A new method for quantitative analysis of interfacial nucleic acid hybridization reaction and its application

Abstract

The application discloses a new method for quantitatively analyzing interface nucleic acid hybridization reaction and application thereof, and comprises the following steps: S1, constructing a DNA tetrahedron interface; S2, imaging the DNA tetrahedron interface through a total internal reflection fluorescence microscope; S3, realizing real-time monitoring of a hybridization event of the DNA tetrahedron interface by adopting double-color co-localization and analyzing a fluorescence signal; and S4, analyzing hybridization kinetics on the DNA tetrahedron interface by recording changes of the fluorescence signal with time. The application uses the DNA tetrahedron interface for single-molecule fluorescence imaging, can realize real-time monitoring of a single nucleic acid molecule hybridization event, improve detection sensitivity and specificity, accurately analyze kinetic parameters of a hybridization reaction, help realize more accurate performance evaluation of a nucleic acid sensor, has important significance for design and optimization of the nucleic acid sensor, and thus plays a greater role in the fields of clinical diagnosis, environmental monitoring and food safety.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and more specifically to a novel method for quantitative analysis of interfacial nucleic acid hybridization reactions and its applications. Background Technology

[0002] Interfacial nucleic acid hybridization is a molecular recognition process that occurs on solid surfaces, allowing specific nucleic acid probes to be immobilized on these surfaces to capture and recognize complementary target nucleic acid sequences. This reaction has wide applications in biosensors, gene expression analysis, and clinical diagnostics. Although existing methods for evaluating interfacial hybridization reactions, such as fluorescence spectroscopy, surface plasmon resonance (SPR), electrochemical sensing, and microarray analysis, are widely used, most of these methods rely on endpoint analysis—measurements performed after the reaction is complete—and cannot achieve real-time monitoring of nucleic acid hybridization events, limiting a deeper understanding of hybridization kinetics. Furthermore, they are significantly insufficient in quantitative analysis, especially in distinguishing between specific and non-specific binding events at the single-molecule level.

[0003] Some interfacial nucleic acid hybridization analysis methods face a series of limitations in quantitative analysis. First, traditional nucleic acid hybridization analysis methods typically rely on batch measurements and cannot provide real-time monitoring capabilities at the single-molecule level. This limits in-depth understanding of the dynamic processes of individual hybridization events, especially in biosensor performance evaluation. Second, interfacial nucleic acid hybridization is affected by the interfacial microenvironment, including surface charge and chemical properties, which reduce the efficiency and sensitivity of interfacial nucleic acid hybridization. Furthermore, existing interfacial nucleic acid hybridization methods struggle to effectively distinguish between specific and non-specific binding events, potentially leading to false positives and affecting the accuracy and reliability of detection.

[0004] Single-molecule fluorescence imaging technology can monitor the behavior of individual nucleic acid molecules in real time, helping to distinguish between specific and non-specific binding events and providing single-molecule-level kinetic parameter measurements, thereby gaining a deeper understanding of the interactions between nucleic acid molecules. Although single-molecule fluorescence imaging technology has advantages in evaluating interfacial hybridization reactions, it suffers from limitations in precise control over interface design, such as uneven probe distribution, insufficient stability, or poor accessibility to the target, which affect hybridization efficiency and signal reliability. Summary of the Invention

[0005] The purpose of this invention is to provide a new method for quantitative analysis of interfacial nucleic acid hybridization reactions and its application, thereby solving the problem that existing technologies cannot achieve real-time monitoring of nucleic acid hybridization events, which limits the in-depth understanding of hybridization kinetics.

