Droplet microarray chip based on molecular logic operation and construction method
By constructing a droplet microarray chip based on molecular logic operations, the challenge of multi-enzyme synergistic detection in the BER pathway was solved, achieving efficient and accurate multi-enzyme synergistic detection and logic operations, supporting high-throughput biological analysis and cell research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING MEDICAL UNIVERSITY
- Filing Date
- 2026-04-14
- Publication Date
- 2026-07-10
Smart Images
Figure CN122357261A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biochemical analysis and detection technology, specifically to a droplet microarray chip based on molecular logic operations and its construction method. Background Technology
[0002] Genome integrity is fundamental to the normal functioning of life, and its stability is directly related to cellular function and overall health. However, throughout life, DNA molecules are continuously subjected to multiple forms of damage, including endogenous metabolic byproducts (such as reactive oxygen species) and exogenous environmental factors (such as ultraviolet radiation and chemical mutagens). If this damage is not repaired in a timely and accurate manner, mutations will gradually accumulate, leading to genomic instability and subsequently inducing the development of various major diseases, including malignant tumors, age-related diseases, and hereditary diseases. Therefore, establishing an efficient and accurate system for detecting and assessing DNA damage is of great significance for early disease diagnosis and precision treatment.
[0003] Among various DNA damage repair pathways, base excision repair (BER) is a highly conserved and crucial mechanism primarily responsible for repairing DNA base damage caused by oxidation, deamination, alkylation, and other factors. The BER pathway is accomplished through the synergistic action of a series of specific repair enzymes, including DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. These enzymes function sequentially according to strict temporal and spatial regulations, working together to complete steps such as damage recognition, excision, filling, and ligation, thereby restoring DNA integrity. Previous studies have shown that abnormalities in the activity of any key enzyme in the BER pathway can lead to impaired or erroneous repair processes, resulting in the accumulation of gene mutations. Therefore, a systematic analysis of the synergistic activities of multiple key enzymes in the BER pathway is of great significance for understanding DNA repair mechanisms and the diagnosis of related diseases.
[0004] Currently, methods for detecting DNA repair enzyme activity mainly include gel electrophoresis, fluorescent probe detection, enzyme-linked immunosorbent assay (ELISA), and high-throughput sequencing. While these methods have achieved the detection of single or a few enzyme activities to some extent, they generally suffer from limited throughput, complex operation, and difficulty in achieving multi-enzyme synergistic analysis. Furthermore, these methods typically rely on a single indicator or a simple combination of indicators, failing to reflect the complex multi-enzyme cascade reactions in the BER pathway, thus limiting their application in the diagnosis of complex diseases.
[0005] In recent years, molecular logic circuits constructed based on DNA nanotechnology have provided a novel approach for multi-target detection. This type of method utilizes toehold-mediated strand displacement (TMSD) to construct programmable logic units, using specific DNA repair enzymes as input signals. These enzymes trigger subsequent strand displacement reactions through the recognition and cleavage of specific substrates, thereby amplifying and outputting the signal. Existing studies have constructed various logic gates for enzyme activity detection, achieving to some extent the logical integration of multiple input signals. However, most existing DNA logic circuits are limited to simple single-layer or dual-input systems with low logical complexity, making it difficult to meet the requirements for fine-grained analysis of the complex synergistic relationships among multiple enzymes in the BER pathway. Furthermore, these systems typically operate in homogeneous solution environments, lacking effective spatial isolation, making them prone to intermolecular crosstalk and signal diffusion loss, thus affecting the accuracy and sensitivity of detection.
[0006] On the other hand, the development of microarray and microdroplet technologies has provided new technical means for high-throughput bioanalysis. Among them, droplet microarray technology constructs a two-dimensional array of droplets on a substrate surface with a specific wettability distribution, achieving spatial separation of microscale reaction systems. This technology can effectively prevent material exchange and cross-interference between different reaction units, thereby significantly improving the independence and accuracy of detection. Existing research has utilized superhydrophilic-superhydrophobic patterned surfaces to achieve precise positioning and high-density arrangement of microdroplets, and applied them to protein analysis, cell culture, and drug screening. However, existing droplet microarray technologies mostly focus on the analysis of single biological processes and lack system design that deeply integrates with molecular logic circuits, making it difficult to achieve parallel computing and high-dimensional analysis of complex biological information.
