A programmable DNA hydrogel-based digital imaging detection platform and application

Abstract

The application belongs to the field of nucleic acid functional materials, and discloses a digital imaging detection platform based on programmable DNA hydrogel and application. The platform uses lock probes and primers to synthesize super-long single-stranded DNA-Chain-1 and DNA-Chain-2 through double rolling circle amplification, and cross-linking is formed through base complementary pairing to form a micrometer-level porous three-dimensional hydrogel. The modular integration target aptamer coding region in the lock probe is combined with the TS DNA; in the presence of the detected substance, specific binding is promoted to release the TS DNA and activate Cas12a, trigger the transcleavage activity to degrade the fluorescent probe, and convert the target recognition event into countable discrete fluorescent points. Finally, quantitative analysis is realized through fluorescence microscopic imaging and point counting. The application overcomes matrix interference, and can realize the extended detection of various pollutants by replacing the aptamer, and is suitable for on-site digital multi-target screening.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid functional materials, specifically relating to a digital imaging detection platform and its application based on programmable DNA hydrogel. Background Technology

[0002] Food and agricultural product safety is crucial to public health, agricultural production, and sustainable social development. With the increasing complexity of the food supply chain, the low-level residues of mycotoxins, pesticides, algal toxins, and other small-molecule contaminants in grains, beverages, and agricultural products have attracted widespread attention. These contaminants are typically characterized by low concentrations, uneven distribution, strong matrix interference, and indistinct early signals. Traditional detection methods often rely on large instruments, complex sample pretreatment, or long reaction times, making it difficult to meet the needs of rapid on-site screening, early risk warning, and highly sensitive quantitative analysis. Therefore, developing a detection method that combines high sensitivity, good anti-interference capabilities, ease of operation, and platform scalability is of great significance for food and agricultural product safety monitoring.

[0003] The development of nucleic acid functional materials has provided new design ideas for biosensing technology. DNA is not only a carrier of genetic information, but also, based on its complementary base pairing and programmable sequence properties, can be used to construct nanomaterials with specific structures and functions. Among them, DNA hydrogels are a class of three-dimensional network materials formed by functional nucleic acid molecules through self-assembly, hybridization cross-linking, or isothermal amplification. They are characterized by mild reaction conditions, good biocompatibility, tunable structural composition, and easy introduction of functional modules. Compared with traditional polymer hydrogels or inorganic porous materials, the outstanding advantage of DNA hydrogels lies in the designability of their constituent units, thus enabling the integration of various biosensing functions while maintaining a flexible three-dimensional network structure.

[0004] Based on this characteristic, DNA hydrogels not only serve as three-dimensional scaffolds but also possess the potential to be developed into programmable functional materials. Through the rational design of nucleic acid sequences, cross-linking sites, and network structures, the formation mode, spatial structure, and functional site distribution of DNA hydrogels can be regulated, thus providing a designable material basis for different sensing systems. However, existing DNA hydrogel sensing systems still have certain limitations. Most platforms primarily use DNA hydrogels as immobilization carriers for pre-fabricated probes, stimulus-response materials, or signal amplification scaffolds, with their functions largely limited to structural support and reaction bearing, failing to fully leverage their advantages of programmable sequences and integrated functional modules. While some systems introduce recognition probes or response elements, the lack of effective coordination between target recognition, signal conversion, and signal amplification leads to insufficient triggering efficiency for low-abundance targets, limiting detection sensitivity and stability. Furthermore, traditional DNA hydrogel detection relies heavily on overall fluorescence intensity, color changes, or gel state changes as readout methods, making them susceptible to complex sample matrices, background signals, and operational differences, resulting in insufficient quantitative accuracy. In addition, existing systems are mostly designed for single targets, and functional module replacement is not convenient, limiting their widespread application in the detection of various food and agricultural product contaminants. Therefore, there is an urgent need to build a programmable DNA hydrogel detection platform that can integrate target recognition, signal conversion, enzyme amplification, and digital readout. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and provide a programmable DNA hydrogel, a digital imaging detection platform and its applications.