[0006] To solve the above problems, the present invention adopts the following technical solution:

[0007] According to a first aspect of the present invention, a novel method for quantitatively analyzing interfacial nucleic acid hybridization reactions is provided, comprising the following steps: S1: constructing a DNA tetrahedral interface, comprising: constructing a DNA tetrahedron with a single-stranded DNA probe extending from one vertex for capturing a target DNA sequence, and fixing the DNA tetrahedron onto a coverslip surface to construct the DNA tetrahedral interface; S2: single-molecule observation of the DNA tetrahedral interface, comprising: imaging the DNA tetrahedral interface using a total internal reflection fluorescence microscope to study the distribution pattern of the DNA tetrahedron on the interface; S3: real-time monitoring of DNA tetrahedral interface hybridization events, comprising: labeling the TDN probe and the target DNA with two different fluorescent molecules respectively, employing dual-color colocalization and analysis of fluorescence signals to achieve real-time monitoring of DNA tetrahedral interface hybridization events; S4: quantitative analysis of DNA tetrahedral interface hybridization kinetics, comprising: analyzing the hybridization kinetics on the DNA tetrahedral interface by recording changes in fluorescence signals over time.

[0008] Preferably, in step S1, the DNA tetrahedron binds to the coverslip surface through any one of the following mechanisms: biotin-avidin interaction, PEG-biotin-SA interaction, or electrostatic binding. For the PEG-biotin-SA binding mechanism, please refer to https: / / pubs.acs.org / doi / 10.1021 / acs.jpclett.8b02992; for the electrostatic binding mechanism after plasma cleaning, please refer to https: / / www.sciencedirect.com / science / article / pii / S2590238524005472.

[0009] Preferably, in step S1, biotin molecules for interface fixation are modified at three vertices of the DNA tetrahedron, and a single-stranded DNA probe extends from the other vertex to capture the target nucleic acid sequence. The extended single-stranded DNA probe can be further modified with fluorescent groups for dual-color co-localization imaging.

[0010] Preferably, step S1 further includes: characterizing the DNA tetrahedrons using polyacrylamide gel electrophoresis to confirm their purity and yield; characterizing the DNA tetrahedrons using atomic force microscopy to confirm their correct assembly from a morphological perspective; and performing a detailed analysis of the structure and size of the DNA tetrahedrons based on AFM imaging data to obtain statistical data on height and particle size.

[0011] Preferably, step S2 further includes optimizing the TDN probe density and target DNA concentration to achieve optimal hybridization efficiency and signal quality. The optimal concentration of the TDN probe is 80-120 pM, with 100 pM being the most preferred, to achieve the highest hybridization efficiency and signal quality.

[0012] Preferably, in step S3, the TDN probe is labeled with the red fluorescent molecule AF647, and the target DNA is labeled with the green fluorescent molecule CY3B. After the TDN probe and the target DNA are incubated, the fluorescence signal is collected through the TIRFM real-time observation interface, and the changes of the two colors of fluorescence signal over time are recorded.

[0013] Preferably, step S4 further includes: performing statistical analysis on the binding time and dissociation time to extract key kinetic parameters, including: binding rate and dissociation rate, etc.

[0014] Preferably, in step S4, the hybridization experiment of the TDN probe and the target DNA is performed at the optimal working concentration, which is: 100 pM TDN probe and 20 nM target DNA.

[0015] Preferably, the new method further includes the step of:

[0016] S5: By comparing the hybridization kinetics of DNA tetrahedral interfaces with those of single-stranded DNA interfaces, we evaluate the potential of DNA tetrahedral interfaces in improving the performance of nucleic acid sensors.

[0017] According to a second aspect of the present invention, a novel method for quantitatively analyzing interfacial nucleic acid hybridization reactions is provided for evaluating the performance of nucleic acid sensors.

[0018] To address the limitation of existing technologies in real-time monitoring of nucleic acid hybridization events, which restricts a deeper understanding of hybridization kinetics, this invention provides a single-molecule fluorescence imaging method based on the TDN interface for quantitative analysis of the kinetics of interfacial nucleic acid hybridization reactions and evaluation of nucleic acid sensor performance. This method aims to overcome the limitations of existing technologies through the following approaches: 1) Real-time monitoring of single nucleic acid molecule hybridization events using single-molecule fluorescence imaging technology, providing more accurate kinetic data; 2) Reducing the influence of the interfacial microenvironment on the nucleic acid hybridization process using DNA tetrahedra, improving detection sensitivity; 3) Effectively distinguishing between specific and non-specific binding events using probe-target co-localization imaging, improving detection accuracy and reliability; 4) Providing a scientific basis for evaluating nucleic acid sensors through quantitative analysis of kinetic parameters, promoting the development of biosensor technology.

