Preparation method of MOF fluorescence sensor and its application in detecting single base mismatch.

Abstract

This invention belongs to the field of bio-optoelectronic sensing technology. Traditional detection of single-base mismatched dsDNA involves complex pretreatment experiments. To address these issues, this invention provides a method for preparing an MOF fluorescence sensor and its application in detecting single-base mismatches. First, lyophilized powders of polyA and polyC are diluted to 5 × 10⁻⁶ with NaAc-HAc buffer. ‑6 M-2×10 ‑5 M; subsequently, the solution was heated to denature the secondary structure, and then rapidly annealed at room temperature; next, a polyC and polyA solution was taken at a molar ratio of 3:2 and thoroughly mixed with MOF, and incubated at 37±3℃ for 24±4 h; the solution was washed and centrifuged to remove free or loosely adsorbed polyC and polyA on MOF@polyA / polyC. This invention provides a simple, label-free, and economical non-hybridization method for directly identifying single-base mutations in double-stranded DNA.

Description

Technical Field

[0001] This invention belongs to the field of bio-optoelectronic sensing technology, specifically relating to a method for preparing an MOF fluorescence sensor and its application in detecting single-base mismatches. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are common genetic variations caused by single base substitutions, often leading to the sudden appearance of novel traits in offspring never seen before by their ancestors, such as disease susceptibility, drug resistance, and multiple pregnancies. Therefore, early detection of gene mutations has significant biological value and commercial applicability, providing mutation types for genetic research and effective strategies for high-quality breeding.

[0003] Gel electrophoresis, PCR sequencing, microarray technology, mass spectrometry, TaqMan assay, and enzymatic methods are commonly used as the gold standard for SNP quantification. However, these methods suffer from drawbacks such as complex experimental procedures, high personnel training costs, and expensive equipment, especially their relatively low sensitivity and selectivity at room temperature. DNA-based sensors utilize fluorescence instruments, photoelectric instruments, and electrochemical workstations to analyze the hybridization of DNA target sequences with immobilized single-stranded oligonucleotide arrays to detect mutations. Examples of DNA-based sensors include those based on Hg... 2+ Sensors based on the specific binding mechanism of ions to T:T and Ag + Fluorescent sensors utilize the specific binding mechanism of ions to C:C. Since DNA-based sensor analyses ultimately rely on molecular recognition principles related to DNA hybridization, the pairing energy difference between single-base mismatches and perfect complementarity with the mutant target strand is very small, resulting in limited sensitivity for detecting single-base mismatches. Furthermore, many real-world samples contain double-stranded DNA rather than single-stranded DNA, meaning that pretreatment to unwind double-stranded DNA is necessary in practical applications, a cumbersome and complex process that severely limits the applicability of these methods.

[0004] Sensors based on Mut-S as DNA base pair mismatch locators can directly detect double-stranded DNA in samples without any pretreatment; however, the high cost of Mut-S severely hinders the widespread use of this technology. Electrochemical biosensors based on the specific recognition mechanism of naphthidine carbamate dimers and GG mismatches are inexpensive, but this method is mainly applicable to GG mismatches and has no effect on the detection of other mismatches. Other methods using PNA or fluorescent dyes suffer from reduced practicality due to the complex synthesis of PNA and the poor specificity of fluorescent dyes. Therefore, designing a simple, label-free, and economical non-hybridization method to directly identify single-base mutations in double-stranded DNA in samples is an urgent problem to be solved. Summary of the Invention

[0005] Traditional detection of single-base mismatch dsDNA involves complex pretreatment experiments such as sample preparation, primer design, and target gene pre-amplification, or its signal readout mainly depends on the hybridization between the ssDNA probe and the ssDNA target, rather than direct extraction from the real sample. To address these issues, this invention provides a method for preparing an MOF fluorescent sensor and its application in detecting single-base mismatches.

[0006] This invention is achieved through the following technical solution:

[0007] On one hand, this invention provides a method for preparing a MOF-based fluorescence sensor, comprising the following steps: firstly, lyophilized powders of polyA and polyC are diluted to 5 × 10⁻⁶ with NaAc-HAc buffer. -6 M-2×10 -5 M; Subsequently, the solution was heated at 95±1℃ to denature the secondary structure, and then rapidly renatured at room temperature; Next, polyA and polyC solutions were taken at a molar ratio of 3:2 and thoroughly mixed with MOF, and incubated at 37±3℃ for 24±4h; Washed and centrifuged to remove free or loosely adsorbed polyC and polyA on MOF@polyA / polyC.

