AuNPs-DNA three-dimensional nano device, preparation method thereof and application of AuNPs-DNA three-dimensional nano device in FEN1 enzyme detection

Abstract

The invention discloses an AuNPs-DNA three-dimensional nano device, a preparation method thereof and application of the AuNPs-DNA three-dimensional nano device in FEN1 enzyme detection.The SR DNA with the 3'end modified with FAM and the 5 'end modified with-SH serves as a signal output molecule, meanwhile, long-chain DNA walker is modified on the surfaces of gold nanoparticles to serve as a walking foot of the DNA nano device, SR can be hybridized with the DNA walker, and a lambda exonuclease (lambda-EXO) cleavage site is generated; in the presence of a DNA protection fragment (P), P and DW are hybridized to form double strands, hybridization of DW and SR is prevented, and because an enzyme recognition site of SR is protected, no fluorescence signal is emitted. According to the constructed three-dimensional DNA nano device, in the presence of FEN1, due to the fact that FEN1 cuts 5 'Flap, lambda-EXO cutting sites are generated, DW is hybridized with SR after being released, more lambda-EXO cutting sites are generated, fluorescence signals are released, and sensitive, simple and convenient detection of FEN1 can be achieved through the fluorescence signals through a fluorescence biosensor.

Description

Technical Field

[0001] This invention belongs to the field of quantitative detection of FEN1 activity, specifically relating to an AuNPs-DNA three-dimensional nanodevice, its preparation method, and its application in FEN1 enzyme detection. Background Technology

[0002] Flap endonuclease 1 (FEN1) is a key 5' flap nuclease in DNA replication, repair, and recombination, responsible for cleaving the RNA primer in Okazaki fragment synthesis and processing DNA secondary structures. Numerous clinical and basic studies have shown that abnormal FEN1 activity is closely related to the occurrence, progression, and drug resistance of various malignant tumors, making it an important biomarker for early cancer diagnosis, prognostic assessment, and drug screening. Therefore, developing a sensitive, rapid, and low-cost FEN1 activity detection platform is of great value for tumor biology research and clinical translation.

[0003] Gold nanoparticles can be used for the detection of many substances. They can detect trimethoprim and chloride ions under different conditions and methods. Related derivatives of gold nanoparticles, such as gold nanoparticle-β-cyclodextrin, glutathione-gold nanoparticles (GSH-AuNPs), and gold-modified carbon fiber microelectrodes (CFMEs), can be applied to the detection of aniline pollutants, cadmium ions, and catechins, respectively. In addition to the detection of the above substances, gold nanoparticles can also participate in the establishment of clenbuterol hydrochloride detection methods, improve the external quantum efficiency of perovskite light-emitting diodes, and analyze the mechanisms of macrophage endocytosis and exocytosis at the cellular level. AuNP-based optical biosensing methods include four types: light scattering biosensing, electrochemiluminescence biosensing, fluorescence biosensing, and colorimetric biosensing.

[0004] DNA is a high molecular polymer, and most DNA exists in a double helix structure. The DNA sequence stores the genetic information of an organism, and the genetic information is transmitted through semi-conservative replication. Using the DNA strand as a template, it can be transcribed into RNA, and using mRNA as a template, it can be translated into protein. DNA carries genetic information, and the correct replication of genetic information can maintain the normal functioning of the body. However, if errors occur in the replication, transcription, or translation of genetic information outside the genetic codon, it may cause diseases or other uncomfortable symptoms in the body.

[0005] To improve detection sensitivity, researchers have developed various signal amplification strategies, such as catalytic hairpin assembly (CHA), rolling circle amplification (RCA), hybridization chain reaction (HCR), and strand displacement amplification (SDA). Others have achieved signal amplification detection by constructing three-dimensional DNA nanodevices.