[0006] The technical solution adopted in this invention is:

[0007] In a first aspect, the present invention provides a programmable DNA hydrogel, wherein the DNA hydrogel is formed by hybridization and entanglement of DNA-Chain-1-TS sequences and DNA-Chain-2 sequences through complementary base pairing, forming a three-dimensional network structure:

[0008] DNA-Chain-1-TS: This sequence is obtained by sequence-specific hybridization and rolling circle amplification of Padlock-1 sequence and Primer-1 sequence to obtain DNA-Chain-1 sequence, and then sequence-specific hybridization with TS DNA sequence to obtain DNA-Chain-1-TS.

[0009] DNA-Chain-2: This sequence was obtained by sequence-specific hybridization and rolling circle amplification of Padlock-2 and Primer-2 sequences.

[0010] The Padlock-1 sequence comprises, from the 5' end to the 3' end, the following: a first hybridization arm region, a target-specific aptamer coding region, and a second hybridization arm region.

[0011] The programmable DNA hydrogel has an interconnected porous three-dimensional network structure with micron-sized pores.

[0012] Optionally, the Primer-1 sequence is as shown in SEQ ID NO.3.

[0013] Optionally, the Primer-2 sequence is as shown in SEQ ID NO.5.

[0014] Optionally, the Padlock-2 sequence is as shown in SEQ ID NO.6.

[0015] Optionally, the TS DNA sequence is as shown in SEQ ID NO.7.

[0016] Specifically, the coding region of the target-specific aptamer is a replaceable region. By replacing this coding region, the DNA-Chain-1 sequence obtained by rolling circle amplification carries recognition sites for different targets, wherein the targets can be selected from ochratoxin A, microcystin-LR, aflatoxin B1, or zearalenone.

[0017] Optionally, the inverse complementary sequence of the coding region of the target-specific aptamer sequence is shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12.

[0018] Optionally, when the target is ochratoxin A, the Padlock-1 sequence is as shown in SEQ ID NO.4.

[0019] Secondly, the application of the programmable DNA hydrogel provided in the first aspect of this invention in the preparation of a digital imaging detection platform for detecting small molecule contaminants, wherein the detection of the small molecule contaminants includes the following steps:

[0020] (1) Target recognition and binding: The sample to be tested is mixed with DNA-Chain-1-TS and incubated;

[0021] (2) Gelification and signal amplification: DNA-Chain-2, Cas12a / crRNA ribonucleoprotein complex and fluorescent probe were added to the reaction system of step (1), and crosslinked to form DNA hydrogel under incubation conditions;

[0022] (3) Imaging and quantification: The DNA hydrogel generated in step (2) is subjected to fluorescence microscopy imaging. The concentration of small molecule pollutants in the sample is calculated by counting the number of discrete fluorescent spots in the image.

[0023] The beneficial effects of this invention are:

[0024] (1) This invention constructs a programmable DNA hydrogel sensing platform based on double rolling circle amplification, which designs and regulates the hydrogel network structure through nucleic acid sequence, and integrates aptamer recognition, signal conversion, Cas12a amplification and fluorescence readout modules into the same three-dimensional system.

[0025] (2) This invention establishes a modular and expandable CRISPR / Cas12a digital imaging detection method, which can convert target recognition events into countable fluorescent spots, realize highly sensitive quantitative detection of low-abundance pollutants, and can be extended to the analysis of various food pollutants by changing the recognition element. Attached Figure Description

[0026] Figure 1 The following are characterizations of the DNA hydrogel, with a scale bar of 500 nm: (A) Schematic diagram of DNA hydrogel synthesis; (B) 12% PAGE analysis of RCA products; (C) Photographs of the DNA hydrogel under natural light (left) and ultraviolet light (right, 365 nm, SYBR Green I staining); (D) Change in DNA hydrogel volume with RCA reaction time; (E) SEM image of the DNA hydrogel; (F) Rheological properties of the DNA hydrogel.

