Preparation method and application of gold and silver nanocluster coupled DNA fluorescent nanoprobe

Abstract

The invention discloses a preparation method and application of a gold and silver nanocluster coupled DNA fluorescent nanoprobe. Firstly, bovine serum albumin (BSA) is used as a template for reducing Au < 3 + > and Ag < + > to form a metal core NC of the nanocluster, histidine (His) and arginine (Arg) are sequentially added to be physically adsorbed to the surface of the NC, and the nanocluster NCHR with the particle size being about 10 nm is obtained. His molecules can stabilize the NC structure and effectively enhance the fluorescence intensity through hydrogen bonds formed between the His molecules and BSA, electrostatic interaction and the like. Guanidyl in the Arg molecule has a cell membrane penetrating function and can promote rapid uptake of NCHR by cells. The NCHR coupled DNA hairpin h with the optimal fluorescence intensity and cellular uptake effect is selected, and the fluorescence signal of the virus gene can be detected by co-incubating the NCHR-h and the cells for 1 hour, so that the detection time is shortened, and sensitive and rapid virus detection is realized. The extracellular detection range is 10 to 400 nM, and the lowest detection limit is 5.3 nM.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical diagnostic nanomaterials and their preparation, and more specifically, to a preparation method and application of a gold-silver nanocluster coupled DNA fluorescent nanoprobe. Background Art

[0002] In recent years, detection technology based on fluorescent nanomaterials has gradually attracted widespread attention from researchers in the field of virus detection due to its flexible sensing mechanism, high specificity, high sensitivity, simple use and practicality (Chem. Soc. Rev., 2022, 51, 5805). In addition, viral nucleic acid detection has higher specificity and accuracy than antigen detection. Therefore, using fluorescent nanomaterials as detection response units to couple DNA sequences targeting viral genomes to construct DNA fluorescent nanoprobes is expected to overcome the shortcomings of existing detection technologies, such as the long detection cycle and high testing cost of quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR); the low sensitivity and high false positive rate of lateral flow immunoassays of colloidal gold nanoparticles in the early stages (Curr. Opin. Solid State Mater. Sci., 2022, 26, 100966). Gold and silver nanoclusters have good photostability, resistance to photobleaching, high fluorescence intensity, large Stokes shift and other optical properties. The synthesis method is green and safe, and easy to modify the surface (Aggregate, 2021, 2, 114–132). Therefore, we considered using gold and silver nanoclusters coupled to DNA hairpins targeting the SARS-CoV-2 genome to construct fluorescent nanoprobes to achieve efficient and sensitive detection of the virus.

[0003] Studies have shown that the vibration and rotation of molecules in the template that constitutes the gold and silver nanoclusters will cause non-radiative decay, thereby affecting the emission intensity of the nanoclusters. Therefore, ligands with groups (such as -COOH, -NH2) or electron-rich atoms (such as N, O) can efficiently promote fluorescence and form hydrogen bonds and other interactions with the template to inhibit energy loss on the gold core surface and increase fluorescence intensity (Nat Commun, 2023, 14, 658). Therefore, we consider using histidine (His) as a small molecule ligand to improve the fluorescence efficiency of gold and silver nanoclusters. In addition, the guanidine group on the side chain of arginine (Arg) can strongly interact with the cell membrane phospholipid head group, thereby improving the cell uptake efficiency (ACS Appl. Mater. Interfaces, 2024, 16, 5451–5461). Therefore, we modified Arg to the surface of the nanocluster, hoping that the fluorescent nanomaterial can pass through the cell membrane at a faster speed to achieve enrichment in the cell, so as to quickly bind to the target to be detected and achieve sensitive detection.

