A method for in-situ synthesis of gold nanoclusters and manganese metal-organic framework fluorescent aptamer sensors from DNA nanoflowers.

Abstract

This invention relates to a method for the in-situ synthesis of gold nanoclusters (AuNCs) and manganese metal-organic frameworks using DNA nanoflowers as templates. AuNCs with high fluorescence signals were synthesized in situ on DNA nanoflowers via a hydrothermal reduction method. The manganese metal-organic framework immobilizes hairpin chains 2, serving as sites for local catalytic hairpin assembly and amplifying the target signal. The added target analyte binds to the aptamer hairpin chain 1, influencing the catalytic hairpin self-assembly reaction and further affecting the number of hairpin chains acting on the DNA nanoflowers. The 3' end of the hairpin chain 3, rich in G bases, quenches the AuNCs, generating a signal change, thereby achieving sensitive detection of the target analyte. This invention demonstrates high sensitivity, high accuracy, high stability, and excellent practical application capabilities for the detection of aflatoxin B1 in food.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a method for preparing a gold nanocluster (AuNCs) and manganese metal-organic framework (Mn-MOF) fluorescent aptamer sensor synthesized in situ using DNA nanoflowers as templates. Background Technology

[0002] Aflatoxins are a class of secondary metabolites produced by fungi in the genera *Aspergillus* and *Aspergillus parasiticus*, possessing strong toxicity and carcinogenicity. Aflatoxin B1 (AFB1) is the most widespread and toxic of the aflatoxins, causing varying degrees of harm to the human liver, immune system, and reproductive system. Aflatoxins mainly enter the human food chain through contamination of grains, feed, and other foods. Due to their strong heat resistance and chemical stability, aflatoxins are difficult to completely remove or reduce in content during traditional food processing and storage. Therefore, strengthening the detection of aflatoxins in food is of paramount importance to protect public health and food safety.

[0003] Currently, chromatographic analysis methods, including liquid chromatography, liquid chromatography-tandem mass spectrometry, and gas chromatography-tandem mass spectrometry, are mainly used for the detection of AFB1. These methods can achieve high sensitivity. However, these methods require sophisticated equipment and technical expertise, and sample processing is complex, time-consuming, and costly, making them unsuitable for large-scale food safety testing. Therefore, developing a simple, efficient, and rapid detection method is one of the effective ways to address the problem of AFB1 exceeding the standard in food.

[0004] Fluorescent aptamer sensors are molecular recognition sensors based on aptamers. They utilize fluorescent groups as signal transducers to achieve high sensitivity and selectivity for target molecules, offering advantages such as fast detection speed, ease of operation, and high sensitivity. Aptamers are molecular probes that can specifically bind to targets. Compared to antibodies, aptamers have advantages such as simple structure, ease of synthesis and modification, wide applicability, and ease of preparation and storage. Furthermore, aptamers are not limited by the type, size, or source of the target molecule, allowing for the design and screening of various target molecules, including non-natural molecules. Fluorescent aptamer sensors typically involve labeling the aptamer or its complementary strand, using the conformational change caused by the addition of the target molecule to activate or quench the fluorescence signal. However, fluorescent labeling of aptamers often has drawbacks, such as high cost and reduced specificity. Therefore, this invention, for the first time, utilizes DNA nanoflowers to synthesize AuNCs with high fluorescence intensity in situ. The label-free AuNCs fluorescent probe reduces costs while maintaining high stability.

[0005] DNA nanoflowers are highly ordered self-assembled DNA structures prepared using DNA nanotechnology. Their morphology resembles a flower, exhibiting high spatial precision and programmability. DNA nanoflowers are spontaneously assembled from circular DNA template strands via polymerase-driven rolling circle replication (RCR). Their structural formation and stability depend on the DNA sequence and pairing rules, allowing for multi-level morphological and structural functions through precise design. DNA nanoflowers are widely used in biomedical fields such as drug delivery and molecular diagnostics. However, their lack of molecular recognition capabilities significantly limits their application in food safety detection, particularly for small molecule mycotoxins. To address this, this invention precisely designs the base sequence of a circular DNA template strand and replicates a large number of complementary hairpin strand sequences on the DNA nanoflower via RCR amplification, enabling specific recognition with the hairpin strands involved in the AFB1 detection region. This invention utilizes a precisely designed three-hairpin strand structure to catalyze the hairpin assembly reaction, improving the sensitivity of the detection system. When hairpin 3 (H3) binds to the nanoflower, the G-rich base (6G) at its 3' end approaches the AuNCs on the nanoflower, thereby quenching the fluorescence signal. This eliminates the need for additional quenching labeling groups or quenching materials, reducing the cost and complexity of the sensor. Furthermore, this invention utilizes Mn-MOF as the catalytic hairpin assembly site, increasing the concentration of local reactants and further improving the efficiency of the catalytic reaction.