[0008] Furthermore, the MOF was synthesized using a solvothermal method, specifically as follows: First, 2-aminoterephthalic acid and FeCl3·6H2O were weighed and mixed into DMF, and stirred thoroughly to ensure complete dissolution; then, the mixture was poured into a high-pressure reactor and placed in an oven at 120°C for 20 hours; after the reaction was completed, the mixture was cooled to room temperature and filtered, then washed multiple times with DMF and ethanol to remove unreacted compounds; finally, the obtained solid was dried to form purified MIL-88-NH2.

[0009] Furthermore, the concentrations of both polyA and polyC are 10. -5 M.

[0010] On the other hand, the present invention provides the application of the fluorescence sensor described herein in breeding:

[0011] Detection of GT single-base mismatch dsDNA: The prepared MOF@polyA / polyC and GT single-base mismatch DNA samples were incubated at 25-45℃ for 12±3h, washed and centrifuged, then NaAc-HAc was added, pH 5-6, and then H2O2 and OPD were added and reacted for 30-45 minutes.

[0012] Furthermore, the MOF@polyA / polyC and GT single-base mismatched DNA samples were incubated at 37°C for 12 hours.

[0013] Furthermore, after the MOF@polyA / polyC and GT single-base DNA samples react, the reaction continues with OPD and H2O2 for 40 minutes.

[0014] Furthermore, the MOF@polyA / polyC was dispersed in NaAc-HAc buffer at pH 5.5.

[0015] Furthermore, the concentration of the GT single-base mismatch DNA sample is 10. -9 M~10 -5 M.

[0016] Beneficial effects:

[0017] This invention selects GT mismatched dsDNA of FecB-related genes as a research model. Polyadenine and polycytosine, acting as intelligent gatekeepers, were modified on MIL-88-NH2 to construct a fluorescence-off-limits platform for direct analysis of single molecules. No pretreatment is required for dsDNA with base mismatches. This fluorescence platform exhibits high selectivity, effectively distinguishing GT mismatches from CT, TT, and AT. Furthermore, the platform maintains a relatively stable fluorescence signal in the presence of various metal ions, proteins, small organic molecules, and complex biomolecules. The maximum fluctuation of this method does not exceed 5%. Linearity experiments show that this method can effectively detect 10% of FecB-related genes in buffer or diluted serum. -9 M~10 -5 The method significantly expands the ground truth mismatch of M by one or two orders of magnitude compared to previously reported results. The detection limit is as low as 0.247 nM while maintaining a large linear range. In stability experiments, the platform signal decreased by less than 10% after 7 days, demonstrating the superior performance and promising application prospects of this method. Attached Figure Description

[0018] Figure 1 SEM (A), TEM (B), EDS (C), and FTIR (D, inset XRD) images of MIL-88B-NH2.

[0019] Figure 2 Fluorescence intensities of OPD-H2O2-MOF@polyA / polyC prepared with different ratios of polyA and polyC (1:0, 1:1, 3:2, and 2:3). Error bars represent SD (n=3).

[0020] Figure 3 Selectivity evaluation of OPD-H2O2-MOF@polyA / polyC prepared at ratios of 1:0 and 3:2 (AT, fully complementary DNA, 10 -6 M.TT, TT single-base mismatched DNA, 10 -6M.CT, CT single-base mismatched DNA, 10 -6 M.GT(FecB), GT single-base mismatch DNA, 10 -6 M); the error bars represent SD (n=3).

[0021] Figure 4 The effects of different factors on fluorescence signal: A. Effect of adsorption concentration ratio on fluorescence signal; B. Effect of pH on fluorescence signal; C. Effect of desorption temperature on fluorescence signal; D. Effect of catalytic reaction time on fluorescence signal changes; Error bars represent SD (n=3).

[0022] Figure 5 Characterization of MOF@polyA / polyC: SEM image of MOF@polyA / polyC (A), TEM image of MOF@polyA / polyC (inset: magnified view of TEM image of MOF@polyA / polyC) (B), EDS of MOF@polyA / polyC (inset: phosphorus mapping) (C), FTIR of MOF@polyC / polyA (bottom half of the figure: zeta potential of MOF and MOF@PolyA / PolyC) (D).

[0023] Figure 6 Zeta potential, fluorescence intensity, and visually observed color change of MOF on the OPD-H2O2-MOF fluorescence platform (a), Zeta potential, fluorescence intensity, and visually observed color change of MOF@polyC / polyA on the OPD-H2O2-MOF@polyC / polyA fluorescence platform (b), and Zeta potential, fluorescence intensity, and visually observed color change of MOF@polyC / polyA+T on the OPD-H2O2-MOF@polyC / polyA+T fluorescence platform (c).

[0024] Figure 7 The supernatant fluorescence emission spectra of MOF@PolyA / PolyC, MOF@PolyA / PolyC+T, MOF@PolyA / PolyC+T+H2O2, MOF@PolyA / PolyC+T+OPD, MOF@PolyA / PolyC+OPD+H2O2, and MOF@PolyA / PolyC+H2O2+OPD+T are shown.

