or mesenchymal tissue of the liver; the former is called primary liver cancer, which is a highly prevalent and extremely dangerous malignant tumor; the latter is called sarcoma, which is less common compared to primary liver cancer. Secondary or metastatic liver cancer refers to malignant tumors originating from multiple organs throughout the body that invade the liver. It is commonly seen as liver metastasis from malignant tumors of the stomach, bile ducts, pancreas, colorectal region, ovary, uterus, lung, and breast.

[0003] Limitations of Current Technology: Liver cancer is one of the top three causes of cancer death worldwide, and early screening and diagnosis are crucial for improving patient survival. However, current routine clinical diagnostic methods have significant limitations: First, the sensitivity of serum markers such as alpha-fetoprotein (AFP) is low, around 40%-60%, and is easily affected by benign lesions such as hepatitis / cirrhosis; second, imaging techniques such as ultrasound and CT / MRI have insufficient resolution for early lesions smaller than 1 cm and cannot provide molecular pathological information; third, tissue biopsy is invasive and makes dynamic monitoring and heterogeneity analysis difficult.

[0004] Therefore, there is an urgent need to develop non-invasive liquid biopsy technology based on peripheral blood to achieve early detection and accurate molecular subtyping of liver cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a convenient, flexible, highly sensitive, specific, and non-invasive liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operations.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operation includes: a probe reagent based on DNA logic operation and a nuclease-free signal amplification reagent. The probe reagent includes nucleic acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.4.

[0007] In the probe reagent, the sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 are used in a molar ratio of 1:0.5-2:0.5-2:0.5-2.

[0008] This invention utilizes high-throughput sequencing of nucleic acid aptamers to simultaneously identify multiple highly expressed, liver cancer-specific proteins in liver cell carcinoma-associated macrophages (PBMCs). Subsequent comprehensive analysis using DNA logic operations effectively distinguishes between healthy individuals and cancer patients. Compared to traditional methods based on single nucleic acid aptamer detection, this invention's multiplex detection method significantly improves the accuracy and anti-interference capability for identifying PMBC surface proteins. Due to its excellent sensitivity, specificity, and scalability, this method provides a new approach for accurately identifying and analyzing PMBC surface proteins in liver cancer patients. Furthermore, this multi-parameter input logic diagnostic method is not limited to early liver cancer screening but can also be extended to the early screening of other malignant tumors and diseases, making it widely applicable in early clinical diagnosis. Based on the programmability of DNA, this invention designs logic gates that directly output signals through logic calculations, thereby simplifying the data processing process and making the signals more intuitive.

[0009] Preferably, the nuclease-free signal amplification reagent includes any one or more nucleic acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.8.

[0010] More preferably, the nuclease-free signal amplification reagent further includes F-Cy5 and M1-Cy3, wherein the sequence of F is as shown in SEQ ID NO.7, and F-Cy5 is a Cy5 group attached to the 3' end of the F sequence; the sequence of M1 is as shown in SEQ ID NO.8, and M1-Cy3 is a Cy3 group attached to the 5' end of the M1 sequence.

[0011] More preferably, in the nuclease-free signal amplification reagent, the sequences SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 are used in a molar ratio of 1:0.5-2:0.5-2:0.5-2.

[0012] More preferably, in the nuclease-free signal amplification reagent, the SEQ ID NO.5 sequence, F-Cy5, and M1-Cy3 are used in a molar ratio of 1:0.5-2:0.5-2.

[0013] Preferably, the probe reagent is formed by base complementary pairing of the nucleic acid sequences shown in SEQ ID NO.1 to SEQ ID NO.4.

[0014] Preferably, the probe reagent further includes a buffer solution; or, the nuclease-free signal amplification reagent includes a buffer solution.

[0015] More preferably, the buffer solution is a TE buffer solution.

[0016] Preferably, the kit also includes a PBMC extraction agent, which is a DPBS solution. PBMCs are peripheral blood mononuclear cells.

[0017] More preferably, the DPBS solution contains FBS and / or nicotinyl dimethylaminoproline hydrazide; nicotinyl dimethylaminoproline hydrazide is prepared by reacting L-proline methyl ester hydrochloride, 6-methoxynicotinyl chloride, hydrazine hydrate, and (dimethylamino)acetyl chloride. The DNA logic-based probe reagent of the present invention only releases signal chain I when T1-Apt51, T2-GR-30, and T3-IBA are simultaneously present on PBMCs, further generating fluorescence under the action of nuclease-free signal amplification reagents, which can effectively distinguish between healthy individuals and cancer patients. The cell viability of PBMCs can ensure more accurate detection results. In this invention, the PBMC extraction agent is DPBS solution. FBS and / or nicotinyldimethylaminoproline hydrazide can be added to the DPBS solution. When only a small amount of FBS or nicotinyldimethylaminoproline hydrazide is added, the cell viability of PBMCs is low, which affects the detection effect. However, by adding an appropriate amount of FBS or nicotinyldimethylaminoproline hydrazide to the DPBS solution, the cell viability of PBMCs can be improved.