[0019] According to a preferred embodiment of the present invention, a single-molecule fluorescence imaging method based on the TDN interface is proposed for quantitative analysis of the kinetics of interfacial nucleic acid hybridization reactions and evaluation of the performance of nucleic acid sensors (e.g., Figure 1 (As shown in ag). The method includes the following steps:

[0020] I. Structural Design and Assembly of DNA Tetrahedrons

[0021] This invention first designs and assembles a TDN with a specific structure: based on a common DNA tetrahedral structure, three vertices are modified with biotin molecules for interface fixation, and a single-stranded DNA probe extends from the other vertex to capture the target nucleic acid sequence. Through precise DNA strand design, the stability and functionality of the tetrahedron are ensured, and a stable tetrahedral structure is formed through self-assembly using the base complementary pairing principle. Biotin molecules are modified at three vertices of the tetrahedron for interface fixation, and a single-stranded DNA probe extends from one vertex to capture the target nucleic acid sequence. The extended DNA probe can be further modified with fluorescent groups for dual-color co-localization imaging.

[0022] (2) Structural characterization of DNA tetrahedron

[0023] DNA tetrahedra were characterized using polyacrylamide gel electrophoresis (PAGE) to confirm their purity and yield. Atomic force microscopy (AFM) was then used to characterize the DNA tetrahedra, confirming their correct assembly morphologically. Based on the AFM imaging data, a detailed analysis of the DNA tetrahedral structure and size was performed, obtaining statistical data on height and particle size.

[0024] (3) DNA tetrahedral interface treatment and fixation

[0025] The coverslips were first treated with piranha (or a detergent or standard coverslip treatment procedure can be used; see reference https: / / wang-lab.uark.edu / protocol-clean-coverslips-or-slides / ) to enhance their surface activity. They were then dried to ensure a clean, moisture-free surface for subsequent operations. A bovine serum albumin-biotin solution was added to the treated coverslip surface, and after standing, Neutravidin was added. Finally, the biotin-modified DNA tetrahedra were immobilized on the coverslip surface through biotin-avidin interactions.

[0026] II. Single-molecule observation of DNA tetrahedral interfaces

[0027] (1) Concentration optimization

[0028] To ensure the reproducibility and reliability of the experiment, the TDN probe density and target DNA concentration were optimized to achieve the best hybridization efficiency and signal quality. The optimal working concentration was identified, and statistical analysis was performed on the probe density at different concentrations.

[0029] (2) Distribution pattern of probes at the interface

[0030] The interface was imaged using total internal reflection fluorescence microscopy (TIRF) to study the distribution pattern of DNA tetrahedra on the interface, such as their number and spacing, and to confirm that the interface modification density of the probe was appropriate and the distribution was uniform.

[0031] III. Real-time monitoring of DNA tetrahedral interface hybridization events

[0032] By employing two different fluorescently labeled probes and targets and a dual-color co-localization strategy, accurate identification of specific hybridization events can be achieved. This strategy utilizes the spatial overlap of fluorescence signals to confirm hybridization events, improving the accuracy and reliability of detection. Furthermore, by utilizing dual-color co-localization and analyzing the fluorescence signals, specific and non-specific hybridization events can be distinguished, further enhancing detection accuracy.

[0033] IV. Quantitative Analysis of DNA Tetrahedral Interface Hybridization Kinetics

[0034] (1) Fluorescence intensity versus time curve analysis

[0035] By recording changes in fluorescence signals over time, hybridization kinetics at DNA tetrahedral interfaces can be analyzed. Real-time monitoring of fluorescence signals allows for the acquisition of dynamic information on the binding and dissociation processes of hybridization events.

[0036] (2) Statistical analysis combining time and dissociation time

[0037] To better understand the nature of hybridization reactions, statistical analysis was performed on binding and dissociation times to extract key kinetic parameters such as binding rate and dissociation rate.

[0038] V. Evaluation of hybridization kinetics at single-strand interfaces

[0039] By comparing the hybridization kinetics of the DNA tetrahedral interface with that of the single-stranded DNA interface, the potential of the TDN interface in improving the performance of nucleic acid sensors was evaluated. Comparison of the kinetic parameters of different interface structures revealed the advantages of the TDN interface in improving sensitivity and specificity.