[0004] In the present invention, we synthesized a gold-silver nanocluster coupled DNA fluorescent nanoprobe, namely NCHR-h, for efficient and sensitive detection of the novel coronavirus genome. First, bovine serum albumin (BSA) was used as a template to reduce Au 3+ 、Ag + The metal core NC that constitutes the nanocluster; His and Arg are added in sequence and reacted for a period of time to obtain a nanocluster NCHR with a particle size of about 10nm. By optimizing the modification amount of His and Arg, the fluorescence intensity and cellular uptake efficiency of NC are effectively improved; the final optimized NCHR is covalently coupled to the DNA hairpin h, and the synthesized NCHR-h has high fluorescence intensity and fast endocytosis speed, which can be used for rapid, sensitive and highly specific detection of the new coronavirus genome. The extracellular detection range is 10-400nM, and the minimum detection limit is 5.3nM. The nanoprobe can detect the fluorescence signal of the viral gene by incubating it with the cells for 1h, realizing sensitive and rapid virus detection. Summary of the invention

[0005] The purpose of the present invention is to provide a gold-silver nanocluster coupled DNA fluorescent nanoprobe for sensitive detection of the novel coronavirus genome. That is, the gold-silver nanocluster modified with His and Arg is used as a fluorescence detection signal response unit, which is covalently coupled with a DNA hairpin to achieve fluorescence detection of the novel coronavirus genome.

[0006] A method for preparing a gold-silver nanocluster coupled DNA fluorescent nanoprobe, characterized in that the preparation method of the fluorescent nanomaterial NCHR comprises the following steps: dissolving 25 mg BSA in 0.5 mL ultrapure water; adding 0.5 mL of 8 mM chloroauric acid tetrahydrate (HAuCl4·4H2O) solution and 0.125 mL of 5.3 mM silver nitrate (AgNO3) solution to the above solution in sequence at 37°C, reacting for 5 min respectively; adding 0.05 mL of 1 M sodium hydroxide (NaOH) solution to the above solution, reacting for 24 h, and synthesizing gold-silver nanocluster NC; adding 0.1 mL of 15, 22.5, and 30 mM HiS solutions to the solution after the above reaction, reacting for 3 h, and synthesizing NCH1, NCH2, and NCH3 respectively; selecting NCH2 with the best fluorescence intensity for the next step of optimization, hereinafter referred to as NCH. To the NCH solution after the reaction was completed, 0.1 mL of 22.5, 34, and 45 mM Arg solutions were added respectively and the reaction was carried out for 3 h. After the reaction was completed, ultrafiltration purification was performed using a 10 kDa ultrafiltration tube at 14,000 rpm for 5 min, and the products were redispersed in PBS (0.01 M, pH 7.4) solution to synthesize NCHR1, NCHR2, and NCHR3 respectively, and stored at 4°C.

[0007] The preparation method of the fluorescent nanoprobe NCHR-h includes the following steps: NCHR3 with the best fluorescence intensity and the best cell uptake effect is used as the final material, which is hereinafter referred to as NCHR. NCHR is diluted to 1 mg mL with PBS (0.01 M, pH 7.4) solution. -1 , 1.2 mL of solution; add (1-ethyl-(3-dimethylaminopropyl)carbodiimide) to the above solution to make the final concentration in the whole system 1 mg mL -1 , activated for 1h; 100μM DNA was prepared into 10μM with TM buffer (0.1M, pH 7.5) and sterile enzyme-free water, 40μL DNA solution (dispersed in TM buffer (0.01M, pH 7.5)), denatured at 95℃ for 5min, then cooled to room temperature in a step-by-step manner, annealed to form a hairpin structure; the above DNA hairpin solution was added to the activated NCHR solution, reacted at 37℃ in the dark for 12h, ultrafiltration was performed using a 30kda ultrafiltration tube, 14000rpm, 5min to remove uncoupled DNA, and redispersed in PBS (0.01M, pH 7.4) solution to obtain NCHR-h, which was stored at 4℃.