[0007] Furthermore, existing technologies still have certain limitations in material construction. For example, traditional microarray substrates mostly adopt planar structures with limited functionality, making it difficult to simultaneously meet the multifunctional needs of reaction space construction, molecular detection, and cell culture. At the same time, most existing DNA logic circuit systems lack the ability to couple with real biological microenvironments, limiting their application potential in complex biological systems.
[0008] In summary, existing technologies still have the following shortcomings in the field of DNA repair enzyme detection: 1. It is difficult to achieve simultaneous high-throughput detection of multiple enzymes in the BER pathway; 2. The computational complexity of DNA logic circuits is limited, making it difficult to analyze the synergistic effects of multiple enzymes; 3. There is a lack of effective spatial isolation mechanisms, leading to signal crosstalk and detection errors; 4. Existing platforms have limited functionality and are difficult to integrate molecular detection with cellular-level research.
[0009] Based on this, the present invention designs a droplet microarray chip based on molecular logic operations and a construction method thereof to solve the above problems. Summary of the Invention
[0010] In view of the above-mentioned shortcomings of the existing technology, the present invention provides a droplet microarray chip based on molecular logic operations and a method for constructing it.
[0011] To achieve the above objectives, the present invention provides the following technical solution: The method for constructing droplet microarray chips based on molecular logic operations includes the following steps: S1. First, a porous polymer film is prepared in situ on the substrate using digital light processing technology. The prepared patterned porous polymer film is then immersed in a PFOC solution. PFOC reacts with the active groups on the polymer surface to form a patterned surface on the substrate with alternating superhydrophilic and superhydrophobic arrangements, resulting in a superhydrophilic-superhydrophobic patterned porous polymer film. The superhydrophilic-superhydrophobic patterned porous polymer film contains superhydrophilic micro-reaction units arranged in an array, which are separated by superhydrophobic polymer regions. S2. Construct a molecular logic circuit based on enzyme-triggered DNA cleavage and foothold-mediated strand replacement. Pre-encapsulate the molecular logic circuit components in the reaction solution and load the reaction solution into each superhydrophilic microreaction unit to form independent microdroplets.
[0012] Furthermore, the substrate layer is made of optically transparent glass.
[0013] Furthermore, in step S1, the preparation method of the superhydrophilic-superhydrophobic patterned porous polymer film is as follows: a prepolymer solution containing hydroxyethyl methacrylate and a photoinitiator is prepared and uniformly spread between the substrate and the printing platform; then, the array pattern designed by the computer is projected into the prepolymer solution in the form of ultraviolet light through a digital micromirror device, initiating a photopolymerization reaction in the exposed area to form a solidified porous polymer structure; the unexposed area is rinsed with ethanol to remove the prepolymer solution, thus obtaining a patterned porous polymer film; the prepared patterned porous polymer film is immersed in a PFOC solution, where PFOC reacts with the active groups on the polymer surface to undergo an acylation reaction, causing the film area to change from hydrophilic to superhydrophobic; while the glass substrate area not covered by the polymer retains its original superhydrophilic properties, thereby forming a patterned surface with alternating superhydrophilic and superhydrophobic arrangements on the substrate, resulting in a superhydrophilic-superhydrophobic patterned porous polymer film.
[0014] Furthermore, the construction method of AND gate logic circuits includes: designing a three-chain bifurcated substrate, which is formed by hybridization of biotin-modified A1, fluorescent-quenched dual-labeled A2, and A3 modified with AP sites; initially, the fluorescent group and the quenching group are very close, and the fluorescence is quenched; when APE1 and FEN1 are present in the system at the same time, after APE1 cleaves the AP site, the 3' end of A3 is precisely formed into a 1nt overhang structure, which can serve as an effective substrate for FEN1; FEN1 then cleaves the 5' end overhang of A2, releasing the fluorescent group and thus outputting a high fluorescence signal.
[0015] Furthermore, the construction method of the OR gate logic circuit includes: designing a triangular flap structure substrate, which is formed by hybridization of biotin-modified O1, O2 double-labeled with a fluorescent group and a quenching group, and O3 containing a depurinated AP site; wherein, the AP site is located inside O2, and the 3' end of O3 has only one nucleotide protrusion; in the initial state, the FAM fluorescent group at the 5' end of O2 and the internally modified BHQ1 quenching group approach each other through the FRET effect, and the fluorescence is effectively quenched, outputting a low signal "0"; when at least one of APE1 or FEN1 is present in the system, APE1 specifically recognizes the AP site for cleavage, or FEN1 recognizes the flap structure for cleavage. The two enzymes act independently, releasing fluorescent groups to move away from the quenching group, thereby generating a significantly enhanced fluorescence signal and outputting a high signal "1".