[0006] This invention addresses the problems of existing detection technologies by providing a method for preparing a fluorescent sensor using in-situ synthesis of AuNCs from DNA nanoflowers and Mn-MOF, for accurate detection of AFB1 in food. The aim is to overcome the drawbacks of commonly used detection methods, such as complex operation, high cost, and low sensitivity. This method uses in-situ synthesized AuNCs as fluorescent signal tags and replaces quenching groups with G-rich sequences, effectively reducing sensor preparation costs and improving detection sensitivity. Furthermore, the introduction of Mn-MOF as a catalytic hairpin assembly site enhances detection rate and accuracy. This invention enables low-cost, rapid, highly sensitive, and highly accurate detection of AFB1 in food samples, facilitating its widespread application. Summary of the Invention

[0007] A method for preparing a DNA nanoflower in situ synthetic gold nanoclusters and manganese metal-organic framework fluorescent aptamer sensor includes the following steps:

[0008] (1) Preparation of DNA nanoflowers: The template strand (Padlock) and primer strand (Primer) were co-incubated to form a circular template under the action of T4 DNA ligase. The Padlock itself contains the complementary sequence (T-rich) for synthesizing AuNCs and the complementary sequence of the H3 strand. The circular template was added to the Phi29 DNA polymerase system solution, which includes 1X Phi29 buffer, dNTPs and Phi29 polymerase. The mixture was incubated at 30°C. The polymerase-driven rolling cycle amplification generated bifunctional DNA nanoflowers. The bifunctional DNA nanoflowers are DNA nanoflowers with in situ incubation of metal nanoclusters and nucleic acid recognition functions.

[0009] (2) In situ synthesis of AuNCs from DNA nanoflowers: The DNA nanoflowers obtained in step (1) were added to sodium citrate buffer and chloroauric acid solution was added. The mixture was then placed in a magnetic stirrer and incubated to obtain fluorescent signal-tagged DNA nanoflowers@AuNCs.

[0010] (3) Preparation and modification of Mn-MOF: Manganese acetate dihydrate and H2TCPP were mixed evenly in N,N-dimethylformamide (DMF) solution and incubated at room temperature to obtain Mn-MOF. Then, the carboxyl groups on the surface of Mn-MOF were activated with EDC / NHS solution and co-incubated with amino-modified hairpin 2 (H2) chain to obtain Mn-MOF-H2 after ligation.

[0011] (4) Construction of fluorescent aptamer sensor: Samples containing the target analyte were prepared into test solutions of different concentrations. Hairpin chains (H1), Mn-MOF-H2 and hairpin 3 (H3) chains were added. A large amount of Mn-MOF-H2 / H3 was obtained by catalyzing the hairpin self-assembly reaction. After mixing and incubating with DNA nanoflowers@AuNCs prepared in step (2), the H3 chain modified on Mn-MOF was complementary to the DNA nanoflowers. Subsequently, the fluorescence of AuNCs was quenched by the 6G sequence at the 3' end of the H3 chain. The fluorescence intensity change was measured by a fluorescence meter to establish the relationship curve between the fluorescence signal response value and the concentration of the target analyte. The amino-modified hairpin H2 chain is 3' end amino-modified.

[0012] (5) Application in food sample detection: The fluorescence signal of the food sample with unknown concentration is measured in step (4), and the detection result is substituted into the standard curve to obtain the AFB1 concentration in the sample.

[0013] Further specified, in step (1), the amount of Padlock and Primer added is 1-10 μL, the concentration is 1-10 μM, the amount of T4 DNA ligase (5 U / μL) added is 1-5 μL, the amount of Phi29 polymerase (10 U / μL) added is 1-5 μL, and the incubation time is 12-36 h.

[0014] Further specified, in step (2), the concentration of sodium citrate buffer is 50-100mM, the amount of tetrachloroauric acid (1mM) added is 5-20μL, the incubation temperature is 80-90℃, and the incubation time is 20-60min.

[0015] Further specify that in step (3), the amount of manganese acetate dihydrate added is 20-50 mg, the amount of H2TCPP added is 5-20 mg, and the volume of DMF is 20-60 μL.