[0008] The obtained nano fluorescent probe has the following characteristics:

[0009] (1) It has a uniform size, with a particle size of about 10 nm;

[0010] (2) Good fluorescence intensity;

[0011] (3) Good cell membrane penetration effect;

[0012] (4) Good detection performance, with an extracellular detection range of 10-400 nM and a minimum detection limit of 5.3 nM; BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Particle size diagram of NCH1, NCH2 and NCH3 of the present invention.

[0014] Figure 2 : Fluorescence spectra of NC, NCH1, NCH2 and NCH3 of the present invention.

[0015] Figure 3 : Particle size diagram of NCH1, NCH2 and NCH3 of the present invention.

[0016] Figure 4 : Quantitative graph of the modification rate and drug loading rate of Arg in NCHR1, NCHR2 and NCHR3 of the present invention.

[0017] Figure 5: Quantitative graph of cellular uptake of NCH of the present invention and NCHR1, NCHR2 and NCHR3.

[0018] Figure 6 : Extracellular detection standard curve of NCHR-h of the present invention. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the following examples, but the present invention is not limited to the following examples. For example, the concentration and volume of the solution used can be adjusted as required.

[0020] Example 1

[0021] (1) Dissolve 25 mg of BSA in a centrifuge tube containing 0.5 mL of ultrapure water. Stir until completely dissolved at room temperature. Place the centrifuge tube in a 37°C constant temperature metal bath.

[0022] (2) 1.647 mg of HAuCl4·4H2O was dissolved in 0.5 mL of ultrapure water. The solution was added to the centrifuge tube containing the BSA solution and mixed at 37°C for 5 min.

[0023] (3) 0.113 mg of AgNO3 was dissolved in 0.125 mL of ultrapure water. The solution was added to the above centrifuge tube and mixed at 37°C for 5 min.

[0024] (4) 2 mg of NaOH was dissolved in 0.05 mL of ultrapure water, and the solution was added to the above centrifuge tube and reacted at 37° C. for 24 h to synthesize NC.

[0025] (5) Dissolve 0.233 mg of His in 0.1 mL of ultrapure water, add the solution to the centrifuge tube where the reaction is completed, and react at 37°C for 3 h to synthesize NCH1. Figure 1 As shown, the hydrated particle size was measured by Mastersize 3000 dynamic light scattering (DLS) (Malvern Instruments, UK) to be approximately 9 nm and the PDI was 0.191.

[0026] Example 2

[0027] (1)-(4) are the same as steps (1)-(4) in Example 1

[0028] (5) Take 0.349 mg of His and dissolve it in 0.1 mL of ultrapure water. Add the solution to the centrifuge tube where the reaction is finished and react at 37°C for 3 h to synthesize NCH2. Figure 1 The hydrated particle size shown is approximately 9 nm and the PDI is 0.123.

[0029] Example 3

[0030] (1)-(4) are the same as steps (1)-(4) in Example 1

[0031] (5) Take 0.465 mg of His and dissolve it in 0.1 mL of ultrapure water. Add the solution to the centrifuge tube where the reaction is completed and react at 37°C for 3 h to synthesize NCH3. Figure 1 The hydrated particle size shown is approximately 10 nm and the PDI is 0.158.

[0032] (6) Fluorescence intensity was measured using a fluorescence spectrophotometer (F-320, Tianjin Gangdong). Figure 2 As shown, NCH1, NCH2 and NCH3 can all produce broad and high fluorescence emission peaks at an excitation wavelength of 374 nm. Among them, NCH2 has the best fluorescence intensity, so this synthesis condition is selected for the next step of optimization, and is hereinafter referred to as NCH.

[0033] Example 4

[0034] (1)-(5) are the same as steps (1)-(5) in Example 2

[0035] (6) Take 0.392 mg of Arg and dissolve it in 0.1 mL of ultrapure water. Add the solution to the centrifuge tube where the reaction is completed and react at 37°C for 3 h to synthesize NCHR1. After the reaction is completed, use a 10 kDa ultrafiltration tube, 14000 rpm, 5 min ultrafiltration purification, redisperse in PBS (0.01 M, pH 7.4) solution, and store at 4°C. Figure 3 The hydrated particle size shown is approximately 9 nm and the PDI is 0.169.