[0018] Preferably, the DPBS solution contains 0.03-0.5 wt% nicotinyl dimethylaminoproline hydrazide; or, the DPBS solution contains 0.1-10 wt% FBS.

[0019] More preferably, in the preparation of nicotinyl dimethylaminoproline hydrazide, nicotinyl proline ester is prepared by reacting L-proline methyl ester hydrochloride and 6-methoxynicotinyl chloride, and then reacted with hydrazine hydrate to generate nicotinyl proline hydrazide. Finally, nicotinyl proline hydrazide is reacted with (dimethylamino)acetyl chloride to generate nicotinyl dimethylaminoproline hydrazide.

[0020] More preferably, in the preparation of nicotinic acid proline ester, L-proline methyl ester hydrochloride is added to a saturated sodium bicarbonate solution, followed by the addition of 6-methoxynicotinic acid chloride solution. The reaction is carried out at 0-10°C for 1-4 h, and then at 20-40°C for 12-48 h. After the reaction is completed, the mixture is extracted with ethyl acetate, the organic layer is washed with a saturated sodium bicarbonate solution, dried, and the organic solvent is removed by rotary evaporation to obtain nicotinic acid proline ester.

[0021] More preferably, in the preparation of nicotinylproline ester, the amount of L-proline methyl ester hydrochloride used is 4-16 wt% of the saturated sodium bicarbonate solution, and the 6-methoxynicotinyl chloride solution is a mixture of 6-methoxynicotinyl chloride and dioxane, containing 2-20 wt% 6-methoxynicotinyl chloride; the amount of 6-methoxynicotinyl chloride solution used is measured by 6-methoxynicotinyl chloride, and the molar amount of 6-methoxynicotinyl chloride used is 80-150% of the molar amount of L-proline methyl ester hydrochloride used.

[0022] More preferably, in the preparation of nicotinylproline hydrazide, nicotinylproline ester is added to methanol, and then hydrazine hydrate is added. The mixture is reacted at 20-40°C for 8-24 hours. The solvent methanol is removed by rotary evaporation, and the mixture is washed and dried to obtain nicotinylproline hydrazide.

[0023] More preferably, in the preparation of nicotinylproline hydrazide, the amount of nicotinylproline ester used is 3-15 wt% of methanol, and the amount of hydrazine hydrate used is 2-8 wt% of nicotinylproline ester. During washing, a mixture of chloroform and saturated brine is used, and the organic layer is separated. During drying, the organic layer is dried with anhydrous magnesium sulfate, and the solvent is removed by rotary evaporation to obtain the product.

[0024] More preferably, in the preparation of nicotinyl dimethylaminoproline hydrazide, nicotinyl proline hydrazide is added to chloroform, then (dimethylamino)acetyl chloride is added, and the mixture is treated at 20-40°C for 2-10 h. The solvent is removed by vacuum drying to obtain nicotinyl dimethylaminoproline hydrazide.

[0025] More preferably, in the preparation of nicotinyl dimethylaminoproline hydrazide, the amount of nicotinyl proline hydrazide used is 5-20 wt% of chloroform, and the amount of (dimethylamino)acetyl chloride used is 20-40 wt% of nicotinyl proline hydrazide.

[0026] This invention discloses an early liver cancer screening model based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operations, comprising: Mix PBMC with DNA logic-based probe reagents and nuclease-free signal amplification reagents, detect the fluorescence signal, and output any one of results (1) to (2) based on the detection results: Result (1): The presence of fluorescent signal indicates a positive result for liver cancer; Result (2): No fluorescence signal indicates negative result for liver cancer.

[0027] Preferably, PBMC is obtained by extraction with a PBMC extractant, which is a DPBS solution.

[0028] This invention discloses the use of nucleic acid aptamers Apt51, GR-30, and IBA, and their corresponding target proteins, as early screening targets for liver cancer.

[0029] The nucleic acid aptamers used in this invention are short-chain oligonucleotides screened using SELEX technology. They can specifically bind to target molecules, such as liver cancer-related membrane proteins like GPC3 and ASGPR1. Compared to traditional antibodies, they have the following advantages: high affinity and flexible chemical modification, supporting multiple target parallel labeling, with Kd reaching the nM level; programmability: functional DNA sequences can be attached to the aptamer ends to achieve integrated "recognition-signal conversion" design.