[0025] Figure 8 Various metal ions (50mM Na) + Ca 2+ Al 3+ K + Cu 2+ Mg 2+Fluorescence intensity of Fecb-related gene GT mismatch dsDNA was detected in the presence of (A); in proteins and organic molecules (glucose 10 μg / mL, urea 20 μg / mL, OMP31 / BP26 / BSA, 3 μg / mL) (B) and in complex biological systems (C). Error bars represent SD (n=3).

[0026] Figure 9 Add different concentrations (10) to buffer (A) or 5% serum (C). -9 M to 10 -5 Fluorescence spectra of the target (M), dynamic response range of the biosensor to the target concentration and calibration plot (inset), and linear relationship between fluorescence intensity changes and target concentration in buffer (B) or 5% serum (D).

[0027] Figure 10 Selection evaluation of MOF@polyA / polyC for various base mismatch dsDNAs, (A) Various single-base mismatch dsDNAs of the same chain length (30 base pairs) (AT, perfectly complementary dsDNA, 10 -6 M;TT, single base mismatch dsDNA, 10 -6 M;CT, single base mismatched dsDNA, 10 -6 M;GT, single-base mismatched dsDNA, 10 -6 M); (B) GT mismatched dsDNA with the same chain length and different mismatch numbers or mismatched dsDNA with the same mismatch number and different chain lengths (GT-3 / 10, GT mismatched dsDNA with a 30-base-pair chain length and three mismatch numbers, with a 10-base-pair interval between GT mismatches, 10 -6 M;GT-5 / 5, GT mismatch dsDNA, with a 30-base-pair strand length and five mismatches, GT mismatch interval of 5 base pairs, 10 -6 M;GT-5 / 10, GT mismatch dsDNA, with a 66-base-pair strand length and five mismatches, the interval between GT mismatches is 10 base pairs, 10 -6 M. The error bars represent SD (n=3).

[0028] Figure 11 The fluorescence responses of MOF@polyA / polyC were stored at 4 °C for 0, 1, 2, 5, 7, and 10 days. Error bars represent SD (n=3). Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments.

[0030] Experimental reagents: Ferric chloride (FeCl3·6H2O) was purchased from Sigma-Aldrich, 2-aminophthalic acid (NH2-BDC) from Sinopharm Chemical Reagent Co., Ltd., and N,N-dimethylformamide (DMF) from Aladdin Reagents (Shanghai, China). OMP31 and BP26 were donated by the College of Animal Science and Technology, Shihezi University. All oligonucleotide sequences listed in Table 1 were synthesized and purified by high-performance liquid chromatography (HPLC) at Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). All reagents were of analytical grade and could be used without further purification. All aqueous solutions were prepared using pure water produced by a Milli-Q water purification system (resistance >18 MΩ / cm). NaAc-HAc buffer (pH 5.5, 0.2 mM) was used in this experiment. Scanning electron microscopy (SEM) was performed using a JEOL JSM-7500FSEM instrument (Hitachi High-Technology Co., Ltd., Japan), transmission electron microscopy (TEM) was performed using a JEM-2100TEM instrument (Hitachi High-Technology Co., Ltd., Japan), and Fourier transform infrared spectroscopy (FTIR) was performed using a Bruker Tensor 70 spectrometer (Bruker Optics, Germany). All fluorescence measurements were performed on an FL-2700 fluorescence spectrophotometer (Shimadzu, Tokyo, Japan) with the following instrument parameters: excitation and emission slit width 10 nm, excitation wavelength set to 435 nm.

[0031] Table 1. Oligonucleotide sequences used in this invention

[0032]

[0033]

[0034] The underlined part represents a mismatched base.

[0035] The oligonucleotide sequences used in this invention were designed using a BLAST search of GenBank DNA sequences (http: / / www.ncbi.nlm.nih.gov / Genbank / index.html). Furthermore, we found no homology with other disease genes.

[0036] Experimental Section

[0037] 1.1 Synthesis and Characterization of MIL-88-NH2(MOF)

[0038] MIL-88-NH2 (MOF) was synthesized using a solvothermal method. The specific procedure was as follows: First, 0.2506 g of 2-aminoterephthalic acid (NH2-BDC) and 0.3740 g of FeCl3·6H2O were accurately weighed and mixed into 30 ml of DMF. The mixture was stirred thoroughly for 2 hours until completely dissolved. Then, the solution was poured into a 50 ml high-pressure reactor lined with Teflon and placed in an oven at 120°C for 20 hours. After the reaction was complete, the mixture was cooled to room temperature and filtered. It was then washed repeatedly with DMF and ethanol, and filtered again to remove unreacted compounds. Finally, the resulting solid was dried to form purified MIL-88-NH2. Activation step: The product was washed with acetone and dried in a vacuum oven at 30°C, followed by further analysis.