[0016] Furthermore, the construction method of the NAND gate logic circuit includes: designing two three-strand complexes: the first complex is the AND gate substrate, formed by hybridization of biotinylated NA1, BHQ1-modified NA2, and NA3 containing the AP site; the second complex is the chain substitution substrate, formed by hybridization of FAM-labeled NA4 with its partially complementary chains NA5 and NA6. In the initial state, the fluorescence of NA4 is not quenched, and a high signal "1" is output; when only APE1 or only FEN1 is present, the fluorescence remains high signal "1" because the complete chain substitution cascade cannot be triggered after cleavage or the cleavage activity is weak; only when APE1 and FEN1 are present simultaneously, APE1 first cleaves the AP site of NA3, producing a precise 1-nt protrusion, and FEN1 then cleaves and releases the 5' flap of NA2. Subsequently, the NA3 fragment binds to the NA4 foothold and substitutes for NA5, exposing the second foothold. The NA2 fragment then binds to and substitutes for NA6, causing BHQ1 to approach FAM and quench, outputting a low signal "0".
[0017] Furthermore, the reaction solution is loaded into each superhydrophilic microreaction unit using a drop-by-drop or sliding liquid distribution method.
[0018] Furthermore, the reaction solution includes molecular logic circuit components, the sample to be tested, and a reaction buffer system.
[0019] Furthermore, the superhydrophilic microreaction unit has a diameter of 2 mm, and each superhydrophilic microreaction unit is separated by a superhydrophobic polymer region with a width of 0.5 mm.
[0020] To better achieve the objectives of this invention, the present invention also provides a droplet microarray chip, comprising a substrate layer, a functional layer located on the substrate layer, and molecular logic circuitry. The functional layer is a superhydrophilic-superhydrophobic patterned porous polymer film prepared in situ on the substrate using digital light processing technology and then prepared by PFOC acylation reaction. The superhydrophilic-superhydrophobic patterned porous polymer film contains superhydrophilic microreaction units arranged in an array, which are separated by superhydrophobic polymer regions. Each superhydrophilic microreaction unit encapsulates an independent microdroplet, and the molecular logic circuitry is encapsulated within the independent microdroplet, so that each independent microdroplet forms an independent molecular computing and sensing unit.
[0021] Compared with the prior art, the beneficial effects of this invention are as follows: by constructing a multi-input molecular logic circuit system, this invention uses different DNA repair enzymes as independent biological input signals to achieve a systematic characterization of their synergistic relationship, thereby breaking through the limitations of the traditional single-index detection mode and improving the ability to evaluate the overall functional status of the BER pathway.
[0022] This invention designs a scalable molecular logic computing architecture. By introducing multi-level cascade reactions and a programmable DNA strand substitution mechanism, it realizes multi-input, multi-level logic operations, significantly improving the computational complexity and information processing capability of the logic system. This enables precise differentiation of different enzyme expression patterns, meeting the needs of complex disease subtyping and accurate diagnosis.
[0023] This invention constructs a microreaction unit array with spatial isolation function. By constructing a structure with a specific wettability distribution on the substrate surface, the independent separation and precise positioning of microdroplets are achieved, thereby effectively avoiding mutual interference between different reaction systems, reducing background noise, and improving the accuracy and stability of detection.
[0024] This invention enables a large number of independent reactions to be carried out in parallel on the same chip, achieving simultaneous analysis of multiple samples and multiple indicators, significantly improving detection efficiency and data acquisition capabilities, and meeting the needs of high-throughput bioanalysis and clinical testing.
[0025] This invention can not only be used for in situ detection of DNA repair enzymes, but also support cell culture and real-time imaging, thereby enabling correlation analysis between molecular-level information and cellular-level behavior and enhancing the biological relevance of detection results.
[0026] In summary, this invention achieves a systematic improvement in multi-enzyme synergistic detection capability, logic computation complexity, spatial isolation control, high-throughput analysis, and adaptation to biological environments by combining droplet microarray chips with molecular logic circuits. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.