[0016] Further specifying, the Padlock sequence mentioned in steps (1) and (4) is one of 5'-P-ATG ATA TGA TCG TTGTCA CTG CCT GCT TTT TTT TTT TAT GAT GG-3' or 5'-P-ATG ATA TGA TCG TTG TCA CTGCCT GCT TTT TTT TTT TTT TTT TTT TTT TTA TGA TGG-3' or 5'-P-ATG ATA TGA TCG TTG TCACTG CCT GCT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TAT GAT GG-3' or 5'-P-ATG ATA TGATCG TTT TTT TTT TAT GAT CGT TGT CAC TGC CTG CTT TTT TTT TTT TTT ATG ATG G-3'; the Primer sequence is 5'-CGA TCA TAT CAT CCA TCA TAA AAA-3'; and the H1 sequence is 5'-GTT GGG. The H2 sequence is 5'-CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CAC TAACAT ATT CAA GGG CAC GAG ACA CAG AG-3'; the H3 sequence is 5'-TTG AAT ATG TTA GGC AGG CAG TGA ATA TGT TAG ATG ATC GTT GTC ACT GCC TGC CTA ACA TAT TCA AGC GAA GCTC-NH2-3'; the H3 sequence is 5'-TTG AAT ATG TTA GGC AGG CAG TGA CAA CGA TCA TCT AACAT ATT CAA GGG CAC GAG ACA CAG AGA TGA TCG TTG TCA CTG CCT GCG GGG GG-3'.

[0017] Compared with the prior art, the present invention has the following significant advantages:

[0018] 1. The signal tag of the present invention is AuNCs synthesized in situ from DNA nanoflowers, which have higher signal intensity and stability compared with AuNCs synthesized by traditional methods using biological proteins, amino acids, single-stranded DNA, etc. as templates.

[0019] This invention utilizes a precisely designed three-hairpin chain structure to achieve a catalytic hairpin assembly reaction, thereby improving the sensitivity of the detection system.

[0020] This invention utilizes Mn-MOF as the site for catalytic hairpin assembly, which increases the concentration of local reactants and further improves the efficiency of the catalytic reaction.

[0021] This invention utilizes the base sequence (6G) of the hairpin chain itself as a signal switch, which can effectively quench the fluorescence signal of AuNCs without the need for the introduction of additional quenching materials, thus reducing the complexity and cost of the system.

[0022] The sensing strategy based on this invention has the characteristics of high sensitivity, good accuracy and high stability, which is conducive to the promotion and application of the invention. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating a method for preparing a fluorescent aptamer sensor for in-situ synthesis of gold nanoclusters and manganese metal-organic frameworks from DNA nanoflowers, and for detecting aflatoxin B1.

[0024] Figure 2 The fluorescence intensity of the sensor constructed in Example 1 of this invention is shown in the presence of 10 ng / mL AFB1 (dashed line) and 100 ng / mL AFB1 (solid line).

[0025] Figure 3 This is a comparison chart showing the selectivity of the fluorescent aptamer sensor constructed in Example 1 of the present invention for AFB1 in the presence of other interfering toxins.

[0026] Figure 4 This is a scanning electron microscope image of the DNA nanoflower prepared by the steps of Example 1 of the present invention, wherein the scale bar of the inlay is 500 nm.

[0027] Figure 5 This is a scanning electron microscope image of the manganese metal-organic framework prepared by the steps of Example 1 of this invention. Implementation

[0028] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0029] A method for preparing a fluorescent aptamer sensor based on the in-situ synthesis of gold nanoclusters and manganese metal-organic frameworks from DNA nanoflowers, and its application in the synergistic detection of aflatoxin B1, is described below. Figure 1 As shown.

[0030] A method for preparing a fluorescent aptamer sensor based on the in-situ synthesis of gold nanoclusters and manganese metal-organic frameworks from DNA nanoflowers, and its application in the synergistic detection of aflatoxin B1, includes the following steps:

[0031] (1) Preparation of circular template: Take 1 μL of Padlcok (1 μM) and 1.5 μL of Primer (1 μM) into 20 μL of 1XT4 ligase buffer, anneal at 95℃ for 5 min in a PCR instrument and gradually cool to room temperature, add 2 μL of T4 DNA ligase (5 U / μL) to the above system, and incubate overnight at 16℃ for 12 h.

[0032] (2) Preparation of DNA nanoflowers: Add 1.5 μL of Phi29 DNA polymerase, 1 μL of dNTP and 3 μL of 10X Phi29 buffer to the system in step (1), mix thoroughly and incubate at 30°C for 24 h.

[0033] (3) In situ synthesis of AuNCs from DNA nanoflowers: Take 10 μL of DNA nanoflower solution, 10 μL of chloroauric acid solution (1 μM), and 10 μL of sodium citrate buffer (100 mM, pH = 6.0) from step (2), add water to make up to 200 μL, and incubate at 90 °C for 30 min in a magnetic stirrer.