[0040] The key inventive point of this invention lies in combining the DNA tetrahedral (TDN) interface with single-molecule fluorescence imaging technology to achieve accurate quantitative analysis of single nucleic acid hybridization reactions. Previous existing technologies were limited to using the DNA tetrahedral interface for electrochemical sensing, only allowing endpoint readout and thus unable to quantitatively analyze single nucleic acid hybridization events. Currently, no other research teams have reported on evaluating the use of the DNA tetrahedral interface for DNA hybridization reaction kinetics at the single-molecule level. This invention achieves quantitative analysis of the kinetics of interfacial nucleic acid hybridization reactions through such a DNA tetrahedral interface and evaluates the performance of nucleic acid sensors. Specifically, traditional electrochemical sensing interfaces provide average values, while the DNA tetrahedral interface constructed in this invention is a nucleic acid sensing interface. Using single-molecule imaging technology, the kinetics of single hybridization reactions can be accurately quantitatively analyzed, thereby more accurately evaluating the performance of nucleic acid sensors.

[0041] This invention proposes an innovative method for quantitative analysis of interfacial nucleic acid hybridization reactions by combining single-molecule fluorescence imaging technology and tetrahedral (TDN) interface technology. Compared with existing technologies, this method has the following significant advantages and effects:

[0042] This invention utilizes single-molecule fluorescence imaging technology to directly detect hybridization events of individual nucleic acid molecules, significantly improving detection sensitivity and enabling accurate identification of low-abundance target nucleic acids. In particular, the introduction of the TDN interface overcomes the influence of traditional interfaces on the probe molecule binding process, further enhancing detection sensitivity and reliability.

[0043] This invention employs a dual-color co-localization strategy, which effectively distinguishes between specific and non-specific binding events, enhancing the specificity of the detection. By applying this strategy, the invention ensures the accuracy of detection results while also improving the ability to identify target molecules in complex samples.

[0044] The method of this invention can monitor the nucleic acid hybridization process at the interface in real time and directly observe the dynamic changes in molecular interactions. This real-time monitoring capability not only makes it possible to analyze the kinetic parameters of hybridization events (such as binding rate and dissociation rate) and realize the quantitative analysis of interfacial nucleic acid hybridization reactions, but also provides key information for a deeper understanding of the dynamic mechanism of nucleic acid hybridization reactions.

[0045] Constructing interfaces using DNA tetrahedrons is a novel method for nucleic acid immobilization. It not only enhances the accessibility of probes on the surface but also provides good controllability and high-precision orientation. This avoids entanglement between probes and local aggregation of self-assembled monolayers, thereby reducing non-specific adsorption and further enhancing sensing performance such as sensitivity and rapid response dynamics.

[0046] This invention not only enables quantitative analysis of the kinetics of interfacial nucleic acid hybridization reactions, but also evaluates the performance of nucleic acid sensors, providing new perspectives and strategies for the design and optimization of biosensors.

[0047] In summary, this invention utilizes the DNA tetrahedral interface for single-molecule fluorescence imaging. This novel technology not only enables real-time monitoring of hybridization events in individual nucleic acid molecules, improving detection sensitivity and specificity, and accurately quantifying the kinetic parameters of hybridization reactions, but also facilitates more accurate performance evaluation of nucleic acid sensors. This is of great significance for the design and optimization of nucleic acid sensors, thereby enabling them to play a greater role in fields such as clinical diagnosis, environmental monitoring, and food safety. Attached Figure Description

[0048] Figure 1 The method for quantitative analysis of interfacial nucleic acid hybridization reactions is shown; (a) probes on a bare interface, (b) probes on a TDN interface, (c) single-molecule imaging visualization of the binding and dissociation of probes and targets, (d) single-molecule fluorescence colocalization, (e) fluorescence time trajectory of probes on a bare interface, (f) fluorescence time trajectory of probes on a TDN interface, and (g) comparison of hybridization kinetics between the two interfaces.