[0030] Nucleic acid aptamers are oligonucleotide sequences approximately 15-60 nt in length. They possess a range of advantages, including broad target scope, high affinity, strong specificity, and ease of synthesis. They can precisely recognize metal ions, small molecules, amino acids, peptides, proteins, and cells, earning them the title of "chemists' antibodies." As a class of recognition molecules, nucleic acid aptamers are used in molecular medicine to identify disease biomarkers and diseased cells, making them suitable for clinical applications as molecular probes and therapeutic drugs.

[0031] A key advantage of nucleic acid aptamers compared to other molecular probes is their nucleic acid composition, which enables them to perform complex DNA computations. Through DNA strand substitution reactions, nucleic acid aptamers can perform biomolecular computations using various Boolean logic gates, processing multiple input signals into a single output. This allows for the accurate detection of multiple biomarkers in single cells, thereby improving the sensitivity and specificity of cancer diagnosis. In vitro screening, also known as SELEX (Systematic Evolutionary Index Enrichment), uses DNA / RNA oligonucleotide sequences generated through in vitro screening. These sequences possess high affinity and specificity, allowing them to bind to various targets such as proteins and small molecules. As a powerful molecular recognition tool, nucleic acid aptamers have broad application prospects in molecular diagnostics, bioimaging, targeted drug delivery, and biomarker discovery. This computational capability of DNA provides new avenues for disease diagnosis and treatment, such as pattern recognition, DNA robotics, and DNA molecular networks.

[0032] 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.

[0033] This invention discloses the potential and integration needs of DNA molecular computing: 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. 1. Multiple logic judgments: Through the "AND gate" design, the signal is triggered only when ≥2 markers are identified simultaneously, significantly improving specificity; 2. Signal amplification capability: Cascade hybridization chain reaction (HCR) or catalytic hairpin assembly (CHA) can convert single-molecule events into detectable macroscopic signals; 3. Microfluidic integration: The DNA computing unit can be combined with a microfluidic chip to achieve automated single-cell manipulation and parallel analysis.

[0034] This invention employs nucleic acid aptamer-specific labeling technology and constructs a liver cancer-targeted aptamer library. High-affinity aptamers targeting liver cancer cell surface markers such as GPC3, ASGPR1, and CD44v6, as well as intracellular metabolites such as ATP, are screened using SELEX technology, with a Kd value less than or equal to 2 nM. Phosphothioester modification is also performed to enhance nuclease resistance. Furthermore, this invention utilizes aptamer-sequencing probe coupling, introducing the sequencing adapter Read2 sequence and a unique molecular identifier (UMI) at the 3' end of the aptamer to achieve direct library construction and sequencing based on aptamer binding signals at the single-cell level.

[0035] This invention combines the highly specific targeting recognition capability of nucleic acid aptamers with the intelligent signal processing capability of DNA molecular logic operations, realizing an integrated "recognition-encoding-computation" operation on the surface of a single cell. This overcomes the limitations of traditional stepwise detection, which requires capture before analysis, and significantly improves detection efficiency.

[0036] In this invention, cocamidopropyl hydroxysulfonate betaine can be added to the DPBS solution used for extracting PBMCs. The content of cocamidopropyl hydroxysulfonate betaine in the DPBS solution is 0.01-0.1 wt%. The DPBS solution contains appropriate amounts of FBS and nicotinyl dimethylaminoproline hydrazide, and cocamidopropyl hydroxysulfonate betaine works synergistically with them to further improve the cell viability of PBMCs.

[0037] This invention employs a DNA logic-based probe reagent 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 EDA amplification. After extracting PBMCs from whole blood, it can detect whether PBMCs simultaneously possess T1-Apt51, T2-GR-30, and T3-IBA. The DNA logic-based probe reagent of this invention only releases signal chain I when PBMCs simultaneously possess T1-Apt51, T2-GR-30, and T3-IBA, further generating fluorescence under the action of the nuclease-free signal amplification reagent, effectively distinguishing healthy individuals from cancer patients. This invention has the following advantages: in vitro detection, high sensitivity, good detection effect, signal amplification, and non-invasiveness.

[0038] 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

[0039] Figure 1 This is a graph showing the total number of nucleic acid aptamers.

[0040] Figure 2 This is a dimensionality-reduced clustering group diagram.

[0041] Figure 3 This is a distribution map of nucleic acid aptamers.

[0042] Figure 4 This diagram shows the expression of cell subpopulations and various classic genes.

[0043] Figure 5 This figure shows the aptamer binding status in single-cell high-throughput sequencing of PMBC in liver cancer patients.

[0044] 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.