[0027] Figure 2 The fluorescence response analysis of different concentrations of OTA is performed with a scale bar of 50 μm. Specifically, it includes: (A) endpoint fluorescence images after treatment with different concentrations of OTA; (B) statistical analysis of the number (N) of fluorescence spots induced by different concentrations of OTA; and (C) calibration curve between fluorescence spot count and the logarithm of OTA concentration.

[0028] Figure 3 This is a validation of the analytical performance and programmability of the programmable DNA hydrogel sensing platform detection system, with a scale bar of 50 μm. Specifically: (A) Detection specificity analysis: at the same concentration of 10 pg·mL -1 (a) Comparison of fluorescence spot signals triggered by OTA and interfering toxins (MC-YR, MC-LR, AFB1, and ZEN); (b) Analysis of OTA recovery in complex cereal matrices (corn and beer) using the standard addition method; (c) Stability testing of the biosensor at an OTA concentration of 10 pg·mL. -1(D) Programmability verification of the programmable DNA hydrogel sensing platform system, where the horizontal axis represents the type of identification element and the vertical axis represents the type of analyte to be tested (ad represents OTA, MC-LR, AFB1 and ZEN respectively). Detailed Implementation

[0029] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0030] Example 1

[0031] The oligonucleotide sequences used in this invention are shown in Table 1. The DNA hydrogel of this invention is constructed using a dual-rolling circle amplification (dual-RCA) strategy. Figure 1 A).

[0032] In the table, Apt-OTA is the specific aptamer sequence for ochratoxin A (OTA) and serves as the front-end target recognition unit in this system. Because Padlock-1 contains an Apt-OTA-related sequence, the resulting DNA-Chain-1, after rolling circle amplification, carries repetitively distributed OTA recognition sites. The subsequently introduced TS DNA can bind to the Apt-OTA recognition region in DNA-Chain-1 through partial sequence complementarity, thereby forming DNA-Chain-1-TS, providing the basis for subsequent OTA-triggered signal transduction.

[0033] Table 1. DNA sequences involved in the embodiments of the present invention.

[0034]

[0035] The preparation method and characterization of the DNA hydrogel in this embodiment are as follows:

[0036] 1) Preparation of CrRNA

[0037] First, the CrRNA template DNA was constructed. CrRNA-TS (SEQ ID NO.1) and CrRNA-CS (SEQ ID NO.2) were prepared to a final concentration of 500 nM and mixed with 1×NEBuffer r2.1, followed by DEPC-treated water to a final volume of 20 µL. The mixture was heated at 95°C for 5 min to fully denature the oligonucleotides, and then slowly cooled to 4°C to anneal the two complementary strands, forming a double-stranded DNA template for CrRNA transcription. This template was stored at -20°C for later use.

[0038] Subsequently, CrRNA was transcribed in vitro using this double-stranded DNA template. The total volume of the transcription reaction was 20 μL, containing 100 UT7 RNA polymerase, 0.5 mM NTP, 5 μL 500 nM template DNA, 20 U RNase inhibitor, 1 mM DTT stabilizer, and 1×T7 RNA reaction buffer. The solution was incubated at 37°C for 2 h to complete CrRNA transcription. The system was then heated to 65°C and held for 10 min to terminate the transcription reaction. To remove residual DNA template, 4 µL of the transcription product was taken, 2 U DNase I and 1×DNase I reaction buffer were added, and the total volume was brought to 20 µL with DEPC-treated water. The system was then incubated at 37°C for 30 min to allow DNase I to fully digest the residual DNA template. Finally, the reaction was terminated by heating to 65°C and holding for 10 min. The resulting CrRNA product was stored at -20°C for later use.