[0028] Figure 1 This is a schematic diagram of the construction method of the droplet microarray chip based on molecular logic operations of the present invention. Figure 2 This is a flowchart of the experimental fabrication method of the droplet microarray chip based on molecular logic operations according to the present invention; Figure 3 A schematic diagram of a molecular logic circuit constructed based on four biological enzymes; Figure 4 The results of the detection experiments for different molecular logic circuits; Figure 5 This is an example of clinical sample testing. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0030] Example 1: As Figure 1-5 As shown, the method for constructing a droplet microarray chip based on molecular logic operations includes the following steps: S1. First, a porous polymer film is prepared in situ on the substrate using digital light processing (DLP) technology, and a superhydrophilic-superhydrophobic alternating pattern is constructed by PFOC (perfluorooctyltrichlorosilane) acylation reaction to obtain a superhydrophilic-superhydrophobic patterned porous polymer film. The superhydrophilic-superhydrophobic patterned porous polymer film contains superhydrophilic micro-reaction units arranged in an array, and the superhydrophilic micro-reaction units are separated by superhydrophobic polymer regions. The substrate layer is made of optically transparent glass, providing a hydrophilic surface and a good optical detection window.
[0031] Specifically, a prepolymer solution (precursor solution) containing hydroxyethyl methacrylate (HEMA) and a photoinitiator is prepared and uniformly spread between the substrate and the printing platform. Then, a computer-designed array pattern is projected into the prepolymer solution using ultraviolet light through a digital micromirror device (DMD). This initiates a photopolymerization reaction in the exposed areas, forming a cured porous polymer structure. The unexposed areas are rinsed with ethanol to remove the prepolymer solution, resulting in a precisely patterned porous polymer film. Subsequently, utilizing the hydroxyl sites on the polymer film itself, the prepared patterned porous polymer film is immersed in a perfluorooctanoic acid (PFOC) solution and treated overnight at room temperature. PFOC reacts with the active groups on the polymer surface, causing the film region to change from hydrophilic to superhydrophobic. The glass substrate region not covered by the polymer retains its original superhydrophilic properties, thus forming a patterned surface with alternating superhydrophilic and superhydrophobic arrangements on the substrate, resulting in a superhydrophilic-superhydrophobic patterned porous polymer film.
[0032] In this embodiment, the microarray is arranged in a 7×22 pattern, with a total of 154 superhydrophilic microreaction units. Each unit has a diameter of about 2 mm, and the units are separated by superhydrophobic polymer regions with a width of 1 mm.
[0033] This invention enables the formation of high-fidelity, hierarchically porous polymer structures at the micrometer to millimeter scale. This porous structure provides abundant reaction sites and sufficient space for subsequent chemical modification, facilitating the efficient introduction of long-chain perfluorinated groups into the polymer backbone via PFOC acylation. This results in alternating superhydrophilic and superhydrophobic regions, achieving precise confinement of the droplet space and preventing droplet fusion. This not only ensures efficient droplet fixation within the hydrophilic micropores but also prevents cross-contamination of adjacent droplets through hydrophobic boundaries, thereby enabling high-throughput, multi-droplet parallel analysis. It provides a reliable experimental platform for applications such as microchemical reactions, high-throughput screening, drug testing, and bioanalysis, effectively improving the operational efficiency, data accuracy, and applicability of droplet microarray chips.
[0034] The surface of the droplet microarray chip, characterized by optical absorption and contact angle analysis, revealed successful polymer film formation, effective chemical modification, and significantly enhanced wettability, exhibiting superhydrophobic-superhydrophilic properties that meet the design requirements for microdroplet manipulation. Scanning electron microscopy and energy dispersive spectroscopy further confirmed the continuity and hierarchy of the porous network structure, as well as the fact that the perfluorinated chains are distributed only on the polymer surface, achieving spatially selective functionalization. Optical microscopy combined with bright-field imaging and grayscale analysis verified the structural fidelity and spatial uniformity of the polymer array, ensuring high-precision conversion of digital design into a physical chip.
[0035] S2. Construction of molecular logic circuits This invention constructs a molecular logic circuit based on enzyme-triggered DNA cleavage and foothold-mediated strand substitution (TMSD) targeting key enzymes in the base excision repair (BER) pathway—uracil DNA glycosylase (UDG) and depurine / depyrimidine endonuclease 1 (APE1).