[0006] FEN1 possesses branched endonuclease activity that cleaves the 5' Flap in the DNA double-strand structure. Following cleavage, the FAM signal modified on the DNA nanodevice is released, generating a fluorescent signal, enabling sensitive detection of FEN1 and exploring methods for early tumor diagnosis. This thesis utilizes three-dimensional (3-D) DNA nanodevices assembled from gold nanoparticles (AuNPs) and DNA to investigate their application in FEN1 detection. This method is simple, highly sensitive, and beneficial for rapid clinical diagnosis. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an AuNPs-DNA three-dimensional nanodevice, its preparation method, and its application in FEN1 enzyme detection. In this invention, FEN1 acts on branched DNA double strands, cleaves the 5' Flap, causes DW to hybridize with SR to initiate a Walker cycle, and releases FAM fluorescence. The activity of FEN1 is determined based on the change in fluorescence signal intensity. This method has the advantages of high sensitivity and good accuracy.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for fabricating AuNPs-DNA three-dimensional nanodevices includes the following steps: 1) SR DNA with 3' end modified with FAM and 5' end modified with -SH, and a DNA walker with 3' end modified with -SH were added to a BSPP-modified AuNPs solution and incubated at 25 ± 5℃ for 10-15 hours. After incubation, NaCl solution was added and sonicated for 5-15 seconds. The steps of adding NaCl and sonication were repeated 4-6 times, once every 1 hour. After reacting at 25 ± 5℃ for 20-25 hours, the mixture was further incubated with thiol PEG for 1-3 hours to block the active sites on the surface of AuNPs to obtain AuNPs-DNA three-dimensional nanodevices. The molar ratio of AuNPs to 3' end modified with -SH DNA walker was 1:(25-35); the molar ratio of 3' end modified with FAM and 5' end modified with -SH SR DNA to 3' end modified with -SH DNA walker was (8-12):1. 2) Centrifuge the material prepared in step 1) to wash away the unmodified DNA strands, and then wash with ultrapure water. After washing, reconstitute the synthesized AuNPs-DNA three-dimensional nanodevices in pH 7.4 Tris-NaCl buffer as a stock solution.

[0009] Furthermore, the DNA walker sequence from the 5' end to the 3' end of the 3' end modified with -SH is as follows: TTg gTC CAT CgCCTC AgC CCg TAT AgT gTA ATg TCT ATC ATT ACA Tgg TAT gAT ATT TTT TTTTTT TTTTTT TTT TTT TT-SH.

[0010] The SR DNA with FAM modified at the 3' end and -SH modified at the 5' end has the following sequence from the 5' end to the 3' end: SH-ATA Cgg gCTgAg gCg ATg gCA TTT-FAM.

[0011] Further, the preparation process of AuNPs is as follows: heating to bring 0.8~1.2 mM chloroauric acid aqueous solution to a boil, adding freshly prepared 35~40 mM sodium citrate aqueous solution while the chloroauric acid aqueous solution is boiling, stirring and maintaining boiling for 10~20 minutes, turning off the heater, and allowing it to cool naturally to room temperature, then storing the prepared AuNPs solution in a brown bottle at 4±2℃, with a molar ratio of chloroauric acid to sodium citrate of (1~1.5):1, and the particle size of AuNPs is 10~20 nm.

[0012] Furthermore, the process of modifying AuNPs with BSPP is as follows: 1 mg of BSPP [(bis(p-sulfonylphenyl)phenylphosphine dipotassium salt dihydrate)] is added to every 900 μL of gold nanoparticle solution, and the reaction is carried out at room temperature for 10-15 h.

[0013] Furthermore, the concentration of the NaCl solution was 3 mol / L, and the concentration of the three-dimensional DNA nanodevices in the stock solution was 10 nM.

[0014] The AuNPs-DNA three-dimensional nanodevices were prepared by the above method.

[0015] The above-mentioned AuNPs-DNA three-dimensional nanodevices are used in the detection of FEN1 enzyme for non-disease diagnostic purposes.