[0039] 2) Preparation of ring template

[0040] Taking the preparation of the circ-1 circular template as an example, Primer-1 (10 µM) and Padlock-1 (10 µM) were mixed in 1×T4 DNA ligase buffer. Subsequently, the mixture was subjected to programmed thermal annealing to promote sequence-specific hybridization between the primers and the lock-lock probe.

[0041] The specific annealing procedure is as follows: first, denature at 95°C for 5 min; then, at 0.1°C·s -1 The temperature was slowly reduced to 65°C and held at 65°C for 5 min; then the temperature was further reduced at a rate of 0.1°C·s. -1 The temperature was slowly reduced to 25°C and held at 25°C for 5 minutes; finally, it was rapidly reduced to 4°C.

[0042] After successful hybridization between Primer-1 and Padlock-1, T4 DNA ligase was added to a 20 µL reaction mixture to bring the final concentration to 20 U·µL. -1 The ligation reaction was carried out at 25°C for 4 h, followed by heating at 65°C for 10 min to inactivate T4 DNA ligase, yielding a circ-1 circular template.

[0043] The circ-2 ring template was prepared using the same method, that is, Primer-1 and Padlock-1 were replaced with Primer-2 and Padlock-2, and the circ-2 template was obtained by following the above annealing and linking procedures.

[0044] 3) Preparation of double rolling circle amplification DNA hydrogel

[0045] For the rolling circle amplification reaction system, 4 μL circ-1, 50 mM NaCl, 1 mM dNTPs, and 0.1 mg·mL⁻¹ were added. -1 Recombinant albumin, 1×phi29 DNA polymerase buffer, and 0.25 U·μL -1 Phi29 DNA polymerase was mixed, and the total volume was brought to 40 μL using DEPC-treated water. The reaction system was incubated at 37°C for 10 h to perform the RCA reaction, yielding DNA-Chain-1.

[0046] Meanwhile, DNA-Chain-2 was synthesized under the same conditions using circ-2 as a template, and the resulting product was stored at -20°C for later use.

[0047] Subsequently, 10 μL of annealed 10 μM TS DNA was mixed with 40 μL DNA-Chain-1 and incubated in 1× annealing buffer (10 mM Tris, 100 mM NaCl, 10 mM KCl) at 50°C for 20 min, followed by incubation at 0.1°C·s⁻¹. -1 The temperature was slowly reduced to 25°C and incubated for another 10 min. After the reaction was complete, unbound or unassembled free TS DNA was removed using a 100 K NMWLAmicon Ultra-0.5 centrifuge filter according to the manufacturer's instructions. Subsequently, the retained DNA-Chain-1 was redispersed in 40 μL of DEPC-treated water to obtain DNA-Chain-1-TS.

[0048] Finally, 40 μL of DNA-Chain-1-TS and 40 μL of DNA-Chain-2 were mixed, and the total volume was brought to 100 μL using DEPC-treated water. The mixture was incubated at 37°C with continuous shaking at 600 rpm for 30 min to obtain the DNA hydrogel.

[0049] 4) Characterization of DNA hydrogels:

[0050] The RCA products were validated by 12% polyacrylamide gel electrophoresis. Figure 1 As shown in Figure B, circ-1 and circ-2 exhibit significant migration lag compared to the linear precursor, indicating that the cyclization reaction proceeded successfully. The RCA products, due to their extremely high molecular weight, remained in the sample wells, confirming the successful synthesis of ultralong single-stranded DNA. When the two RCA products were mixed under gentle oscillation conditions, the DNA strands tangled and cross-linked through complementary sequence hybridization, forming a visible three-dimensional DNA hydrogel.