[0039] To confirm the successful synthesis of MOF, a series of characterization procedures were performed. For example... Figure 1 As shown in A and B, the MOF has a regular octahedral structure with a particle size of approximately 200 nm and good dispersibility. Figure 1 As shown in Figure C, the elements C, N, O, and Fe are displayed in energy-dispersive spectroscopy (EDS). Figure 1 As shown in D, 3437cm -1 The band at 1572 cm⁻¹ is due to the symmetrical and asymmetric stretching of the amino group, confirming the presence of an amino group in the synthesized MIL-88B-NH₂. -1 and 1421cm -1 The strong characteristic bands at 1331 cm⁻¹ are related to the asymmetric and symmetric stretching vibrations of the coordinating carboxyl group, respectively, indicating the presence of a dicarboxylic acid linker in the synthesized structure; simultaneously, at 1331 cm⁻¹... -1 and 1246cm -1 The peak at 765 cm⁻¹ can be attributed to characteristic CN stretching, indicating the presence of aromatic amines; -1 The peak at 570 cm⁻¹ belongs to the CH bending vibration of the aromatic ring. Furthermore, at 570 cm⁻¹... -1 The nearby apparent peaks are related to the Fe-O vibration. All these infrared characteristic peaks indicate the successful preparation of NH2-MIL-88(Fe). Figure 1 As shown in Inset D, the characteristic diffraction peaks of the prepared MIL-88B-NH2 appear at approximately 9.2° (002), 13.1° (102), 16.7° (103), 18.5° (200), 19.1° (201), and 26.3° (204), consistent with the simulated XRD pattern of the MIL-88B-NH2 structure established from the crystallographic information file (Cambridge Crystallographic Data Centre). This further demonstrates that we have successfully synthesized MIL-88B-NH2.

[0040] 1.2 Preparation of MOF@polyA / polyC

[0041] (1) Concentrations of polyA / polyC:

[0042] First, different concentrations of polyA and polyC (3:2, 100 μL, 10 μL) were mixed. -7 M~2×10 -5 M) was added to six electron tubes respectively, and then mixed with MOF (600 μL, 1 mg / mL). After 24 hours, washing, centrifugation, and volume determination were performed. Then, 50 μL of MOF@polyA / polyC was pipetted and mixed with 10 μL of target (10 -6 Mix the solution (M) at 37°C for 12 h. Then, centrifuge the mixture, bring it to a final volume with NaAc-Hac (100 μL, pH 5.5, 0.2 M), and add 20 μL of H2O2 and 10 μL of NaAc-Hac (100 μL, pH 5.5, 0.2 M). -3 M) and OPD (80 μL, 10 -3 M) was added to MOF@polyA / polyC and reacted for 40 minutes. Next, fluorescence measurements were performed.

[0043] (2) The ratio of polyA to polyC:

[0044] MOF (600 μL, 1 mg / mL) and different proportions of polyA (10 -5 M) and polyC(10 -5 Add M)(1:0, 1:1, 3:2, 2:3) to four e-tubes respectively, and incubate at 37°C for 24 h; then centrifuge these mixtures and wash with deionized water. Next, bring the volume to 600 μL with NaAc-HAc buffer. Finally, add 50 μL of the above solution to four e-tubes respectively, and mix with 10 μL of target (10... -6 Mix at 37°C for 12 h. After washing and centrifugation, add NaAc-HAc (100 μL, pH 5.5, 0.2 M), H2O2 (20 μL and 10 μL). -3 M) and OPD (80 μL, 10 -3 Incubate for 40 minutes (M). Next, conduct a fluorescence experiment.

[0045] After the above experiments, the optimal concentrations of polyA and polyC were determined to be 10. -5 M, the optimal ratio is 3:2. First, centrifuge the lyophilized polyA and polyC powders at 4000 rpm for 1 minute, then dilute to 10 with NaAc-HAc buffer. -5M. Subsequently, the solution was heated at 95°C for 5 minutes, and then rapidly allowed to renature slowly at room temperature. Next, 40 μL of polyC and 60 μL of polyA were thoroughly mixed with 600 μL of MOF (1 mg / mL) at 37°C for 24 hours, followed by washing and centrifugation to remove free or loosely adsorbed polyC and polyA from the MOF@polyA / polyC mixture. Finally, the MOF@polyA / polyC mixture was dispersed in 600 μL of NaAc-HAc buffer for further use.

[0046] 1.3 Detection of GT-Mismatched dsDNA

[0047] Using FecB-related genes and GT mismatched dsDNA as the research model, the optimal pH, catalytic time, and temperature were determined through the following optimization experiments.