[0036] Taking the "AND logic" as an example, a triple-branched substrate structure is designed, formed by hybridization of biotin-modified A1, fluorescent (e.g., FAM)-quenched (e.g., BHQ1) dual-labeled A2, and A3 modified with an AP site. Initially, the fluorescent group and the quenching group are very close, quenching the fluorescence. When APE1 and FEN1 are present simultaneously, APE1 cleaves the AP site, precisely forming a 1nt overhang structure at the 3' end of A3, which serves as an effective substrate for FEN1. FEN1 then cleaves the 5' overhang of A2, releasing the fluorescent group and thus outputting a high fluorescence signal. If only a single enzyme is present, cleavage cannot be fully performed, and the fluorescence signal remains quenched. Therefore, the "on" of the fluorescence signal strictly depends on the synergistic presence of the two enzymes, realizing the "AND gate" logic function.
[0037] Similarly, by changing the probe structure and recognition site, various logic circuits such as "OR gate" and "NAND gate" can be constructed to achieve fine differentiation of different enzyme expression patterns. Figure 3 (and Table 1): For example, the construction method of an OR gate logic circuit includes: designing a triangular flap structure substrate, formed by hybridization of biotin-modified O1, O2 double-labeled with a fluorescent group and a quenching group, and O3 containing an apurinol (AP) site; wherein, the AP site is located inside O2, and the 3' end of O3 has only one nucleotide protrusion; initially, the FAM fluorescent group at the 5' end of O2 and the internally modified BHQ1 quenching group approach each other through the FRET effect, and the fluorescence is effectively quenched, outputting a low signal "0"; when at least one of APE1 or FEN1 is present in the system, APE1 specifically recognizes the AP site for cleavage, or FEN1 recognizes the flap structure for cleavage, and the two enzymes can act independently, releasing fluorescent groups to move away from the quenching group, thereby generating a significantly enhanced fluorescence signal and outputting a high signal "1"; this design achieves a positive response to three input combinations of (0,1), (1,0), and (1,1). Thus, as long as any input enzyme is present, fluorescence is turned on, realizing the "OR gate" logic function.
[0038] For example, the construction method of the NAND gate logic circuit includes: designing two triple-stranded complexes: the first complex is the AND gate substrate, formed by hybridization of biotinylated NA1, BHQ1-modified NA2, and NA3 containing the AP site; the second complex is the strand substitution substrate, formed by hybridization of FAM-labeled NA4 with its partially complementary strands NA5 and NA6. Initially, NA4 fluorescence is not quenched, outputting a high signal "1". When only APE1 or only FEN1 is present, the fluorescence remains at a high signal "1" due to the inability to trigger a complete strand substitution cascade or weak cleavage activity, respectively; only when APE1 and FEN1 are present simultaneously, APE1 first cleaves the AP site of NA3, producing a precise 1-nt protrusion, and FEN1 then cleaves and releases the 5' flap of NA2. Subsequently, the NA3 fragment binds to the NA4 flap and substitutes for NA5, exposing the second flap. The NA2 fragment then binds to and substitutes for NA6, causing BHQ1 to approach FAM and quench, outputting a low signal "0". Thus, fluorescence is turned off only when both enzymes are present, realizing the "NAND gate" logic function.
[0039] Table 1. Logic Gate Related Sequences
[0040] S3. Fluorescence detection and signal reading The aforementioned molecular logic circuit components are pre-encapsulated in a reaction solution, and the reaction solution is loaded into each superhydrophilic microreactor unit by dropwise or sliding liquid distribution to form independent microdroplets, thereby constructing a droplet microarray chip containing 154 independent microreactors.
[0041] The reaction solution includes molecular logic circuit components, the sample to be tested, and a reaction buffer system; Droplet-by-droplet dispensing allows for the precise placement of multiple solutions in different wells, with fluorescence imaging showing that the droplets do not interfere with each other, enabling high-throughput parallel analysis. Sliding dispensing utilizes the discontinuous dehumidification properties of the polymer membrane to rapidly break down and capture large droplets into microwells, achieving efficient arraying of single solutions. Side-view analysis of the chip shows high droplet uniformity, further demonstrating the repeatability and stability of droplet manipulation.