[0045] Figure 7 This is a schematic diagram of logic diagnosis based on DNA AND gate logic operation.

[0046] Figure 8 This is a schematic diagram of the AND gate logic operation section and signal amplification in the kit.

[0047] Figure 9 This is an infrared spectrum.

[0048] Figure 10 This is a cell viability graph. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] The research basis of this invention is as follows: 1. Isolation of PBMCs from blood samples Centrifuge fresh whole blood in an anticoagulant tube at 3000 rpm for 10 min, discard the supernatant plasma, and add 2% FBS / DPBS to a final volume of 20 mL. Gently invert to mix, and then slowly pipette the diluted sample into a SepMate™ tube containing Ficoll (density gradient buffer) passed through the central well of the SepMate™ insert. The Ficoll buffer should completely submerge the insert. Centrifuge at 1200 g for 10 min at room temperature. For samples stored for more than 24 hours, a centrifugation time of 20 min is recommended. Immediately transfer the white film layer (rich in PBMCs) to a new tube and add 10 mL of 2% FBS. Wash PBMCs with DPBS, centrifuge at 300g for 8 min at room temperature, and discard the supernatant. If the precipitate is light pink, erythrocyte lysis is not necessary; if it is dark red, erythrocyte lysis is required: add 1×DPBS and erythrocyte lysis buffer (Solepro) at a ratio of 1:3, gently mix with pipette tip, incubate on ice for 15 min, gently inverting the container periodically to mix, centrifuge at 500g for 5 min at 4°C, and discard the supernatant; resuspend the cells with an appropriate amount of binding buffer according to the cell density, and count the live and dead cells using a cell counting chamber to determine cell viability and cell concentration.

[0052] 2. Nucleic acid aptamer incubation and loading. Based on the concentration determination in the previous step, 1 million cells were placed in 400 μL of binding buffer, and 200 nM of nucleic acid aptamer was added. The mixture was incubated on ice for 30 min, with gentle inversion and mixing at intervals. After incubation, the cells were centrifuged at 800 g for 3 min at room temperature, the supernatant was discarded, and the cells were washed once with wash buffer. The cells were resuspended in an appropriate amount of wash buffer, and a cell counting chamber was used to determine cell viability and concentration. 22,000 cells were then used for the next step of instrumental analysis. The nucleic acid aptamer used in this step was the one with the nucleotide sequence disclosed in CN119506289A.

[0053] 3. Nucleic acid aptamer single-cell high-throughput sequencing library preparation and sequencing Library construction and sequencing were performed according to the DNBelab C series high-throughput single-cell RNA library preparation kit and the BGI sequencer instruction manual provided by BGI.

[0054] Bioinformatics analysis revealed that the total number of nucleic acid aptamers was as follows: Figure 1 As 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 2As 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 high-throughput sequencing of single-cell PBCs from liver cancer patients, the binding of nucleic acid aptamers was analyzed. Among 293 different nucleic acid aptamers, the top three were Apt51, GR-30, and IBA. Apt51 (sequence shown in SEQ ID NO. 9) targets CD49c; GR-30 (sequence shown in SEQ ID NO. 10) targets PTPRF; and IBA (sequence shown in SEQ ID NO. 11) targets alkaline phosphatase heterodimer. First, this invention extends a domain from the ends of the nucleic acid aptamers Apt51, GR-30, and IBA, respectively, and names these probes as T1-Apt51, T2-GR-30, and T3-IBA. Subsequently, using the base pairing principle, this invention designs DNA sequences cT1, cT2, cT3, and I for these three domains. These four sequences are used to construct AND logic gates. The sequences of cT1, cT2, and cT3 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and I, respectively. This invention employs a nuclease-free isothermal amplification technique, i.e., entropy-driven signal amplification, and designs DNA sequences for S-II, W, F, F-Cy5, M1, and M1-Cy3. The sequence of S-II is shown in SEQ ID NO.5, the sequence of W in SEQ ID NO.6, the sequence of F in SEQ ID NO.7, the sequence of F-Cy5 is TCCCGAGTGAGACTCGGTGG-GGTC-AACATTTCTCCAACTAACTTACGT-Cy5, the sequence of M1 in SEQ ID NO.8, and the sequence of M1-Cy3 is Cy3-ACGTAAGTTAGTTGGAGAAATGTT-GACCCCACCGAGTCTCACTCGGGAGTCAG. Specific sequences are shown in Table 1.