[0048] (1) pH optimization:

[0049] MOF@polyA / polyC was dispersed in 50 μL of NaAc-HAc at different pH values ​​(pH 5-6), then added to 10 μL of GT single-base sample, and incubated at 37 °C for 12 h. After washing and centrifugation, NaAc-HAc (pH 5-6, 0.2 M) was added to bring the volume to 100 μL, followed by the addition of 20 μL and 10 μL of H2O2. -3 M) and OPD (80 μL, 10 -3 The reaction was carried out for 40 minutes (M). Next, fluorescence measurement was performed.

[0050] (2) Optimization of catalytic time and temperature:

[0051] Except for the MOF@polyC / polyA and GT single-base mismatches, the incubation temperature was changed from 37℃ to 4℃, 25℃, and 45℃. The fluorescence catalytic reaction time was changed from 40 minutes to 10 minutes, 20 minutes, 50 minutes, and 60 minutes, and then the fluorescence signal was measured. All other conditions were performed under optimal conditions.

[0052] (3) Biosensing of MOF@polyA / polyC in buffer and diluted serum

[0053] Biosensing of MOF@polyA / polyC in buffer: Different concentrations of MOF@polyA / polyC (50 μL, 1 mg / mL) and GT mismatched dsDNA (10 μL, 10 mg / mL) were prepared. -5 M~10 -9 Add NaAc-HAc (100 μL, pH 5.5, 0.2 M) to several centrifuge tubes and incubate at 37°C for 12 h. After washing and centrifugation, add NaAc-HAc (100 μL, pH 5.5, 0.2 M) and H2O2 (20 μL, 10 M).-3 M) and OPD (80 μL, 10 -3 The reaction was carried out for 40 minutes (M). Next, fluorescence measurement was performed.

[0054] MOF@polyA / polyC biosensing in different interfering molecules: First, in various interfering molecules (Na + Ca 2 + Al 3+ K + Cu 2+ Mg 2+ A series of samples were prepared in 50 mM of glucose (10 μg / mL), urea (20 μg / mL), OMP31, BP26, and BSA at 3 μg / mL or diluted serum (1%, 5%, and 10%). (GT mismatched dsDNA, 10) -6 M), then mixed with MOF@polyA / polyC (50 μL, 1 mg / mL) at 37 °C for 12 hours, washed and centrifuged, and then NaAc-HAc (100 μL, pH 5.5, 0.2 M) and H2O2 (20 μL, 10 M) were added. -3 M) and OPD (80 μL, 10 -3 The reaction was carried out for 40 minutes (M). Next, fluorescence measurement was performed.

[0055] Conclusions and Data

[0056] (1) Preparation and characterization of MOF@polyA / polyC:

[0057] The objective of this invention is to detect GT-mismatched dsDNA. Once G / T appears in the FecB gene, the original inner helix conformation of A / T is disrupted, and then G or T flips out of the dsDNA and regains its ability to bind to C or A. Based on the above theory, polyA with 30 adenine deoxynucleotides and polyC with 30 cytosine deoxynucleotides were designed as probes (Table 1), mixed in a 3:2 ratio, and then added to MOF. These probes were modified onto the MOF surface simply through electrostatic adsorption and π-π conjugation. To demonstrate the successful construction of MOF@polyA / polyC, detailed structural characterization was performed. Typical transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images show that MOF@polyA / polyC is a uniform octahedral shape with an average diameter of 200 nm. Figure 5 A and B). Compared with MOF ( Figure 1No significant changes were observed after polyA and polyC modification (A and 1B), which indirectly indicates that only a very thin DNA membrane was formed on the MOF, which is beneficial for the MOF to recover its catalytic activity in the later stage.

[0058] Energy-dispersive X-ray spectroscopy (EDS) revealed the elemental composition distribution of MOF@polyA / polyC. Besides C, O, N, and Fe from the MOF itself, [the following elements were also present]. Figure 1 In addition to C), phosphorus, as part of ssDNA, appears in the MOF@polyA / polyC spectrum. Figure 5 C). In the infrared spectrum, besides the characteristic peaks of the MOF itself ( Figure 1 In addition to D), 710cm was also produced. -1 and 1130cm -1 Vibrational peaks caused by nearby ssDNA pentose ( Figure 5 D). Since MOFs themselves contain a large number of positively charged amino groups, theoretically, after modification with negatively charged phosphates via ssDNA, MOF@polyA / polyC would be tightly surrounded by negatively charged molecules. Therefore, in the study of zeta potential ( Figure 6 (a) and (b) we found that the charge of the MOF changed from positive to negative before and after ssDNA modification. These findings confirm that polyA and polyC were successfully anchored on the surface of MIL-88B-NH2.