[0042] The droplet microarray chip was incubated at 37°C for 30 minutes to promote the full progress of enzymatic and chain displacement reactions. Subsequently, the chip was imaged using a fluorescence microscope or a high-throughput fluorescence scanner to collect the fluorescence intensity of each microdroplet under the corresponding excitation light. The fluorescence signal value of each microdroplet was extracted by image analysis software and compared with a preset logic truth table to determine the activity state and synergistic relationship of the target enzyme in each reaction unit.
[0043] Because each microdroplet is independent, multiple enzyme combinations and samples of different concentrations can be detected in parallel on the same chip, enabling high-throughput, multi-index simultaneous analysis. Figure 4 ).
[0044] By constructing a droplet microarray chip based on patterned wettability regulation and combining it with programmable DNA molecular logic circuits, we have achieved simultaneous analysis of the activities of multiple key enzymes in the base excision repair pathway, efficient processing of complex biological information, and in-situ monitoring at the cellular level.
[0045] Each independent microdroplet serves as an independent molecular computing and sensing unit, enabling parallel detection of multi-enzyme activity and analysis of complex logical relationships through spatial resolution reading of fluorescence signals. Simultaneously, this droplet microarray chip supports cell culture and in-situ imaging through its open droplet structure, thus bridging the gap between molecular logic operations and the biological microenvironment.
[0046] Example 2: A droplet microarray chip based on molecular logic operations, comprising a substrate layer, a functional layer on the substrate layer, and a molecular logic circuit. The functional layer is a superhydrophilic-superhydrophobic patterned porous polymer film prepared in situ on the substrate by digital light processing (DLP) technology and then prepared by PFOC (perfluorooctyltrichlorosilane) acylation reaction. The superhydrophilic-superhydrophobic patterned porous polymer film contains superhydrophilic microreaction units arranged in an array. The superhydrophilic microreaction units are separated by superhydrophobic polymer regions. Each superhydrophilic microreaction unit encapsulates an independent microdroplet. The molecular logic circuit is encapsulated in the independent microdroplet, so that each independent microdroplet forms an independent molecular computing and sensing unit.
[0047] Experimental example: Clinical sample testing and validation To verify the clinical application potential of the droplet microarray chip, this embodiment further utilizes the aforementioned AND gate logic circuit to detect clinical serum samples. Figure 5 Serum samples were collected from pathologically confirmed breast cancer patients (n=30) and healthy controls (n=30). The serum samples were mixed with DNA logic circuit reaction solution and then loaded onto a droplet microarray chip, with three parallel reaction units for each sample. Fluorescence signals were detected after incubation.
[0048] The results showed that the fluorescence signal originated from the synergistic activity of two enzymes, UDG and APE1, in serum, and the activity levels of both enzymes were significantly upregulated in the patient group. This result confirms the feasibility of the platform of this invention for multi-enzyme synergistic analysis and disease subtyping in complex biological samples.
[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for constructing a droplet microarray chip based on molecular logic operations, characterized in that, Includes the following steps: S1. First, a porous polymer film is prepared in situ on the substrate using digital light processing technology. The prepared patterned porous polymer film is then immersed in a PFOC solution. PFOC reacts with the active groups on the polymer surface to form a patterned surface on the substrate with alternating superhydrophilic and superhydrophobic arrangements, resulting in a superhydrophilic-superhydrophobic patterned porous polymer film. The superhydrophilic-superhydrophobic patterned porous polymer film contains superhydrophilic micro-reaction units arranged in an array, which are separated by superhydrophobic polymer regions. S2. Construct a molecular logic circuit based on enzyme-triggered DNA cleavage and foothold-mediated strand replacement. Pre-encapsulate the molecular logic circuit components in the reaction solution and load the reaction solution into each superhydrophilic microreaction unit to form independent microdroplets.
2. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 1, characterized in that, The substrate is made of optically transparent glass.
3. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 1, characterized in that, In step S1, the preparation method of the superhydrophilic-superhydrophobic patterned porous polymer film is as follows: a prepolymer solution containing hydroxyethyl methacrylate and a photoinitiator is prepared and uniformly spread between the substrate and the printing platform; then, the array pattern designed by the computer is projected into the prepolymer solution in the form of ultraviolet light through a digital micromirror device, and a photopolymerization reaction is initiated in the exposed area to form a solidified porous polymer structure; the unexposed area is rinsed with ethanol to remove the prepolymer solution, and a patterned porous polymer film is obtained; the prepared patterned porous polymer film is immersed in a PFOC solution, and the PFOC reacts with the active groups on the polymer surface to undergo an acylation reaction, so that the film region changes from hydrophilic to superhydrophobic; The glass substrate area not covered by the polymer retains its original superhydrophilic properties, thereby forming a patterned surface with alternating superhydrophilic and superhydrophobic arrangements on the substrate, resulting in a superhydrophilic and superhydrophobic patterned porous polymer film.
4. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 3, characterized in that, The construction method of AND gate logic circuit includes: designing a three-chain bifurcated substrate, which is formed by hybridization of biotin-modified A1, fluorescent-quenched dual-labeled A2, and A3 modified with AP site; initially, the fluorescent group and the quenching group are very close, and the fluorescence is quenched; when APE1 and FEN1 are present in the system at the same time, after APE1 cuts the AP site, the 3' end of A3 is precisely formed into a 1nt overhang structure, which can be used as an effective substrate for FEN1; FEN1 then cuts the 5' end overhang of A2, releasing the fluorescent group, thereby outputting a high fluorescence signal.
5. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 4, characterized in that, The method for constructing an OR gate logic circuit includes: designing a triangular flap structure substrate, which is formed by hybridization of biotin-modified O1, O2 double-labeled with a fluorescent group and a quenching group, and O3 containing a depurinated AP site; wherein, the AP site is located inside O2, and the 3' end of O3 has only one nucleotide protrusion; in the initial state, the FAM fluorescent group at the 5' end of O2 and the internally modified BHQ1 quenching group approach each other through the FRET effect, and the fluorescence is effectively quenched, outputting a low signal "0"; when at least one of APE1 or FEN1 is present in the system, APE1 specifically recognizes the AP site for cleavage, or FEN1 recognizes the flap structure for cleavage. The two enzymes act independently, releasing fluorescent groups to move away from the quenching group, thereby generating a significantly enhanced fluorescence signal and outputting a high signal "1".
6. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 5, characterized in that, The construction method of NAND gate logic circuit includes: designing two three-strand complexes: the first complex is the AND gate substrate, formed by hybridization of biotinylated NA1, BHQ1-modified NA2, and NA3 containing AP sites; the second complex is the chain substitution substrate, formed by hybridization of FAM-labeled NA4 with its partially complementary chains NA5 and NA6. In the initial state, NA4 fluorescence is not quenched, and a high signal "1" is output; when only APE1 or only FEN1 is present, the fluorescence remains high signal "1" because the cleavage cannot trigger a complete chain substitution cascade or the cleavage activity is weak; only when APE1 and FEN1 are present simultaneously, APE1 first cleaves the AP site of NA3, producing a 1-nt protrusion, and FEN1 then cleaves and releases the 5' flap of NA2. Subsequently, the NA3 fragment binds to the NA4 foothold and substitutes for NA5, exposing the second foothold. The NA2 fragment then binds to and substitutes for NA6, causing BHQ1 to approach FAM and quench, outputting a low signal "0".
7. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 1, characterized in that, The reaction solution was loaded into each superhydrophilic microreaction unit using a drop-by-drop or sliding liquid distribution method.
8. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 1, characterized in that, The reaction solution includes molecular logic circuit components, the sample to be tested, and a reaction buffer system.
9. The method for constructing a droplet microarray chip based on molecular logic operations according to claim 1, characterized in that, The superhydrophilic microreaction unit has a diameter of 2 mm, and each superhydrophilic microreaction unit is separated by a superhydrophobic polymer region with a width of 0.5 mm.
10. A droplet microarray chip obtained by the construction method according to any one of claims 1 to 9, characterized in that, The device includes a substrate layer, a functional layer on the substrate layer, and molecular logic circuitry. The functional layer is a superhydrophilic-superhydrophobic patterned porous polymer film prepared in situ on the substrate using digital light processing technology and then prepared by PFOC acylation reaction. The superhydrophilic-superhydrophobic patterned porous polymer film contains superhydrophilic microreaction units arranged in an array. The superhydrophilic microreaction units are separated by superhydrophobic polymer regions. Each superhydrophilic microreaction unit encapsulates an independent microdroplet, and the molecular logic circuitry is encapsulated in the independent microdroplet, so that each independent microdroplet forms an independent molecular computing and sensing unit.