[0034] (4) Preparation and modification of manganese metal-organic frameworks (Mn-MOF): 27 mg of manganese acetate dihydrate was mixed thoroughly in 30 mL of N,N-dimethylformamide (DMF); 10 mg of H2TCPP was added to 10 mL of DMF and mixed thoroughly before being slowly added to the above manganese acetate solution. After mixing thoroughly, the mixture was incubated at room temperature for 12 h. The product was centrifuged and washed three times with anhydrous ethanol. EDC / NHS solution (50 mM) and H2 chain (10 mM) were added to Mn-MOF, and the mixture was incubated at room temperature for 2 h to obtain Mn-MOF-H2.

[0035] (5) Construction of fluorescent aptamer sensor: Samples containing the target analyte were prepared into test solutions of different concentrations. The aptamer chains H1, Mn-MOF-H2 and H3 were added. A large amount of Mn-MOF-H2 / H3 was obtained by catalytic hairpin self-assembly reaction. After mixing and incubating with DNA nanoflowers@AuNCs prepared in step (3), the H3 chain modified on Mn-MOF was complementary to the DNA nanoflowers. Then, the fluorescence of AuNCs was quenched by the 6G sequence at the 3' end of the H3 chain. The fluorescence intensity change was measured by a fluorescence meter to establish the relationship curve between the fluorescence signal response value and the concentration of the target analyte.

[0036] (6) Application in food sample detection: The fluorescence signal of the food sample with unknown concentration is measured in step (5), and the detection result is substituted into the standard curve to obtain the AFB1 concentration in the sample.

[0037] like Figure 2 As shown, the fluorescence intensity of the sensor constructed in Example 1 of this invention is the fluorescence intensity of the sensor in the absence of AFB1 (dotted line), and in the presence of 10 ng / mL AFB1 (dashed line) and 100 ng / mL AFB1 (solid line).

[0038] Example

[0039] A method for preparing a fluorescent aptamer sensor for in-situ synthesis of gold nanoclusters and manganese metal-organic frameworks from DNA nanoflowers and its application in detecting aflatoxin B1 includes the following steps:

[0040] (1) To verify the specific recognition of AFB1 by the prepared fluorescent aptamer sensor based on DNA nanoflowers@AuNCs and Mn-MOF, AFB1 standard was added to Tris buffer to make the concentration of AFB1 in the sample 10 ng / mL; other interfering toxin (OTA, DON, FB1, ZEN) standard solutions were prepared using 50 mM Tris buffer, with a concentration of 200 ng / mL for each. The signal intensity corresponding to the ordinate is the signal value of different target substances and blank sample at 438 nm. The detection system constructed in Example 1 was used to detect the above-mentioned different interfering toxin standards, and the detection results are as follows. Figure 3 As shown, this illustrates that the method of the present invention has high selectivity for AFB1.

[0041] Example

[0042] A method for preparing a fluorescent aptamer sensor for in-situ synthesis of gold nanoclusters and manganese metal-organic frameworks from DNA nanoflowers and its application in detecting aflatoxin B1 includes the following steps:

[0043] (1) Actual sample processing: Take 0.5g of food spiked sample (corn flour, peanut flour), add it to 10mL of methanol / water solution (ratio 7:3), place it on a shaker and shake for 20 minutes, then take it out and place it in a centrifuge for 10 minutes at a speed of 10000 rpm. After centrifugation, take out the supernatant to obtain the food sample extract.

[0044] (2) Sample detection: The fluorescence intensity signal was measured according to the steps in Example 1, and the concentration of AFB1 in the sample was obtained by substituting it into the standard curve.

[0045] (3) When using peanut powder as the actual sample for determination, with an addition amount of 10 ng / mL as the baseline, 0.1 times and 10 times the baseline amount of AFB1 standard were added to the peanut powder respectively. Take 2 μL of sample solution and measure the fluorescence intensity signal according to steps (1)-(5) of Example 1. Substitute the signal into the standard curve detected in Example 1 to obtain the AFB1 concentration in the sample. Each sample was measured three times and the average value was taken. The RSD and recovery rate were calculated as shown in the table below:

[0046]

[0047] The prepared fluorescent aptamer sensor has been verified to exhibit high accuracy, good sensitivity, and excellent reproducibility and stability in the detection of AFB1. Furthermore, detection results on actual samples (such as peanuts) demonstrate that the prepared sensor has significant practical application value.

