A controllable assembly method of gold nanoparticles-DNA probe based on protein-assisted freezing acceleration

Abstract

The application discloses a controllable assembly method of a nano-gold-DNA probe based on protein-assisted freezing acceleration, which is realized by mixing a nano-gold solution with sufficient protein solution and thiol-modified DNA solution, and then performing freezing, thawing and centrifugation steps. The addition of sufficient protein forms a "protein crown" on the surface of the nano-gold, which protects the nano-gold from aggregation during the freezing process by steric hindrance. The thiol-modified DNA can be connected to the surface of the nano-gold through an Au-S bond. The process is greatly accelerated by the "limiting effect" of the ice crystals formed in the freezing process. Meanwhile, the density of the DNA on the surface of the nano-gold can be controlled by adjusting the amount of the added DNA. The assembly method has the advantages of simple operation, short time consumption and adjustable DNA density, and provides a new general strategy for the assembly and control of probes in the field of biochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nano-gold-DNA probe, in particular to a controllable assembly method of nano-gold-DNA probe based on protein-assisted freezing acceleration. BACKGROUND

[0002] The probe assembled by connecting DNA to nano-gold (AuNPs) has many unique properties, which makes it have wide application in the field of nano-material assembly, biosensing and gene regulation. Further, by attaching DNA and other biomolecules to AuNPs at the same time, multifunctional nano-probes can be assembled, which integrates the specific recognition ability of DNA chain and the unique characteristics of biomolecules. In order to realize the attachment of negatively charged DNA to negatively charged AuNPs, the commonly used method is salt-aging method, which needs to add salt (such as sodium chloride) to the AuNPs solution step by step to reduce the electrostatic repulsion between AuNPs and DNA, but this process usually needs to continue for tens of hours. In order to overcome this limitation, researchers have proposed some methods to promote the assembly of AuNPs-DNA, including freezing-guided method, microwave water removal method, etc. These methods shorten the assembly time, but often require a large amount of even excess DNA to prevent AuNPs from aggregating. However, it must be emphasized that in the field of biochemical sensing, high-density DNA attachment on AuNPs often weakens the sensitivity of the sensor, in addition, the use of a large amount of DNA also greatly increases the detection cost. Therefore, it is of great research and application value to develop a fast and controllable assembly method of AuNPs-DNA probe, which is of great significance to promote the development of biochemical sensing, nano-materials and other fields. SUMMARY

[0003] The present application aims at the deficiencies of the prior art, and provides a controllable assembly method of nano-gold-DNA probe based on protein-assisted freezing acceleration.

[0004] The purpose of the present application is achieved by the following technical scheme: a controllable assembly method of nano-gold-DNA probe based on protein-assisted freezing acceleration, mixing AuNPs solution with sufficient protein solution and thiol-modified DNA solution to obtain a mixture; then freezing the mixture to complete freezing, and then taking out the mixture and thawing to liquid; after thawing, removing the unattached protein and DNA by centrifugation, and redissolving the precipitate to obtain the assembled AuNPs-DNA probe.

[0005] Further, the AuNPs solution comprises nano-gold with a diameter of 15 nanometers synthesized by the method of reducing chloroauric acid with trisodium citrate or nano-gold with a diameter of 4 nanometers synthesized by the method of reducing chloroauric acid with sodium borohydride.

[0006] Further, the protein solution for assisting contains enzymes and proteins, including horseradish peroxidase, glucose oxidase or bovine serum albumin.

[0007] Further, the thiol-modified DNA solution contains thiol-modified or disulfide-modified DNA single strands, which are not limited in sequence order, sequence length and concentration.

[0008] Further, the molar ratio of protein to AuNPs is 200:1 or above.

[0009] Further, the sufficient amount of protein forms a "protein crown" on the surface of AuNPs, which protects AuNPs from aggregation during freezing by steric hindrance, and accelerates the connection of thiol-modified DNA to the surface of AuNPs through Au-S bond based on the "confinement effect" of ice crystals formed during freezing.

[0010] Further, the density of DNA on the surface of AuNPs required is regulated by the amount of DNA added.

[0011] Advantages of the present application: The present application realizes the rapid and controllable assembly of gold nanoparticle-DNA probes. Compared with the existing assembly method of gold nanoparticle-DNA probes, the present application overcomes the shortcomings of long synthesis time and uncontrollable surface DNA amount of the existing method. According to the above advantages, the method of the present application can be widely applied in the fields of biochemical sensing, nanomaterials and drug delivery, etc. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is a schematic diagram of the principle of the method of the present application;

[0013] Figure 2 is an absorption spectrum of the probe assembled by the method of the present application;

[0014] Figure 3 is a hydration particle size diagram of the probe assembled by the method of the present application;

[0015] Figure 4 is a real object diagram of the probe assembled by the method of the present application;

[0016] Figure 5 is a verification result diagram of the controllable assembly of AuNPs-DNA by the method of the present application. DETAILED DESCRIPTION

[0017] The present application will be described in detail below in combination with the drawings and specific examples.