[0051] Further staining with SYBR Green I revealed a significant fluorescence response, indicating that the DNA hydrogel network is primarily cross-linked through complementary base pairing. Figure 1 C). Considering both the integrity of the hydrogel network and the need to avoid over-amplification, a reaction time of 10 h for RCA is deemed appropriate. Figure 1 D). Scanning electron microscopy results showed that the DNA hydrogel had an interconnected porous structure and formed micron-sized pores ( Figure 1 E). This porous network provides space for the introduction of sensing components and provides a structural basis for subsequent target recognition and signal generation. The mechanical properties of the network were characterized using rheological tests. The results show that the storage modulus G' is consistently higher than the loss modulus G'', indicating that the hydrogel possesses stable solid-like behavior and macroscopic structural integrity. Furthermore, the relatively low G' value of approximately 3.5 Pa indicates that the hydrogel remains highly hydrated and flexible. Figure 1 F).

[0052] Example 2

[0053] Take 5 μL of the sample solution to be tested and mix it with 40 μL of DNA-Chain-1-TS prepared in Example 1, and incubate at 25°C and 300 rpm for 30 min. Then, add 40 μL of DNA-Chain-2, IS-10, FAM-BHQ1 probe, Cas12a / CrRNA RNP, RNase inhibitor and 10× reaction buffer prepared in Example 1 in sequence, and incubate the reaction at 37°C and 600 rpm.

[0054] After the reaction, the resulting DNA hydrogel was transferred to a single-hole glass slide with a diameter of 12 mm and a depth of 0.054 mm for imaging using an inverted fluorescence microscope. Fluorescent spots were excited by a 498 nm laser, and the emitted photons were collected and imaged through a 10× eyepiece and a 40× objective lens, with an exposure time set to 500 ms. Fluorescent spots within a 600×600 pixel region of interest were counted using ImageJ software, with a size threshold set to only count fluorescent spots larger than 30×30 pixels. The final total number of fluorescent spots was obtained by analyzing five images.

[0055] Under optimized conditions, the analytical performance of the DNA hydrogel for OTA was evaluated by statistically analyzing the number of fluorescent spots generated by target triggering. For example... Figure 2 As shown, the density of discrete fluorescent dots gradually increases with increasing OTA concentration, indicating that the number of fluorescent dots can reflect changes in target concentration. Quantitative analysis results show that at 2 pg·mL⁻¹… -1 Up to 100 ng·mL -1Within the specified range, there is a good linear relationship between the number of fluorescent spots and the logarithm of OTA concentration. The corresponding linear regression equation is N = 93.5logC + 275.2, where N is the number of fluorescent spots, C is the OTA concentration, and the correlation coefficient R is [missing value]. 2 The value was 0.98, and the limit of detection was 1.5 pg·mL. -1 .

[0056] The analytical performance of this detection system stems from the combination of a three-dimensional DNA hydrogel network and modular recognition elements. In specificity evaluation, the DNA hydrogel only triggered Cas12a trans-cleavage and produced obvious fluorescent spots in the presence of OTA, while showing no significant response to similar toxins such as MC-YR (microcystin-YR), MC-LR (microcystin-LR), AFB1 (aflatoxin B1), and ZEN (zearalenone). This indicates that the analytical specificity of this system is primarily determined by the specific binding between the aptamer and the target. Figure 3 A).

[0057] In actual sample analysis, the standard addition method was used to evaluate the detection capability of DNA hydrogel in corn and beer samples. The results showed that, at different spiking levels, the recovery rate of OTA by DNA hydrogel ranged from 98.19% to 106.82%, with relative standard deviations as low as 2.22% and 3.01%, indicating that this method still has good quantitative accuracy and repeatability in complex food matrices. Figure 3 B).

[0058] Further detection at 0.05 ng / mL was performed using a programmable DNA hydrogel sensing platform prepared independently in five batches. -1 The relative standard deviation of OTA was 4.66%, indicating that the preparation process and fluorescence spot counting strategy of this platform have good stability. Figure 3 C).