[0016] Further, the process is as follows: (1) Hybridize AuNPs-DNA three-dimensional nanodevices with P-DNA in Tris-MgCl2 buffer. After hybridization, add 0~2U of FEN1 enzyme in gradient amounts and incubate in 1×Thermo Pol reaction buffer at 37±2℃ for 3~5 hours. Then add 8~12U of λ-EXO enzyme and react in 1×Lambda Exonuclease Reaction Buffer for 1~3 hours. Finally, adjust the volume with Tris-NaCl buffer and measure the fluorescence. Plot a standard curve with FEN1 enzyme content as the abscissa and fluorescence intensity as the ordinate to obtain the standard curve equation. (2) Add the test sample containing FEN1 enzyme to the reaction solution of AuNPs-DNA three-dimensional nanodevice and P-DNA hybridization in Tris-MgCl2 buffer. Measure the fluorescence according to the detection process in (1). Substitute the measured fluorescence intensity into the standard curve equation to obtain the concentration of FEN1 enzyme in the test sample.

[0017] The p-DNA sequence from the 5' end to the 3' end is as follows: TTT ATA Cgg gCT gAg gCg ATg gAC CAA.

[0018] Furthermore, the molar ratio of AuNPs-DNA three-dimensional nanodevices to P-DNA is 1:(40~60); the amount of FEN1 enzyme added is less than or equal to 2U.

[0019] Furthermore, the pH of the Tris-MgCl2 buffer is 7.4; the pH of the Tris-NaCl buffer is 7.4; and the fluorescence measurement uses an excitation wavelength of 488 nm.

[0020] Compared with the prior art, the present invention has the following features and advantages: (1) such as Figure 1As shown, the SR DNA modified with FAM at the 3' end and -SH at the 5' end in this invention serves as the signal output molecule. Simultaneously, a long-chain DNA walker is modified on the surface of gold nanoparticles to act as the "foot" (walker) of the DNA nanodevice. SR can hybridize with the DNA walker, generating λ-EXO cleavage sites. In the presence of a DNA protection fragment (P), P and DW hybridize to form a double strand, preventing DW from hybridizing with SR. Because the enzyme recognition site of SR is protected, no fluorescent signal is emitted. In the presence of FEN1, the constructed three-dimensional DNA nanodevice generates λ-EXO cleavage sites by cleaving the 5' Flap. After DW is released, it hybridizes with SR, subsequently generating more λ-EXO cleavage sites and releasing a fluorescent signal. This fluorescent signal can be sensitively and easily detected by a fluorescent biosensor.

[0021] (2) This invention utilizes DNA Walker cyclic amplification to generate a large amount of FAM to improve detection sensitivity.

[0022] (3) The present invention has the advantages of low cost, speed, simplicity, sensitivity and high specificity. Attached Figure Description

[0023] Figure 1 This is a flowchart for FEN1 activity detection; Figure 2 TEM image of gold nanoparticles (AuNPs); Figure 3 The image shows the UV-Vis absorption spectrum of gold nanoparticles (AuNPs). Figure 4 This diagram is for verifying the experimental principle. Figure 5 The blocking effect of MCH on the remaining active sites of gold nanoparticles; Figure 6 The blocking effect of thiol PEG on the remaining active sites of gold nanoparticles; Figure 7 The effect of the modification ratio of SR to DW on the detection effect; Figure 8 To illustrate the effect of different FEN1 enzyme activities on the detection system, fluorescence spectra of FEN1 in the range of 0 to 2 U are shown: (a) 0, (b) 0.2 U, (c) 0.25 U, (d) 0.35 U, (e) 0.45 U, (f) 1 U, (g) 2 U. Figure 9 The light intensity change curves caused by different amounts of FEN1 enzyme catalysis (y=82732+66373) c R 2 =0.997). Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be noted that those skilled in the art can make several modifications and improvements without departing from the principle of the present invention, and these should also be considered to fall within the protection scope of the present invention.

[0025] The reagents and instruments used in this experiment are as follows: DNA was purchased from Shanghai Bioengineering Technology Service Co., Ltd., and the fluorescence spectrometer was (Fluoromax-4, HoribaJobin Yvon, Japan).