[0049] Figure 2 The construction and characterization of the DNA tetrahedral interface are shown; (a) structural design and assembly of TDN, (b) polyacrylamide gel electrophoresis, the band on the left is the marker, and the band on the right is the TDN separated band, (c) AFM image of TDN, the scale bar in the large AFM image is 100 nm, the single particle image marked by the white solid box is magnified and displayed on the right side of the large field of view image, the scale bar is 10 nm, (d) height distribution of TDN, and (e) particle size distribution of TDN.

[0050] Figure 3 The diagram shows single-molecule observations of the DNA tetrahedral interface; (a) a schematic diagram of the concentration optimization of the single-strand interface, (b) TIRF images of the concentration optimization of the single-strand interface, from left to right, showing 3.2 pM, 32 pM, 100 pM and 320 pM, respectively, (c) a schematic diagram of the distribution of TDN at the single-molecule interface, (d) the distribution pattern of TDN at the single-molecule interface (12 x 12 μm), and (e) the spacing distribution of TDN.

[0051] Figure 4 The dual-color colocalization strategy is shown to distinguish between non-specific and specific binding events; where (a) 638 channels; (b) 561 channels; (c) colocalization;

[0052] Figure 5The quantitative analysis of hybridization kinetics at the TDN interface and single-strand interface is shown; (a) fluorescence time trajectory, (b) histogram distribution of binding events, (c) binding time, and (d) dissociation time. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0054] Example 1: Construction and characterization of DNA tetrahedral interfaces

[0055] DNA tetrahedral structural design and assembly

[0056] This embodiment details the preparation and characterization process of the TDN interface used for interfacial nucleic acid hybridization analysis. The process includes four key steps: DNA tetrahedron synthesis, structural characterization, interface modification, and probe immobilization. The DNA tetrahedron is formed by the self-assembly of four specifically designed single-stranded DNA (ssDNA) strands. Three of the ssDNA strands are modified with biotin molecules at their 5' ends for interface immobilization, while the 5' end of the fourth ssDNA strand is labeled with an AF647 fluorescent molecule for monitoring hybridization events. The DNA used in the experiment (Table 1) was dissolved in 1×TAEMg. 2+ Prepare a solution of 100 μM in a buffer solution containing (20 mM Tris, 12.5 mM MgCl2, pH 8.0).

[0057] Three biotin-modified 66nt ssDNA strands were mixed with 75nt ssDNA labeled with a fluorescent molecule at a concentration ratio of 1:1:1:1. The mixture was heated to 95°C and held for 10 minutes using a PCR instrument, then rapidly cooled to 4°C to form a DNA tetrahedral structure with a side length of 20 bp. In this structure, a side-chain DNA probe labeled with AF647 extended from one vertex of the DNA tetrahedron, while the other three vertices were modified with biotin, such as... Figure 2 As shown in 'a'.

[0058] Table 1. DNA sequences used in this specification

[0059] (2) Structural characterization of DNA tetrahedron

[0060] The synthesized DNA tetrahedra were purified and characterized by polyacrylamide gel electrophoresis (PAGE). Electrophoresis was performed in 1×TAE buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA-2Na, pH 8.0) at a constant voltage of 100 V for 90 minutes. After electrophoresis, the gel was stained with 0.01% (v / v) Gel Red and scanned using a G:BOX Chemi XL1.4 imaging system. The gel image showed a major band (e.g., ...). Figure 2 As shown in b), the correct assembly of the TDN structure was confirmed. Furthermore, the morphology and size of the DNA tetrahedra were characterized using atomic force microscopy (AFM). DNA tetrahedron samples were deposited on the surface of freshly cleaved mica treated with APTES, and imaging was performed using a Bruker Multimode 8.0 SPM instrument to obtain morphological images of the DNA tetrahedra (e.g., b). Figure 2 (As shown in c). The calculated average height of the DNA tetrahedron is approximately 5.5 nm, and the average apparent lateral length is approximately 12.9 nm (as shown in c). Figure 2 (as shown in de).