[0055] Table 1 DNA Sequence Listing

[0056] 4. Construction of DNA logic gates DNA sequences cT1, cT2, cT3, and I were stored in 1×TE / Mg. 2+ Annealing is performed in a buffer solution, and the DNA self-assembles to form double-stranded logic gates. The buffer solution contains 1×TE / Mg. 2+ The buffer solution contains 40 mM Tris, 1 mM EDTA and 5 mM Mg 2+ cT1 and TE / Mg 2+The buffer solutions used were in the following ratios: 1 μM : 1 mL, cT2 to cT1 molar ratio was 1:1, cT3 to cT1 molar ratio was 1:1, and I to cT1 molar ratio was 1:1. 5. Preparation of clinical samples Human serum samples were collected from Zhejiang Cancer Hospital. This study was approved by the Ethics Committee of Zhejiang Cancer Hospital (Ethics No. IRB-2024-26(IIT)) and complied with all relevant ethical guidelines. All samples (n=20) were anonymized, with only sex, age, and pathological diagnosis information recorded. In the validation experiment, the selected cohort of patients (n=10) were diagnosed with liver cancer, while the healthy donors (n=10) had no history of cancer prior to sample collection.

[0057] 6. Perform DNA logical calculations on clinical samples The extracted PBMCs were incubated with T1-Apt51, T2-GR-30, and T3-IBA at equal concentrations in 200 μL of 1×TE buffer at 37 °C for 1 h. Subsequently, the mixture was washed three times with washing buffer to remove excess aptamers. Next, the treated solution was incubated with 1 μM double-stranded AND, 1 μM M-stranded M-stranded M-stranded M-stranded M-stranded F ... M-stranded M-stranded M-stranded M-stranded M-stranded M-stranded M-str 2+ Incubate in buffer for 30 min. Finally, measure the fluorescence signal using a fluorescence spectrophotometer.

[0058] This invention conducted an in-depth analysis of serum samples from 10 liver cancer patients and 10 healthy donors, and the results are as follows: Figure 6 As shown. The results indicated that, based on fluorescence signal detection, there was a statistically significant difference in PMBC between liver cancer patients and healthy donors (P<0.05), as shown. Figure 6 As shown in Figure a. In further analysis using receiver operating characteristic (ROC) curves, this invention observed that the area under the curve (AUC) distinguishing liver cancer patients from healthy individuals reached 0.82, as shown in Figure a. Figure 6 As shown in Figure b, by using a three-parameter input AND gate logic, this invention can effectively distinguish between liver cancer patients and healthy donors. The results of this invention have demonstrated the great potential of AND gate logic detection in the field of cancer diagnosis.

[0059] Based on the above research results, the present invention proposes the following design: 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.

[0060] Example 1: A liver cancer diagnostic kit based on DNA logic gates combined with nucleic acid aptamer sequencing The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extractants.

[0061] In this embodiment, the PBMC extractant includes a DPBS solution. The DPBS solution contains 2 wt% FBS.

[0062] 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 / Mg2+ The buffer contains 1 mM EDTA, TE / Mg 2+ The buffer solution contains 10 mM Mg 2+ cT1 and TE / Mg 2+ The buffer solution was used in the following ratios: 1 μM: 1 mL, cT2 to cT1 was 1:1, cT3 to cT1 was 1:1, and I to cT1 was 1:1.

[0063] Nuclease-free signal amplification reagent: Mix S-II, W, F, F-Cy5, M1, and M1-Cy3 in TE buffer to obtain the nuclease-free signal amplification reagent. The TE buffer contains 40 mM Tris and 1 mM EDTA. The ratio of S-II to 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.

[0064] A diagnostic method based on nucleic acid aptamer single-cell sequencing combined with DNA logic gates, comprising: S1: Centrifuge 20 mL of fresh whole blood, discard the supernatant plasma, then bring the volume to 20 mL with DPBS solution. Mix well and add to a SepMate™ tube containing Ficoll density gradient buffer passed through the central well of the SepMate™ insert. The density gradient buffer should completely submerge the insert. Centrifuge, collect the white membrane layer, wash with 10 mL of DPBS solution, centrifuge again to remove the supernatant, and obtain PBMCs. For subsequent use, resuspend the cells in an appropriate amount of binding buffer solution according to the cell density to obtain PBMC solution. The DPBS solution contains 2 wt% FBS.

[0065] S2, add the DNA logic gate-based probe reagent and the nuclease-free signal amplification reagent to the PBMC solution and let it stand. The volume ratio of the DNA logic gate-based probe reagent to the PBMC solution is 1:1, and the volume ratio of the nuclease-free signal amplification reagent to the DNA logic gate-based probe reagent is 1:1.

[0066] Result (1): If there is a fluorescent signal, it indicates a positive result for liver cancer.

[0067] Result (2): No fluorescence signal indicates negative result for liver cancer.