[0026] DNA sequences used in the experiment: Name Sequence (5' to 3') DNA Walker (DW) TTg gTC CAT CgC CTC AgC CCg TAT AgT gTA ATg TCT ATC ATT ACA Tgg TAT gAT ATT TTT TTT TTT TTT TTT TTT TTT TT-SH SR SH-ATA Cgg gCT gAg gCg ATg gCA TTT-FAM P TTT ATA Cgg gCT gAg gCg ATg gAC CAA Example 1: The preparation method of AuNPs-DNA three-dimensional nanodevices is as follows: 1) Synthesis of gold nanoparticles: First, all glassware used in the AuNPs synthesis process was soaked in an alkaline bath (prepared by dissolving sodium hydroxide in water and then adding ethanol) for more than 5 hours. After being removed, it was washed with deionized water and then rinsed with aqua regia (concentrated HCl / concentrated HNO3). 3， After soaking for 24 hours with a volume ratio of 3:1, the solution was rinsed thoroughly with deionized water. Next, under vigorous stirring and reflux, when the aqueous solution of chloroauric acid tetrahydrate (1 mM, 50 mL) boiled, freshly prepared sodium citrate solution (38.8 mM, 0.057 g sodium citrate + 5 mL H₂O) was rapidly added, stirred, and kept boiling for 15 minutes. During this period, the solution color changed from yellow to transparent, black, purple, and finally deep red. After 15 minutes, the heater was turned off, and the solution was allowed to cool naturally to room temperature. The resulting AuNPs solution was then stored in a brown bottle at 4 °C, and its TEM image is shown below. Figure 2 As shown, by Figure 2 It is known that the synthesized gold nanoparticles have a diameter of 13 nm, such as Figure 3 As shown, the concentration of the prepared gold nanoparticles was determined to be 10 nM using ultraviolet-visible absorption spectroscopy.

[0027] 2) AuNPs modifies DW and SR: To ensure that DNA-modified AuNPs can be well dispersed under high ionic strength, AuNPs were first modified with BSPP to increase their surface charge, thereby improving the dispersibility of gold nanoparticles in high ionic concentration solutions. The specific steps were as follows: 900 μL of freshly prepared gold nanoparticle solution was taken out, 1 mg of BSPP was added using a weighing spoon, and incubated at room temperature for 12 h. Then, the surface of BSPP-protected AuNPs was modified with SR probes and DW sequences. The 5' end of the SR probe was modified with FAM (fluorescein), and the 3' end was modified with -SH. The 3' end of the DW molecule was also modified with -SH.

[0028] The specific process is as follows: First, DW and SR were diluted with deionized water to prepare a 100 μM stock solution. Further dilution was then performed based on the amount added. The mixture of DW and SR was added to 1 mL of 13 nm AuNPs solution at a molar ratio of 1:30 for AuNPs and 10:1 for SR. The mixture was incubated at 25 °C for 12 hours. After incubation, 20 μL of 3 M NaCl solution was slowly added to the solution, followed by sonication for 10 seconds. This process of adding NaCl and sonication was repeated 5 times, every hour, to ensure maximum DNA loading on the AuNPs. After reacting at 25 °C for 24 hours, 6 μL of 20 mM thiol PEG (thiol-PEG6-methyl, purchased from Sigma-Aldrich Inc.) was added, and the mixture was incubated with the thiol PEG at 25 °C for 2 hours to block the active sites on the AuNPs surface.

[0029] 3) Material Processing: The prepared material was centrifuged for 20 minutes (13200 rpm, 10 ℃) to wash away unmodified DNA strands, followed by washing with ultrapure water. This process was repeated three times in total. After washing, the synthesized AuNPs-DNA three-dimensional nanodevices were reconstituted in a pH 7.4 Tris-NaCl (10 mM Tris, 50 mM NaCl) buffer. The final stock solution contained 10 nM AuNPs-DNA three-dimensional nanodevices and was stored at 4 ℃ for later use.