[0059] (2) OPD-H2O2-MOF@ployA / ployC fluorescence platform

[0060] This invention selects MIL-88B-NH2 as a substrate because it possesses two unique characteristics: enzyme mimicry activity and abundant positive charge. In this case, using OPD as the peroxidase substrate for the peroxidase mimicry, the Fe3-μ3-OXO cluster in MIL-88B-NH2 can undergo an electron transfer reaction with H2O2 to generate Fe... 2+ Meanwhile, H2O2 is activated by the Fenton reaction to generate ·OH, as shown in (1) and (2). Then, OPD is reflected and generated into DAP in the presence of hydroxyl radicals, as shown in (3) and (4). This can accelerate the generation of ·OH, greatly improve the conversion efficiency of non-fluorescent OPD to fluorescent DAP, thereby producing a significant fluorescent signal, and the reaction solution gradually turns deep yellow. Figure 6 a).

[0061] H₂O₂ + Fe³⁻μ³⁻oxo → Fe 2+ +O2+2H (1)

[0062] Fe 2+ +H₂O₂→Fe 3++·OH+HO - (2)

[0063]

[0064]

[0065] Modifying MOFs with polyA and polyC resulted in the catalytic activity of the MOFs being shut down. For OPD-H2O2-MOF@ployA / ployC, the fluorescence intensity of DAP at 535 nm was significantly reduced, and the reaction solution appeared lighter in color (pale yellow) to the naked eye. Figure 6 b). Optimize the concentration and ratio of polyA and polyC, such as... Figure 2 As shown, 10 -7 The fluorescence signal changes of polyA and polyC modified OPD-H2O2-MOF were not ideal; it gradually increased with increasing polyA and polyC concentrations, reaching a low level at 10⁻⁶. -5 The maximum value is reached at time M, therefore 10 is chosen. -5 M represents the optimal concentration of polyA and polyC. Various concentration ratios of polyA and polyC (1:0, 1:1, 2:3, and 3:2) were used, such as... Figure 3 As shown, although the fluorescence signal at a 3:2 ratio is 93% of that at a 1:0 ratio, it is strong enough for this invention. Furthermore, in subsequent selectivity experiments, a 3:2 ratio of MOF@polyA / polyC yielded the best results; therefore, the polyA / polyC ratio was chosen to be 3:2.

[0066] When GT-mismatched dsDNA is introduced, polyA and polyC are withdrawn from the MOF surface due to GT-specific induction. Figure 6 In step c, we found that the electronegativity of MOF@polyA / polyC is decreasing, and the catalytic activity of MOF is recovering. For the OPD-H2O2-MOF@polyA / polyC fluorescence platform, the fluorescence intensity of DAP at 535 nm changed by 84% before and after the addition of analyte. Furthermore, the visually observed color of DAP returned to a deep yellow. In summary, all data demonstrate the feasibility of this invention. In this invention, optimization experiments with pH (5–6) and temperature (4°C, 25°C, 37°C, and 45°C) showed that the OPD-H2O2-MOF@polyA / polyC platform achieved optimal catalytic efficiency at pH 5.5 and 37°C. Figure 4Therefore, pH 5.5 and 37℃ were selected for subsequent experiments. Furthermore, optimization experiments with catalytic times of 10 mins, 30 mins, 40 mins, 50 mins, and 60 mins showed that the fluorescence intensity difference of DAP at 550 nm for MOF@polyA / polyC and MOF@polyA / polyC+T peaked at 40 min and then began to decline. This may be because the fluorescent substrate initially requires time to slowly diffuse to the iron metal centers of MOF@polyA / polyC, gradually forming a reaction pool. Over time, the diffusion rate of the fluorescent substrate dominates the overall reaction rate. Due to the different resistivities of MOF@polyA / polyC and MOF@polyA / polyC+T to the fluorescent substrate, the signal difference increased, reaching its maximum at 40 min. Therefore, 40 min was chosen as the catalytic time for the entire fluorescence platform.

[0067] (3) Feasibility

[0068] To demonstrate the feasibility of this invention, MOF@polyA / polyC, MOF@polyA / polyC+T, MOF@polyA / polyC+H2O2+T, MOF@polyA / polyC+OPD+T, MOF@polyA / polyC+H2O2+OPD, and MOF@polyA / polyC+H2O2+OPD+T were compared and studied under fluorescence spectroscopy detection via the catalytic oxidation reaction of OPD in the presence of H2O2. Figure 7 As shown, the wavelength ranges for MOF@polyA / polyC, MOF@polyA / polyC+T, MOF@polyA / polyC+H2O2+T, and MOF@polyA / polyC+OPD+T are 550-650 nm. This phenomenon indicates that the fluorescence platform cannot function properly in the presence of OPD and H2O2 alone or in the absence of OPD and H2O2. Even in the presence of OPD and H2O2 but without analyte, the fluorescence platform still functions, but with a very low signal, indicating that polyA and polyC act as intelligent gatekeepers, effectively inhibiting the catalytic activity of MOF. Only upon the introduction of analyte does the catalysis of MOF restart, exhibiting a relatively strong fluorescence signal, demonstrating the feasibility of this method.