[0048] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0049] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing gold nanoclusters by in-situ synthesis of DNA nanoflowers, characterized in that, Includes the following steps: (1) Preparation of DNA nanoflowers: The template strand (Padlock) and primer strand (Primer) were co-incubated to form a circular template under the action of T4 DNA ligase; the circular template was added to the Phi29 DNA polymerase system solution, and the bifunctional DNA nanoflowers were generated by the polymerase-driven rolling cycle amplification; the sequence of the template strand (Padlock) is SEQ ID NO.1: 5'-P-ATG ATA TGA TCG TTG TCA CTG CCT GCT TTT TTT TTT TTT TAT GAT GG-3'; SEQ ID NO.2: 5'-P-ATG ATA TGA TCG TTG TCA CTG CCT GCT TTT TTT TTT TTT TTT TTT TTA TGA TGG-3'; SEQ ID NO.3: 5'-P-ATG ATA TGA TCG TTG TCA CTG CCT GCT TTT TTT TTT TTT TTT TTT TTT TTT TTT TAT GAT GG-3'; SEQ ID NO.4: 5'-P- One of ATG ATA TGA TCG TTT TTT TTT TATGAT CGT TGT CAC TGC CTG CTT TTT TTT TTT ATG ATG G -3'; the primer sequence is SEQ ID NO.5: 5'-CGA TCA TAT CAT CCA TCA TAA AAA-3'; the Phi29 DNA polymerase system solution includes Phi29 DNA polymerase, Phi29 buffer, and dNTPs; the amount of template strand (Padlock) and primer strand (Primer) added is 1-10 μL, and the concentration is 1-10 μM; the amount of T4 DNA ligase added is 1-5 μL, and the volumetric activity is 5 U / μL; the amount of Phi29 polymerase added is 1-5 μL, and the volumetric activity is 10 U / μL; the incubation time is 12-36 h; (2) In-situ synthesis of gold nanoclusters: DNA nanoflowers were added to sodium citrate buffer and tetrachloroauric acid solution was added. The mixture was then placed in a magnetic stirrer and incubated to obtain fluorescent signal-tagged DNA nanoflowers @AuNCs.

2. The method for preparing gold nanoclusters by in-situ synthesis of DNA nanoflowers according to claim 1, characterized in that, The bifunctional DNA nanoflowers are DNA nanoflowers with in-situ incubation of metal nanoclusters and nucleic acid recognition functions.

3. A method for preparing a manganese metal-organic framework fluorescent aptamer sensor, characterized in that, Includes the following steps: (1) Preparation of DNA loaded on manganese metal-organic framework Mn-MOF: Manganese acetate and H2TCPP were mixed evenly in N,N-dimethylformamide solution and incubated at room temperature to obtain Mn-MOF; (2) The carboxyl groups on the surface of Mn-MOF were activated with EDC / NHS solution and co-incubated with amino-modified hairpin H2 chains. After ligation, Mn-MOF-H2 was obtained; the amino-modified hairpin H2 chains were 3'-terminally aminoized. (3) Samples containing the target analyte were prepared into test solutions of different concentrations. The hairpin H1 chain, Mn-MOF-H2 and hairpin H3 chains were added, and Mn-MOF-H2 / H3 was obtained by catalytic hairpin self-assembly reaction. When the target analyte is aflatoxin B1, the sequence of the H1 chain is SEQ ID NO.6: 5'-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGCCCT TCG CTA GGC CCA CAC TAA CAT ATT CAA GGG CAC GAG ACA CAG AG-3'; the sequence of the H2 chain is SEQ ID NO.7: 5'-CTC TGT GTC TCG TGC CCT TGA ATA TGT TAG ATG ATC GTT GTC ACTGCC TGC CTA ACA TAT TCA AGC GAA GCT C-NH2-3'; the sequence of the H3 chain is SEQ ID NO.8: 5'-TTGAAT ATG TTA GGC AGG CAG TGA CAA CGA TCA TCT AA CAT ATT CAA GGG CAC GAG ACACAG AGA TGA TCG TTG TCA CTG CCT GCG GGG GG-3'; (4) After mixing and incubating Mn-MOF-H2 / H3 with the DNA nanoflowers@AuNCs prepared in step (2) of claim 1, the H3 chain modified on Mn-MOF binds complementary to the DNA nanoflowers. Then, the fluorescence of AuNCs is quenched by the 6G sequence at the 3' end of the H3 chain. The fluorescence intensity change is measured by a fluorescence meter to establish a relationship curve between the fluorescence signal response value and the concentration of the target substance. The signal value of the actual sample is detected and substituted into the relationship curve to obtain the concentration of the target substance.

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