[0036] Example 5

[0037] (1)-(5) are the same as steps (1)-(5) in Example 2

[0038] (6) Take 0.592 mg of Arg and dissolve it in 0.1 mL of ultrapure water. Add the solution to the centrifuge tube where the reaction is completed and react at 37°C for 3 h to synthesize NCHR2. After the reaction is completed, use a 10 kDa ultrafiltration tube, 14000 rpm, 5 min ultrafiltration purification, redisperse in PBS (0.01 M, pH 7.4) solution, and store at 4°C. Figure 3 The hydrated particle size shown is approximately 10 nm and the PDI is 0.209.

[0039] Example 6

[0040] (1)-(5) are the same as steps (1)-(5) in Example 2

[0041] (6) Take 0.784 mg of Arg and dissolve it in 0.1 mL of ultrapure water. Add the solution to the centrifuge tube where the reaction is completed and react at 37°C for 3 h to synthesize NCHR3. After the reaction is completed, use a 10 kDa ultrafiltration tube, 14000 rpm, 5 min ultrafiltration purification, redisperse in PBS (0.01 M, pH 7.4) solution, and store at 4°C. Figure 3 The hydrated particle size shown is approximately 10 nm and the PDI is 0.175.

[0042] (7) Quantify the amount of Arg loaded in NCHR1, NCHR2, and NCHR3, e.g. Figure 4 It showed that NCHR3 had the best modification rate and drug loading rate, which were 25.4% and 1.1%, respectively.

[0043] (8) A549 cells were used to evaluate the cellular uptake efficiency of NCHR. A549 cells were cultured at 5×10 4 Cells were seeded in confocal culture dishes and incubated for 24 h. NCH, NCHR1, NCHR2, and NCHR3 were prepared with 1640 medium at 200 μg mL -1 1 mL of each solution was added to the above confocal culture dish and incubated for 2 h, and the uptake of the four groups of materials by cells was observed every 30 min using a laser confocal microscope (Leica, Germany). Figure 5 As shown in the figure, the modification with Arg effectively improved the membrane penetration effect of the fluorescent material, among which NCHR3 had the highest cell uptake efficiency under the same incubation time, so NCHR3 was selected as the final fluorescent nanomaterial, hereinafter referred to as NCHR.

[0044] Example 7

[0045] (1)-(6) are the same as steps (1)-(6) in Example 6

[0046] (7) Dilute NCHR with PBS (0.01 M, pH 7.4) to 1 mg mL -1 , 1.2mL of solution; (1-ethyl-(3-dimethylaminopropyl)carbodiimide) was added to the above solution to make the final concentration in the whole system 1mgmL -1 , activated at 37°C for 1h.

[0047] (8) Take 4 μL of 100 μM DNA stock solution and 4 μL of TM buffer solution (0.1 M, pH 7.5) and add them to 24 μL of sterile enzyme-free water to prepare 40 μL of 10 μM DNA solution. Denature at 95°C for 5 min, then cool to room temperature in a stepwise manner to anneal and form a hairpin structure.

[0048] (9) Add the above DNA hairpin solution to the activated NCHR solution, react at 37°C in the dark for 12 h, use a 30 kDa ultrafiltration tube, 14,000 rpm, 5 min ultrafiltration to remove uncoupled DNA, and redisperse in PBS (0.01 M, pH 7.4) to prepare 2 mg mL -1 The solution was NCHR-h, which was stored at 4°C.