[0069] like Figure 6As shown, the initial zeta potentials of MOF, MOF@polyA / polyC+OPD+T, and MOF@polyA / polyC+T are positive, which is consistent with the fact that MOF itself has a large number of amino cations. When PolyA and PolyC are modified on MOF, a large number of DNA phosphate anions accumulate at the MOF interface, covering its original positive charge. The zeta potential becomes negative. After the analyte is introduced, due to hybridization induction, PolyA and PolyC gradually leave the MOF interface, and the electronegativity of MOF begins to gradually decrease. This indirectly proves that the present invention has successfully constructed a catalase-like MOF-based fluorescent probe for switching off fluorescence. The color of diammonium phosphate observed by the naked eye changes according to the pattern of deep yellow-light yellow-deep yellow. In summary, all the data prove that the method of the present invention is feasible.

[0070] (4) Fluorescence sensing of MOF@polyA / polyC in buffer and diluted serum

[0071] The fluorescence signal of MOF@polyA / polyC was tracked at different target DNA concentrations prepared in buffer, and the calibration graph is shown below. Figure 9 As shown in A and B, the average fluorescence signal of MOF@polyA / polyC (n=3) is compared with that of 10 -9 M to 10 -5 The logarithm of DNA concentration within the M range showed a linear correlation with the value of DNA, with a correlation coefficient of 0.996. The limit of detection (LOD) was 0.294 nM, and the corresponding regression equation was F = 1146 + 110.5 log[C]. GT ].

[0072] Although the OPD-H2O2-MOF@ployA / ployC fluorescence platform exhibits good analytical properties in buffer solutions, given the greater complexity of real-world biological samples, including a large number of inorganic compounds, organic compounds, and proteins, the performance of this fluorescence platform must be evaluated in the presence of several possible coexisting interfering substances, including metal ions (Na+, Na+, and So-called "H2O2-MOF@ployA / ployC"). + K + Ca 2+ Mg 2+ Al 3+ Cu 2+ Proteins (BSA, OMP31, BP26), small organic molecules (ascorbic acid, glucose, and urea), and complex biological systems (1%, 5%, and 10% serum). For example... Figure 8 As shown in A, B, and C, the overall fluctuation of the fluorescence signal does not exceed 10%, which indicates that the fluorescence platform is promising for actual sample testing.

[0073] When this fluorescence platform is used to detect GT mutations in 5% serum ( Figure 9At times C and D), the average fluorescence signal of MOF@polyA / polyC was correlated with the logarithm of dsDNA concentration in a ratio of 10. -9 M to 10 -5 The regression shows a good linear relationship within the range of M, with a correlation coefficient of 0.992. The corresponding regression equation is F = 1158 + 107.6lg[C] GT The detection limit of the target object is 0.247 nM.

[0074] Prior to this invention, PCR was considered the gold standard for Fecb quantification. This method requires complex sample preparation, primer design, pre-amplification of the target gene, a well-equipped laboratory, and relatively expensive instruments. This invention, however, enables direct, rapid, and low-cost identification of dsDNA, and demonstrates a wider detection limit and superior detection performance in both buffered and complex biological systems (Tables 2 and 3).

[0075] Table 2 compares the performance of fluorescence detection platforms modified with different materials for direct detection of single-base mismatched DNA.

[0076]

[0077]

[0078] Table 3. Performance comparison of traditional methods for detecting FecB-related gene GT mismatch dsDNA.

[0079]

[0080] (5) Selectivity and stability of MOF@polyA / polyC

[0081] The platform's strong selectivity was demonstrated using four DNA double-stranded sequences: a perfectly complementary dsDNA sequence (AT), a dsDNA sequence with a GT single-base mismatch (GT), a dsDNA sequence with a CT single-base mismatch (CT), and a dsDNA sequence with a TT single-base mismatch (TT). Figure 10 As shown in Figure A, the signal strength of CT and TT is only about half that of GT, and AT is only 22%. This indicates that the platform offers reasonable selectivity.

[0082] However, when the base length of dsDNA is the same, the signal does not increase with the increase of GT mismatch number (GT, GT-3 / 10, GT-5 / 5, GT-5 / 10, ...). Figure 10B) The fluorescence intensity becomes stronger. Among them, DNA with three GT mismatches exhibits the best fluorescence intensity. This indicates that the intensity of hybridization induction is related not only to the number of mismatched base pairs but also to the length of the space between the mismatched base pairs. Furthermore, the probe length also has a certain impact on the OPD-H2O2-MOF / ployA / ployC fluorescence platform. Compared to GT-3 / 10, the fluorescence signal of GT-5 / 10 is reduced by approximately 30%. The main reason may be that the spherical three-dimensional structure of MOF@polyA / polyC is unfavorable for the hybridization induction of long-chain dsDNA and polyA / polyC.