[0068] The diagnostic method described in this invention has high specificity. The DNA logic gate-based probe reagent, designed with "AND" logic, reduces the false positive rate to below 5%, while traditional methods exceed 20%. The diagnostic method of this invention has high sensitivity, capable of capturing more than 2000 aptamers / cells at the single-cell level. The diagnostic method of this invention can perform non-invasive dynamic monitoring based on peripheral blood PBMCs, avoiding the invasiveness of tissue biopsy. The diagnostic method of this invention has a cost advantage; compared with PCR amplification, EDA technology reduces signal amplification costs by 50%.

[0069] Example 2: A liver cancer diagnostic kit based on DNA logic gates combined with nucleic acid aptamer sequencing The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extraction reagents. The AND gate-based probe reagents and nuclease-free signal amplification reagents used in this embodiment are described in Example 1.

[0070] In this embodiment, the PBMC extractant includes a DPBS solution. The DPBS solution contains 0.2 wt% FBS and 0.05 wt% nicotinyl dimethylaminoproline hydrazide. The preparation of nicotinyl dimethylaminoproline hydrazide includes the preparation of nicotinyl proline ester, the preparation of nicotinyl proline hydrazide, and the preparation of nicotinyl dimethylaminoproline hydrazide.

[0071] Preparation of nicotinylproline ester: L-proline methyl ester hydrochloride was added to a saturated sodium bicarbonate solution, followed by the addition of 6-methoxynicotinic acid chloride solution. The reaction was carried out at 5°C for 2 hours, and then at 25°C for 24 hours. After the reaction was completed, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate solution, dried, and the organic solvent was removed by rotary evaporation to obtain nicotinylproline ester. The amount of L-proline methyl ester hydrochloride used was 8.28 wt% of the saturated sodium bicarbonate solution. The 6-methoxynicotinic acid chloride solution was a mixture of 6-methoxynicotinic acid chloride and dioxane, containing 8.58 wt% 6-methoxynicotinic acid chloride. The amount of 6-methoxynicotinic acid chloride solution used was measured by 6-methoxynicotinic acid chloride, and the molar amount of 6-methoxynicotinic acid chloride used was 100% of the molar amount of L-proline methyl ester hydrochloride used.

[0072] Preparation of nicotinylproline hydrazide: Nicotinylproline ester was added to methanol, followed by hydrazine hydrate. The reaction was carried out at 25°C for 12 h. The solvent methanol was removed by rotary evaporation, followed by washing and drying to obtain nicotinylproline hydrazide. The amount of nicotinylproline ester used was 8 wt% of methanol, and the amount of hydrazine hydrate used was 3.6 wt% of nicotinylproline ester. During washing, a mixture of chloroform and saturated brine was used, and the organic layer was separated. During drying, the organic layer was dried with anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation to obtain the product.

[0073] Preparation of nicotinyl dimethylaminoproline hydrazide: Nicotinyl proline hydrazide was added to chloroform, followed by the addition of (dimethylamino)acetyl chloride. The mixture was treated at 25°C for 5 hours, and then dried under vacuum to remove the solvent, yielding nicotinyl dimethylaminoproline hydrazide. The amount of nicotinyl proline hydrazide used was 10 wt% of chloroform, and the amount of (dimethylamino)acetyl chloride used was 32 wt% of nicotinyl proline hydrazide.

[0074] Example 3: A liver cancer diagnostic kit based on DNA logic gates combined with nucleic acid aptamer sequencing The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extraction reagents. The AND gate-based probe reagents and nuclease-free signal amplification reagents used in this embodiment are described in Example 1.

[0075] In this embodiment, the PBMC extractant includes a DPBS solution. The DPBS solution contains 0.2 wt% FBS and 0.2 wt% nicotinyldimethylaminoproline hydrazide.

[0076] Example 4: A liver cancer diagnostic kit based on DNA logic gates combined with nucleic acid aptamer sequencing The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extraction reagents. The AND gate-based probe reagents and nuclease-free signal amplification reagents used in this embodiment are described in Example 1.

[0077] In this embodiment, the PBMC extractant includes a DPBS solution. The DPBS solution contains 0.2 wt% FBS, 0.2 wt% nicotinyl dimethylaminoproline hydrazide, and 0.03% cocamidopropyl hydroxysulfonate.

[0078] Example 5: A liver cancer diagnostic kit based on DNA logic gates combined with nucleic acid aptamer sequencing The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extraction reagents. The AND gate-based probe reagents and nuclease-free signal amplification reagents used in this embodiment are described in Example 1.

[0079] In this embodiment, the PBMC extractant includes a DPBS solution. The DPBS solution contains 0.2 wt% FBS, 0.2 wt% nicotinyl dimethaminoproline hydrazide, and 0.05 wt% cocamidopropyl hydroxysulfonate.