[0049] (10) The target viral sequence to be detected was prepared into a 1 μM solution with sterile enzyme-free water. The total detection reaction system was 400 μL, with a total of 6 groups. 200 μL NCHR-h (2 mg mL -1 ), 40 μL TM buffer solution (0.1 M, pH 7.5) and 0, 4, 20, 40, 80, 160 μL of viral target sequence (1 μM), and the solution of the 6 detection systems was supplemented to 400 μL with sterile enzyme-free water. Incubate at 37°C in the dark for 1 hour. Fluorescence signal detection was performed using a fluorescence spectrophotometer, and parallel samples were tested 3 times. Figure 6 The data showed that the recovery intensity of the fluorescence signal was positively correlated with the concentration of the viral target sequence, with a detection range of 10-400nM, and the calculated minimum detection limit was 5.3nM.

Claims

1. A method for preparing a gold-silver nanocluster coupled DNA fluorescent nanoprobe, It is characterized in that Gold-silver nanoclusters NC were synthesized using bovine serum albumin (BSA) as a template, and histidine His and arginine Arg molecules were modified on their surface through electrostatic interactions to prepare NCHR. NCHR was covalently coupled to a DNA hairpin h targeting the SARS-CoV-2 genome, and the uncoupled DNA was removed by centrifugation to obtain the nanofluorescent probe NCHR-h. The preparation method of fluorescent nanomaterial NCHR comprises the following steps: 1) dissolving BSA in ultrapure water; 2) adding tetrahydrate chloroauric acid HAuCl to the above solution in sequence at 37°C; 4 ·4H 2 O solution and silver nitrate solution, react for 5 minutes respectively; 3) add sodium hydroxide NaOH solution to the above solution, react for 24 hours, and synthesize gold-silver nanoclusters NC; 4) add 15, 22.5, and 30mM His solution to the solution after the above reaction, react for 3 hours, and synthesize NCH1, NCH2 and NCH3 respectively; 4) select NCH2 with the best fluorescence intensity for the next step of optimization, which is hereinafter referred to as NCH; add 22.5, 34, and 45mM Arg solution to the NCH solution after the reaction, react for 3 hours, use 10kda ultrafiltration tube for ultrafiltration purification, and redisperse in PBS solution to synthesize NCHR1, NCHR2 and NCHR3 respectively, and store at 4°C; The concentration of the NaOH solution is 1 M, and the molar ratios of the three groups of NCH are NCH1 = BSA: HAuCl 4 ·4H 2 O:AgNO 3 :His=1:10:1.7:4;NCH2=BSA:HAuCl 4 ·4H 2 O:AgNO 3 :His=1:10:1.7:6; NCH3=BSA:HAuCl 4 ·4H 2 O:AgNO 3 :His=1:10:1.7:8; the molar ratios of the three groups of NCHR are NCHR1=BSA:HAuCl 4 ·4H 2 O:AgNO 3 :His:Arg=1:10:1.7:6:6;NCHR2=BSA:HAuCl 4 ·4H 2 O:AgNO 3 :His:Arg=1:10:1.7:6:9;NCHR3=BSA:HAuCl 4 ·4H 2 O:AgNO 3 :His:Arg=1:10:1.7:6:12; The preparation method of the fluorescent nanoprobe NCHR-h comprises the following steps: 1) diluting NCHR with PBS solution to prepare 1 mg mL -1 2) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the above solution to make the final concentration in the whole system 1mg mL -1 , activated for 1h; 3) denaturing the DNA at 95°C for 5min, then cooling to room temperature in a stepwise manner to anneal and form a hairpin structure. The DNA concentration was 10×10 -6 M; 4) adding the above DNA hairpin solution to the activated NCHR solution, reacting at 37°C in the dark for 12 hours, using a 30kDa ultrafiltration tube to ultrafiltration and purify to remove uncoupled DNA, redispersing in PBS solution to synthesize NCHR-h, and storing at 4°C.

2. The preparation method according to claim 1, Features: The centrifugal speed is 12000-14000rpm, and the centrifugal time is 5-10min.

3. The fluorescent nanoprobe is prepared by the preparation method described in claim 1.

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