[0059] More importantly, this detection system is highly programmable. This characteristic mainly stems from the modular design of the Padlock-1 template strand. Specifically, Padlock-1, from the 5' end to the 3' end, contains: a first hybridization arm, a coding region for the target-specific aptamer, and a second hybridization arm. The hybridization arms at both ends are used for primer annealing, circularization, and subsequent rolling circle amplification (RCA); while the coding region in the middle serves as the amplification template, determining the type of recognition site carried by the product strand (DNA-Chain-1).

[0060] Therefore, by keeping the primers and amplification conditions unchanged, simply replacing the coding region in the middle of Padlock-1 with the reverse complementary sequence of the corresponding aptamer sequence (e.g., SEQ ID NO. 10, 11, 12) of the target analyte (e.g., MC-LR, AFB1, ZEN) can construct a detection system for different pollutants. The results show that ( Figure 3 D) A noticeable fluorescent spot will only be generated when the analyte matches the recognition module. Therefore, this invention can achieve expanded detection of multiple pollutants by replacing the front-end recognition module in Padlock-1.

[0061] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A programmable DNA hydrogel, characterized in that, The DNA hydrogel is formed by the hybridization and entanglement of DNA-Chain-1-TS and DNA-Chain-2 sequences through complementary base pairing, creating a three-dimensional network structure. DNA-Chain-1-TS: This sequence is obtained by sequence-specific hybridization and rolling circle amplification of Padlock-1 sequence and Primer-1 sequence to obtain DNA-Chain-1 sequence, and then sequence-specific hybridization with TS DNA sequence to obtain DNA-Chain-1-TS. DNA-Chain-2: This sequence was obtained by sequence-specific hybridization and rolling circle amplification of Padlock-2 and Primer-2 sequences. The Padlock-1 sequence comprises, from the 5' end to the 3' end, the following: a first hybridization arm region, a target-specific aptamer coding region, and a second hybridization arm region.

2. The programmable DNA hydrogel according to claim 1, characterized in that, The programmable DNA hydrogel has an interconnected porous three-dimensional network structure with micron-sized pores.

3. The programmable DNA hydrogel according to claim 1, characterized in that, The Primer-1 sequence is shown in SEQ ID NO.

3.

4. The programmable DNA hydrogel according to claim 1, characterized in that, The Primer-2 sequence is shown in SEQ ID NO.

5.

5. The programmable DNA hydrogel according to claim 1, characterized in that, The Padlock-2 sequence is shown in SEQ ID NO.

6.

6. The programmable DNA hydrogel according to claim 1, characterized in that, The TS DNA sequence is shown in SEQ ID NO.

7.

7. The programmable DNA hydrogel according to claim 1, characterized in that, The coding region of the target-specific aptamer is a replaceable region. By replacing this coding region, the DNA-Chain-1 sequence obtained by rolling circle amplification can carry recognition sites for different targets, wherein the targets can be selected from ochratoxin A, microcystin-LR, aflatoxin B1 or zearalenone.

8. The programmable DNA hydrogel according to claim 1, characterized in that, The inverse complementary sequence of the coding region of the target-specific aptamer sequence is shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.

12.

9. The programmable DNA hydrogel according to claim 1 or 7, characterized in that, When the target is ochratoxin A, the Padlock-1 sequence is as shown in SEQ ID NO.

4.

10. The application of the programmable DNA hydrogel according to any one of claims 1-9 in the preparation of a digital imaging detection platform for detecting small molecule pollutants, characterized in that, The detection of the small molecule pollutants includes the following steps: (1) Target recognition and binding: The sample to be tested is mixed with DNA-Chain-1-TS and incubated; (2) Gelification and signal amplification: DNA-Chain-2, Cas12a / crRNA ribonucleoprotein complex and fluorescent probe were added to the reaction system of step (1), and crosslinked to form DNA hydrogel under incubation conditions; (3) Imaging and quantification: The DNA hydrogel generated in step (2) is subjected to fluorescence microscopy imaging. The concentration of small molecule pollutants in the sample is calculated by counting the number of discrete fluorescent spots in the image.

Images