[0030] 4) Fluorescence detection: 2 μL of 10 nM AuNPs-DNA three-dimensional nanodevices were taken and hybridized with 1 μL and 1 μM P-DNA in 20 μL of pH 7.4 Tris-MgCl2 buffer (10 mM Tris, 50 mM MgCl2) at 37 °C for 2 h. After hybridization, different concentrations (0, 0.2 U, 0.25 U, 0.35 U, 0.45 U, 1 U, 2 U) of FEN1 enzyme were added and incubated in 25 μL of 1×Thermo Pol reaction buffer (10 mM KCl, 20 mM Tris-HCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8) at 37 °C for 4 h. Then, 10 U of λ-EXO enzyme was added and incubated in 30 μL of 1×LambdaExonucleaseReaction Buffer (pH 8.8). The reaction mixture was prepared in 9.4, 67 mM Glycine-KOH, 2.5 mM MgCl2, and 50 µg / ml BSA at 37°C for 2 hours. Finally, it was diluted to 200 μL with pH 7.4 Tris-NaCl (10 mM Tris, 50 mM NaCl). Fluorescence was measured (emission spectrum was measured at an excitation wavelength of 488 nm, and the peak intensity at 520 nm was read). Figure 8 As shown, by Figure 8 It can be seen that the fluorescence intensity increases with the increase of FEN1 enzyme content. A standard curve was plotted with FEN1 enzyme content on the x-axis and fluorescence intensity on the y-axis, as shown below. Figure 9 As shown, by Figure 9 It can be seen that the equation of the standard curve is y = 82732 + 66373 c R 2 =0.997, FEN1 shows a good linear relationship in the range of 0 to 2 U, and the detection limit is 0.55 mU.

[0031] Fluorescence was measured on the following reaction solutions: AuNPs-DNA three-dimensional nanodevices, AuNPs-DNA three-dimensional nanodevices hybridized with p-DNA, reaction solutions without FEN1 enzyme, reaction solutions with 10 U λ-EXO enzyme added directly, and reaction solutions with 0.45 U FEN1 enzyme followed by 10 U λ-EXO enzyme. Figure 4 As shown, Figure 4 The results showed that the addition of FEN1 had a significant impact on the fluorescence recovery of the system.

[0032] For the sample to be tested, the AuNPs-DNA three-dimensional nanodevice and P-DNA are hybridized and then added to the sample. The sample is then tested according to the fluorescence detection process in step 4) above. The fluorescence result is substituted into the standard curve equation to obtain the FEN1 enzyme content in the sample. If the FEN1 enzyme content is not in the range of 0 to 2 U, it needs to be diluted before testing.

[0033] Example 2 In the fabrication of DNA nanodevices, thiol-based PEG and MCH (thiol hexanol, with a final concentration of 100 nM in the reaction solution) were compared. Fluorescence performance was detected after adding 0.35 U FEN1 enzyme, and other parameters were the same as in Example 1. The blocking effects of both on the remaining active sites on gold nanoparticles were investigated; results are detailed below. Figure 5 , Figure 6 , Figure 5 , Figure 6 The results showed that the thiol PEG blocking effect was relatively good.

[0034] Example 3 By varying the molar ratio of SR to DW to 1:1, 5:1, 10:1, 15:1, and 20:1, and adding 0.35 U FEN1 and not adding FEN1, AuNPs-DNA three-dimensional nanodevices were fabricated. The fluorescence difference between the addition and absence of FEN1 at the same molar ratio was measured. The results are as follows: Figure 7 As shown, Figure 7 The results show that a molar ratio of SR to DW of 10:1 is the optimal condition.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the specific implementation of the present invention and not to limit it. Those skilled in the art should understand that any equivalent substitutions or obvious modifications made to the implementation of the present invention without changing its performance or use, without departing from the spirit of the present invention, should be covered within the scope of protection claimed by the present invention.