[0083] The stability of MOF@polyA / polyC was studied in detail under optimized conditions, such as... Figure 11 As shown, the signal did not change significantly within the intervals of 1 day, 2 days, 5 days, 7 days, and 10 days, indicating that the method is relatively stable and reliable for at least one week.

[0084] In summary, this invention successfully constructed a MIL-88B-NH2-based on-off / on-off fluorescence platform. Its potential applications were demonstrated through analysis of single-base mismatched dsDNA of FecB-related genes in buffered or diluted serum. This platform exhibits excellent analytical performance for the determination of GT mismatched dsDNA, with a low detection limit (0.294 nM) and a wide linear range (10⁻⁶). -9 M-10 -5 The method (M) exhibits high selectivity, sensitivity, and stability. Compared to traditional detection methods, this invention, as an emerging technology, offers advantages such as speed, low cost, and ease of use. Most importantly, it avoids the hassle of pretreatment experiments, including complex enzyme mixtures, specialized experimental equipment, numerous primer optimizations, and reverse transcriptase. Furthermore, dsDNA is easier to extract and more stable than ssDNA. All of these factors indicate that this method is more suitable for on-site DNA diagnostics.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention. sequence list <110> Qingdao University of Science and Technology <120> Preparation method of MOF fluorescence sensor and its application in detecting single base mismatch. <141> 2022-06-13 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <400> 1 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <400> 2 cccccccccc cccccccccc cccccccccc 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <400> 3 agacagaaat atatcagacg gtgttgatga 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <400> 4 agacagaaat atatcggacg gtgttgatga 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 agacagaaat atatccgacg gtgttgatga 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 agacagaaat atatctgacg gtgttgatga 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <400> 7 agacggaaat atatcggacg gtgttggtga 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <400> 8 agacggaaat gtatcaggcg gtggtgatgg 30 <210> 9 <211> 66 <212> DNA <213> Artificial Sequence <400> 9 gctgctagac gtgcgaacat cgccgtatca gagccggtag ctaggacact gatgcgtcga 60 gtacca 66

Claims

1. A method for preparing a MOF-based fluorescent sensor for detecting GT single-base mismatched dsDNA, characterized in that, Includes the following steps: First, the lyophilized powders of polyA and polyC were diluted to 5 × 10⁻⁶ with NaAc-HAc buffer. -6 M~2×10 -5 M; Subsequently, the solution was heated at 95±1℃ to denature the secondary structure, and then rapidly placed at room temperature for slow refolding; Next, polyA and polyC solutions were prepared at a molar ratio of 3:2 and thoroughly mixed with MOF. The mixture was then incubated at 37±3℃ for 24±4h. After washing and centrifugation, free or loosely adsorbed polyC and polyA on MOF@polyA / polyC were removed. The MOF was synthesized using a solvothermal method, specifically as follows: First, 2-aminoterephthalic acid and FeCl3·6H2O were weighed and mixed into DMF, and stirred thoroughly until completely dissolved; then, the mixture was poured into a high-pressure reactor and placed in an oven at 120°C for 20 hours; after the reaction was completed, the mixture was cooled to room temperature and filtered, then washed multiple times with DMF and ethanol to remove unreacted compounds; finally, the obtained solid was dried to form purified MIL-88B-NH2. OPD and H2O2 need to be added during fluorescence sensor detection.

2. The method for fabricating a MOF-based fluorescence sensor according to claim 1, characterized in that, The concentrations of both polyA and polyC are 10. -5 M.

3. The application of the MOF-based fluorescent sensor prepared by the method of claim 1 in the detection of GT single-base mismatched dsDNA for non-disease diagnostic purposes, characterized in that, The prepared MOF@polyA / polyC and GT single-base mismatched DNA samples were incubated at 25–45°C for 12–3 h. After washing and centrifugation, NaAc-HAc was added, pH 5–6, and then H2O2 and OPD were added and reacted for 30–45 minutes.

4. The application according to claim 3, characterized in that, The MOF@polyA / polyC and GT single-base mismatched DNA samples were incubated at 37°C for 12 hours.

5. The application according to claim 3, characterized in that, The reaction time of MOF@polyA / polyC after GT addition with OPD and H2O2 is 40 minutes.

6. The application according to claim 3, characterized in that, The MOF@polyA / polyC was dispersed in NaAc-HAc buffer at pH 5.

5.

7. The application according to claim 3, characterized in that, The concentration of the GT single-base mismatch DNA sample was 10. -9 M~10 -5 M.

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