[0080] Comparative Example 1: A liver cancer diagnostic kit based on DNA logic gates combined with nucleic acid aptamer sequencing The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extraction reagents. The AND gate-based probe reagents and nuclease-free signal amplification reagents used in this embodiment are described in Example 1.

[0081] In this comparative example, the PBMC extraction agent included DPBS solution. The DPBS solution contained 0.2 wt% FBS.

[0082] Comparative Example 2: The diagnostic kit includes DNA logic gate-based probe reagents, nuclease-free signal amplification reagents, and PBMC extraction reagents. The AND gate-based probe reagents and nuclease-free signal amplification reagents used in this embodiment are described in Example 1.

[0083] In this comparative example, the PBMC extractant included a DPBS solution. The DPBS solution contained 0.2 wt% FBS and 0.001 wt% nicotinyl dimethylaminoproline hydrazide. The preparation of nicotinyl dimethylaminoproline hydrazide was carried out according to Example 2.

[0084] Example 6: An intelligent diagnostic model based on nucleic acid aptamer single-cell sequencing combined with DNA logic gates PBMC cells were mixed with DNA logic gate-based probe reagents and nuclease-free signal amplification reagents, and the fluorescence signal was detected. The results were output as follows: Result (1): If there is a fluorescent signal, it indicates a positive result for liver cancer.

[0085] Result (2): No fluorescence signal indicates negative result for liver cancer.

[0086] Example 7: An intelligent diagnostic model based on nucleic acid aptamer single-cell sequencing combined with DNA logic gates PBMCs were extracted using a PBMC extraction reagent. The PBMCs were then mixed with DNA logic gate-based probe reagents and nuclease-free signal amplification reagents. Fluorescence signals were detected, and the results were output. Result (1): If there is a fluorescent signal, it indicates a positive result for liver cancer.

[0087] Result (2): No fluorescence signal indicates negative result for liver cancer.

[0088] Experimental example: 1. Infrared characterization of nicotinyldimethylaminoproline hydrazide The nicotinyl dimethaminoproline hydrazide prepared in Example 2 was characterized by infrared spectroscopy, and the results are as follows: Figure 9 As shown, at 3228cm -1 The absorption peaks for nitrogen and hydrogen are located at 3040 cm⁻¹. -1 The absorption peak of the unsaturated hydrocarbons in the nicotinyl group is at 2932 cm⁻¹. -1The absorption peak at 2848 cm⁻¹ is the carbon-hydrogen absorption peak of the methyl group. -1 The absorption peak at 1702 cm⁻¹ represents the hydrocarbon absorption of the methylene group. -1 The absorption peak at 1042 cm⁻¹ represents the carbon-oxygen double bond. -1 The absorption peaks are for carbon, oxygen, and carbon.

[0089] 2. Cytotoxicity test The test group was set up by adding nicotinyl dimethylaminoproline hydrazide from Example 2 to DMEM medium containing 0.05 wt% nicotinyl dimethylaminoproline hydrazide and 2 wt% FBS. Then, 100 μL of DMEM medium was added to a 96-well plate. Mouse fibroblast L-929 cells were digested with 0.25% trypsin to prepare a cell suspension. 1×10⁻⁶ cells were then added to the DMEM medium. 6 Mouse fibroblast L-929 cells were seeded into 96-well plates and cultured at 37°C with 5% CO2 for 24 h. A control group was set up, whose DMEM medium did not contain nicotinyl dimethylaminoproline hydrazide, and was otherwise identical to the test group.

[0090] The survival rate of mouse fibroblast L-929 cells was detected using the CCK-8 assay. The absorbance at 450 nm was measured using an ELISA reader, and the cell viability was calculated.

[0091] In this invention, the survival rate of the test group relative to the control group was 99.6%, indicating that the nicotinyl dimethaminoproline hydrazide in this invention has no cytotoxicity.

[0092] 3. Cell viability In this invention, the cell viability of PBMCs obtained in S1 of Examples 1-5 and Comparative Examples 1-2 was tested. The PBMCs obtained in S1 were resuspended in an appropriate amount of binding buffer solution, and dead and live cells were counted using a cell counting chamber to determine the cell viability.