[0061] (3) Interface treatment and fixation of DNA tetrahedrons

[0062] Interface modification is a crucial step in ensuring effective fixation of TDN to the coverslip surface. In this embodiment, the slide treatment process is as follows: First, the slide is placed in a piranha solution composed of concentrated sulfuric acid and 30% hydrogen peroxide at a volume ratio of 7:3 to thoroughly remove organic contaminants and inorganic impurities from the slide surface. The slide is immersed in the piranha solution for 30 minutes, and then thoroughly rinsed with deionized water in a fume hood to remove any residual piranha solution. After cleaning, the slide requires surface activation to enhance its affinity for biomolecules. The slide is immersed in an ethanol solution containing 0.5% (v / v) aminopropyltriethoxysilane (APTES) and gently shaken for 1 minute to form a uniform silanized layer on the slide surface. The amino functional groups of APTES will provide active sites for subsequent biotin-avidin interactions. After silanization, the slides were rinsed with deionized water and dried. A 1:1 mixture of 0.1 mg / mL bovine serum albumin (BSA) solution and biotin was then formed to create a BSA-biotin complex. The slides were immersed in the BSA-biotin solution and incubated at room temperature for 30 minutes to form a biotinylated surface. The slides were rinsed with PBS buffer to remove unbound BSA-biotin, and then immersed in a 0.5 mg / mL avidin solution and incubated at room temperature for 5 minutes to form a stable avidin layer, providing high-affinity binding sites for subsequent TDN probe immobilization.

[0063] The synthesized and purified TDN probe was incubated with avidin-modified glass slides. Due to the extremely high affinity between biotin and avidin, TDN was firmly immobilized on the slide surface. Incubation at room temperature for 10 minutes ensured sufficient binding between the TDN probe and avidin. 1x PBS buffer and 1x TE buffer were used. 2+ The probe is thoroughly washed with buffer to remove any unbound probes. These steps establish a stable and controllable TDN interface.

[0064] The preparation and characterization process ensures the uniform distribution and stability of the TDN probe at the interface, providing a foundation for subsequent single-molecule fluorescence imaging experiments. This method enables researchers to monitor and analyze nucleic acid probe-target interactions at the interface at the single-molecule level, thus providing important experimental data and a theoretical basis for developing novel, highly sensitive electrochemical biosensors.

[0065] Example 2: Single-molecule observation of DNA tetrahedral interface

[0066] (1) Concentration optimization

[0067] In this embodiment, we conducted a series of concentration optimization experiments to determine the optimal concentration of the TDN probe to achieve the highest hybridization efficiency and signal quality. The experimental design included four different TDN probe concentrations (e.g., ...). Figure 3 (as shown in a) : 320 pM, 100 pM, 32 pM, and 3.2 pM. Probes of each concentration were fixed to a pretreated glass slide surface, and their density was monitored using TIRFM. Imaging conditions were ensured to be consistent for all concentrations by adjusting the laser intensity (20% 638), exposure time (100 ms), acquisition area (512x512 ROI), and frame rate (10000 frames). Experimental results (as shown in a) Figure 3 As shown in b), the optimal probe density is observed in the field of view at a concentration of 100 pM. Ensuring sufficient space between probes to avoid nonspecific aggregation while maintaining adequate density to improve hybridization efficiency, the optimal working concentration is crucial for the design of subsequent experiments and the practical application of probes.

[0068] (2) Distribution pattern of probes at the interface

[0069] To investigate the distribution pattern of TDN probes at the interface (e.g. Figure 3 As shown in c), we used TIRFM to image the TDN probes fixed on the slide. Fluorescence microscopy images revealed the two-dimensional distribution of the probes at the interface. Using these images, we evaluated the probe uniformity and density, and experimental results (as shown in c) were obtained. Figure 3As shown in d), the TDN probes exhibit a heterogeneous distribution at the interface, with a number of 41 and an average spacing of approximately 1200 nm (as shown in d). Figure 3 (as shown in e in the figure). This distribution pattern provides ideal conditions for subsequent single-molecule observations and kinetic analyses.