Claims

1. A method for preparing AuNPs-DNA three-dimensional nano device, characterized in that, Comprising the following steps: 1) 3' end modified FAM and 5' end modified-SH SR DNA, 3' end modified-SH DNA walker are added to BSPP modified AuNPs solution, incubated at 25±5℃ for 10-15 hours, after incubation, NaCl solution is added and ultrasonic for 5-15 seconds, the step of adding NaCl and ultrasonic is repeated 4-6 times, every 1 hour, after reaction at 25±5℃ for 20-25 hours, further incubated with thiol PEG for 1-3 hours to block the active sites on the surface of AuNPs to obtain AuNPs-DNA three-dimensional nano device; the molar ratio of AuNPs and 3' end modified-SH DNA walker is 1:(25-35); the molar ratio of 3' end modified FAM and 5' end modified-SH SR DNA and 3' end modified-SH DNA walker is (8-12):1; 2) The material prepared in step 1) is centrifuged, and the unmodified DNA chain is washed away, and then ultrapure water is used for washing, and after washing, the synthesized AuNPs-DNA three-dimensional nano device is redissolved in pH 7.4 Tris-NaCl buffer solution as a stock solution.

2. The method of claim 1, wherein the AuNPs-DNA three-dimensional nano device is prepared by the steps of: The preparation process of AuNPs is as follows: heating, boiling 0.8-1.2 mM aqueous solution of chloroauric acid, adding freshly prepared 35-40 mM aqueous solution of sodium citrate under the condition of boiling aqueous solution of chloroauric acid, stirring and keeping boiling for 10-20 minutes, turning off the heater, and naturally cooling to room temperature, then storing the prepared AuNPs solution in a brown bottle, storing at 4±2℃, the molar ratio of chloroauric acid and sodium citrate is (1-1.5):1, and the particle size of AuNPs is 10-20 nm.

3. The method for fabricating AuNPs-DNA three-dimensional nanodevices according to claim 1, characterized in that, The process of BSPP modified AuNPs is as follows: 1 mg of BSPP is added to every 900 μL of gold nanoparticle solution, and the reaction is carried out at room temperature for 10-15 h.

4. The method of claim 1, wherein the AuNPs-DNA three-dimensional nano device is prepared by the steps of: The concentration of NaCl solution is 3 mol / L, and the concentration of three-dimensional DNA nano device in stock solution is 10 nM.

5. The AuNPs-DNA three-dimensional nano device prepared by the preparation method of any one of claims 1-4.

6. The use of the AuNPs-DNA three-dimensional nano device of claim 5 in the detection of FEN1 enzyme for non-disease diagnosis purposes.

7. Use according to claim 6, characterized in that, The process is as follows: (1) The AuNPs-DNA three-dimensional nano device is hybridized with P-DNA in Tris-MgCl2 buffer solution, 0-2 U of FEN1 enzyme with gradient content is added after hybridization, incubated at 37±2℃ for 3-5 hours in 1×Thermo Pol reaction buffer, then 8-12 U of λ-EXO enzyme is added, reacted for 1-3 hours in 1× Lambda Exonuclease Reaction Buffer, and finally diluted with Tris-NaCl buffer solution, the fluorescence is measured, the FEN1 enzyme content is taken as the abscissa, the fluorescence intensity is taken as the ordinate, a standard curve is drawn, and a standard curve equation is obtained; (2) The FEN1 enzyme-containing sample to be measured is added into the reaction solution in which the AuNPs-DNA three-dimensional nano device and the P-DNA are hybridized in the Tris-MgCl2 buffer solution, the detection process in (1) is followed, the fluorescence is measured, and the measured fluorescence intensity is substituted into the standard curve equation to obtain the concentration of the FEN1 enzyme in the sample to be measured.

8. Use according to claim 7, characterized in that, The molar ratio of the AuNPs-DNA three-dimensional nano device to the P-DNA is 1: (40-60), and the added amount of the FEN1 enzyme is less than or equal to 2 U.

9. Use according to claim 7, characterized in that, The pH of the Tris-MgCl2 buffer solution is 7.4, the pH of the Tris-NaCl buffer solution is 7.4, and the fluorescence is measured by using an excitation wavelength of 488 nm.

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