[0093] In this invention, three terminally extended (T) aptamers were first selected to target three specific membrane proteins of hepatocellular carcinoma (PMBC), named T1-Apt51, T2-GR-30, and T3-IBA, respectively. Based on the coupling of T1-Apt51, T2-GR-30, and T3-IBA with the aptamers, a double-stranded DNA based on DNA logic gates, consisting of cT1, cT2, cT35, and I, was prepared. Then, entropy-driven amplification of EDA was used to prepare a DNA matrix containing S-II, W, F, F-Cy5, and M. The nuclease-free signal amplification reagents 1 and M1-Cy3 can detect whether PBMCs simultaneously contain T1-Apt51, T2-GR-30, and T3-IBA after extraction from whole blood. The double-stranded DNA based on DNA logic gates of this invention will only release signal chain I when PBMCs simultaneously contain T1-Apt51, T2-GR-30, and T3-IBA, and further form fluorescence under the action of nuclease-free signal amplification reagents, which can effectively distinguish between healthy people and cancer patients. The cell viability of PBMCs ensures more accurate detection results. In this invention, the PBMC extraction agent is DPBS solution. FBS and / or nicotinyl dimethylaminoproline hydrazide can be added to the DPBS solution. Nicotinyl dimethylaminoproline hydrazide is prepared by reacting L-proline methyl ester hydrochloride and 6-methoxynicotinyl chloride to form nicotinyl proline ester, which then reacts with hydrazine hydrate to generate nicotinyl proline hydrazide. Finally, nicotinyl proline hydrazide is reacted with (dimethylamino)acetyl chloride to form nicotinyl dimethylaminoproline hydrazide. The cell viability in this invention is as follows: Figure 10 As shown, when PBMCs are extracted using a DPBS solution containing FBS, the resulting PBMCs exhibit good cell viability. However, if the FBS content in the DPBS solution is low, the cell viability of the resulting PBMCs is poor. In this invention, when PBMCs are extracted using a DPBS solution containing a low amount of FBS and nicotinamide dimethylaminoproline hydrazide, the cell viability of the resulting PBMCs is even higher. Increasing the amount of nicotinamide dimethylaminoproline hydrazide within a suitable range can improve the cell viability of the obtained PBMCs, while if the amount of nicotinamide dimethylaminoproline hydrazide is too low, the cell viability of the obtained PBMCs is poor. Furthermore, in this invention, a DPBS solution containing a low amount of FBS, nicotinamide dimethylaminoproline hydrazide, and cocamidopropyl hydroxysulfonate betaine can also be used for PBMC extraction. The use of cocamidopropyl hydroxysulfonate betaine and low amounts of FBS and nicotinamide dimethylaminoproline hydrazide can further improve the cell viability of PBMCs.

[0094] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0095] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operation, comprising: The probe reagents are based on DNA logic operations and are nuclease-free signal amplification reagents. The probe reagents include nucleic acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.

4.

2. The liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA "AND" logic operation according to claim 1, characterized in that: The nuclease-free signal amplification reagent includes any one or more nucleic acid sequences as shown in SEQ ID NO.5 to SEQ ID NO.

8.

3. The liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA "AND" logic operation as described in claim 1, characterized in that: The probe reagent is formed by base complementation of the nucleic acid sequences shown in SEQ ID NO.1 to SEQ ID NO.

4.

4. The liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA "AND" logic operation as described in claim 1, characterized in that: The probe reagent further includes a buffer solution; or, the nuclease-free signal amplification reagent includes a buffer solution.

5. The liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA "AND" logic operation as described in claim 1, characterized in that: The kit also includes a PBMC extractant, which is a DPBS solution.

6. The liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operation according to claim 5, characterized in that: The DPBS solution contains FBS and / or nicotinyl dimethaminoproline hydrazide; nicotinyl dimethaminoproline hydrazide is prepared by reacting L-proline methyl ester hydrochloride, 6-methoxynicotinyl chloride, hydrazine hydrate and (dimethylamino)acetyl chloride.

7. The liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operation according to claim 6, characterized in that: The DPBS solution contains 0.03-0.5 wt% nicotinyl dimethylaminoproline hydrazide; or, the DPBS solution contains 0.1-10 wt% FBS.

8. The liver cancer early screening kit based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operation according to claim 6, characterized in that: In the preparation of the nicotinyl dimethylaminoproline hydrazide, L-proline methyl ester hydrochloride and 6-methoxynicotinyl chloride are reacted to form nicotinyl proline ester, which is then reacted with hydrazine hydrate to generate nicotinyl proline hydrazide. Finally, nicotinyl proline hydrazide is reacted with (dimethylamino)acetyl chloride to generate nicotinyl dimethylaminoproline hydrazide.

9. An early liver cancer screening model based on nucleic acid aptamer single-cell high-throughput sequencing combined with DNA AND gate logic operations, including: Mix PBMC with DNA logic-based probe reagents and nuclease-free signal amplification reagents, detect the fluorescence signal, and output any one of results (1) to (2) based on the detection results: Result (1): The presence of fluorescent signal indicates a positive result for liver cancer; Result (2): No fluorescence signal indicates negative result for liver cancer.

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