[0070] Example 3: Real-time monitoring of hybridization events at DNA tetrahedral interfaces

[0071] In this embodiment, we employed a two-color co-localization strategy to monitor hybridization events at the DNA tetrahedral interface in real time. Figure 4 The TDN probe was labeled with the red fluorescent molecule AF647, and the target was labeled with the green fluorescent molecule CY3B. After incubation of the TDN probe and the target, fluorescence signals were acquired at the interface in real time using TIRFM, and the changes of the two colors of fluorescence signals over time were recorded. The red fluorescence signal of the interface-fixed TDN probe was observed in the 638nm channel (e.g., red fluorescence signal of the TDN probe). Figure 4 As shown in a), the 561nm channel represents the green fluorescent signal of the target DNA (as shown in a diagram). Figure 4 (As shown in b). Co-localization events are defined as events in which red and green fluorescence signals spatially overlap (e.g., as shown in b). Figure 4 As shown in c), this indicates the occurrence of specific hybridization. Only green signals are defined as targets that have non-specifically bound to the interface. In our experiments, we observed distinct colocalization "scintillation" events, the frequency and duration of which were used to analyze hybridization kinetics. Through colocalization, we were able to identify specific hybridization events. Specific hybridization events exhibited high binding frequencies and long binding times, while non-specific events manifested as brief, low-intensity fluorescence signals.

[0072] Example 4: Quantitative Analysis of DNA Tetrahedral Interface Hybridization Kinetics

[0073] (1) Fluorescence time trajectory analysis

[0074] In this embodiment, we analyzed the fluorescence time trajectory of the TDN interface probe hybridization with the target DNA to determine binding and dissociation events (e.g., Figure 5(As shown in a) The hybridization experiments of the TDN probe and target DNA were performed at the optimal working concentration. The concentration of the TDN probe was 100 pM, and the concentration of the target DNA was 20 nM. The binding and dissociation processes of each hybridization event were obtained by recording the fluorescence signal using TIRFM. By analyzing the time trajectory, we were able to observe the dynamic interaction between the TDN probe and the target DNA, including the frequency and duration of binding events. Experimental data showed that binding events of the TDN probe and target DNA were characterized by fluorescence intensities close to 1, while dissociation events were characterized by fluorescence intensities close to 0. Within 50 seconds, the fluorescence intensity-time trajectory of the TDN probe showed frequent binding (15 binding events).

[0075] (2) Combining the histogram distribution of events

[0076] The histogram distribution of binding events is constructed by statistically analyzing the time points of all recorded binding events, revealing the frequency and kinetic characteristics of these events. This distribution reveals the frequency of binding events, providing an intuitive understanding of the dynamic characteristics of the hybridization reaction. By analyzing this data, we can determine the distribution pattern of binding events, including their frequency and the width of the distribution. Histograms are shown (e.g.) Figure 5 As shown in b), the binding events of the TDN probe were most frequent in the range of 20 to 40 events. The frequency of binding events gradually decreased with the increase of the number of events, indicating that the binding of the TDN probe to the target DNA has rapid kinetic characteristics. The total number of binding events was 2799, indicating that a large number of binding events were observed in the experiment.

[0077] (3) Statistical analysis combining time and dissociation time

[0078] Statistical analysis of binding and dissociation times was performed by analyzing the time trajectories of individual hybridization events. We recorded the binding and dissociation times for each event and constructed the corresponding statistical distributions (e.g., Figure 5 (as shown in cd in the diagram). By fitting these distributions to an exponential decay model, we obtained the average binding and dissociation times, as well as the rate constant. These parameters provide us with detailed information about the hybridization kinetics, including the stability of the reaction. The TDN probes exhibited a higher binding frequency and a longer average binding time (mean binding time 1.21 s, mean dissociation time 16.81 s), indicating a more stable interaction with the target DNA. Furthermore, the shorter dissociation time of the TDN probes indicates that the probes can rapidly rebind to new target molecules, which is crucial for improving the sensitivity and dynamic range of the sensor.

[0079] Example 5: Evaluation of hybridization kinetics at single-strand interfaces

[0080] To evaluate the advantages of TDN interfaces over traditional single-stranded DNA interfaces, we conducted comparative experiments. Under the same experimental conditions, we compared the hybridization kinetics (e.g., TDN probes and single-stranded DNA probes, sequences of which are shown in Table 1 for ssDNA-biotin) with those of TDN probes. Figure 5 (As shown in Figure ac). By comparing the binding and dissociation rates of the two probes, as well as the average binding time, we found that the TDN interface resulted in an increased number of binding events and accelerated reactions compared to the single-stranded interface. The TDN interface achieved approximately a 2-fold improvement in single-molecule DNA hybridization kinetics. The three-dimensional structure of the TDN probe provides a better spatial orientation, increasing the effective collision probability with the target sequence and thus improving hybridization efficiency, demonstrating the potential of the TDN interface to enhance the performance of nucleic acid sensors. Furthermore, the TDN probe also exhibited lower non-specific binding, further confirming its advantages in improving detection specificity and sensitivity.

[0081] In summary, the novel method for quantitative analysis of interfacial nucleic acid hybridization reactions of the present invention has significant advantages in improving detection sensitivity, specificity, real-time monitoring capability, quantitative analysis capability, and evaluation of nucleic acid sensor performance, providing new possibilities for the development and application of biosensor technology.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A novel method for quantitative analysis of interfacial nucleic acid hybridization reactions, characterized in that, Includes the following steps: S1: Constructing a DNA tetrahedral interface, comprising: constructing a DNA tetrahedron with a single-stranded DNA probe extending from one vertex for capturing a target DNA sequence, and fixing the DNA tetrahedron to the surface of a coverslip to construct a DNA tetrahedral interface; S2: Single-molecule observation of DNA tetrahedral interface, including: imaging the DNA tetrahedral interface by total internal reflection fluorescence microscopy, and then studying the distribution pattern of DNA tetrahedra on the interface. S3: Real-time monitoring of DNA tetrahedral interface hybridization events, which includes: using two different fluorescent molecules to label TDN probes and target DNA respectively, employing dual-color co-localization and analysis of fluorescence signals to achieve real-time monitoring of DNA tetrahedral interface hybridization events; S4: Quantitative analysis of hybridization kinetics at the DNA tetrahedral interface, which includes: analyzing hybridization kinetics at the DNA tetrahedral interface by recording changes in fluorescence signals over time.

2. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 1, characterized in that, In step S1, the DNA tetrahedron binds to the surface of the coverslip via a biotin-avidin interaction.

3. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 2, characterized in that, In step S1, biotin molecules for interface fixation are modified at three vertices of the DNA tetrahedron, and a single-stranded DNA probe extends from the other vertex to capture the target nucleic acid sequence. The extended single-stranded DNA probe can be further modified with fluorescent groups for dual-color co-localization imaging.

4. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 1, characterized in that, Step S1 also includes: characterizing DNA tetrahedra using polyacrylamide gel electrophoresis to confirm their purity and yield; characterizing DNA tetrahedra using atomic force microscopy to confirm their correct assembly from a morphological perspective; and performing a detailed analysis of the structure and size of DNA tetrahedra based on AFM imaging data to obtain statistical data on height and particle size.

5. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 1, characterized in that, Step S2 also includes optimizing the TDN probe density and target DNA concentration to achieve optimal hybridization efficiency and signal quality.

6. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 1, characterized in that, In step S3, the TDN probe is labeled with the red fluorescent molecule AF647, and the target DNA is labeled with the green fluorescent molecule CY3B. After the TDN probe and the target DNA are incubated, the fluorescence signal is collected through the TIRFM real-time observation interface, and the changes of the two colors of fluorescence signal over time are recorded.

7. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 1, characterized in that, Step S4 also includes: performing statistical analysis on the binding time and dissociation time to extract key kinetic parameters, including the binding rate and dissociation rate.

8. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 1, characterized in that, In step S4, the hybridization experiment of the TDN probe and the target DNA is performed at the optimal working concentration, which is: 100 pM TDN probe and 20 nM target DNA.

9. The novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to claim 1, characterized in that, The new method also includes the following steps: S5: By comparing the hybridization kinetics of DNA tetrahedral interfaces with those of single-stranded DNA interfaces, we evaluate the potential of DNA tetrahedral interfaces in improving the performance of nucleic acid sensors.

10. The application of a novel method for quantitative analysis of interfacial nucleic acid hybridization reactions according to any one of claims 1-9 in evaluating the performance of nucleic acid sensors.

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