[0018] Example 1:

[0019] As Figure 1As shown, the present application proposes a controllable assembly method of gold nanoparticles-DNA probe based on protein-assisted freezing acceleration. 300 μL of AuNPs solution 1 is mixed with 5 μL of protein solution 2 and 5 μL of thiol-modified DNA solution 3 in a centrifuge tube, the molar ratio of protein to AuNPs is 200:1 or more, and the thiol-modified DNA solution contains thiol-modified or disulfide-modified DNA single strands, and the sequence order, i.e. the arrangement order of A, G, C, T bases, the length of the sequence, the concentration of the DNA solution, etc. are not particularly limited. Then the mixture is placed in a low temperature condition and frozen to complete freezing, and then the mixture is taken out and thawed to liquid. After thawing, the unattached protein and DNA are removed by centrifugation, and the precipitate is redissolved to obtain the assembled AuNPs-DNA probe 4.

[0020] Specifically, the gold nanoparticles AuNPs solution is: 100 mL of 0.01% mass fraction chloroauric acid solution is added to a round-bottom flask, heated to boiling state. Then, 4 mL of 1% mass fraction trisodium citrate solution is quickly added to the above solution, and heating is continued for 15 minutes, and finally the obtained solution is cooled to room temperature to obtain a gold nanoparticle solution with a diameter of 15 nanometers.

[0021] Specifically, the protein solution is: bovine serum albumin (BSA) solution, BSA powder is dissolved to a concentration of 10 mg / mL using 1×PBS buffer.

[0022] Specifically, the thiol-modified DNA solution is: the sequence of the DNA is: 5'-SH-TTTTTTTTTTCAGACCGAGACAAGTGCAATGCTTTTTTTTTTTTTT-3', and the DNA powder is dissolved to different concentrations using TE buffer.

[0023] Specifically, the low temperature condition is: the mixture is frozen in a -80°C refrigerator for 20 minutes and taken out;

[0024] Specifically, the centrifugation condition is: the mixed solution is centrifuged at a speed of 14000 rpm for 30 minutes by a centrifuge, the supernatant is discarded, and then the precipitate is redissolved in 300 μL of 1×PBS buffer.

[0025] The absorption spectrum of the probe obtained by the above synthesis method is as follows: Figure 2 As shown, the characteristic absorption peak of the assembled probe has a certain red shift compared with the un-assembled AuNPs, which indicates the successful synthesis of the probe and proves the feasibility of the method.

[0026] Example 2:

[0027] As shown in Figure 1As shown, the present application proposes a controllable assembly method of gold nanoparticles-DNA probe based on protein-assisted freezing acceleration. 300 μL of AuNPs solution 1 is mixed with 5 μL of protein solution 2 and 5 μL of thiol-modified DNA solution 3 in a centrifuge tube, the molar ratio of protein to AuNPs is 200:1 or more, and the thiol-modified DNA solution contains thiol-modified or disulfide-modified DNA single strands, and the sequence order, i.e. the arrangement order of A, G, C, T bases, the length of the sequence, the concentration of the DNA solution, etc. are not particularly limited. Then the mixture is placed in a low temperature condition and frozen to complete freezing, and then the mixture is taken out and thawed to liquid. After thawing, the unattached protein and DNA are removed by centrifugation, and the precipitate is redissolved to obtain the assembled AuNPs-DNA probe 4.

[0028] Specifically, the gold nanoparticles AuNPs solution is: 100 mL of 0.01% mass fraction chloroauric acid solution is added to a round-bottom flask, heated to boiling state. Then, 4 mL of 1% mass fraction trisodium citrate solution is quickly added to the above solution, and heating is continued for 15 minutes, and finally the obtained solution is cooled to room temperature to obtain a gold nanoparticle solution with a diameter of 15 nanometers.

[0029] Specifically, the protein solution is: horseradish peroxidase (HRP) solution, the HRP stock solution is dissolved in 1×PBS buffer to a concentration of 10 mg / mL.

[0030] Specifically, the disulfide-modified DNA solution is: the sequence of the DNA is: 5'-HS-SH-TTTTTTTTTTCAGACCGAGACAAGTGCAATGCTTTTTTTTTTTTTT-3', and the DNA powder is dissolved in TE buffer to different concentrations.

[0031] Specifically, the low temperature condition is: the mixture is frozen in a -80°C refrigerator for 20 minutes and taken out;

[0032] Specifically, the centrifugation condition is: the mixed solution is centrifuged at a speed of 14000 rpm for 30 minutes by a centrifuge, the supernatant is discarded, and then the precipitate is redissolved in 300 μL of 1×PBS buffer.

[0033] The dynamic light scattering results of the probe obtained by the above synthesis method are as follows Figure 3 As shown, the hydrated particle size of the assembled probe is larger than that of the un-assembled AuNPs, which indicates the successful synthesis of the probe and proves the feasibility of the method.

[0034] Example 3:

[0035] As shown in Figure 1As shown, the present application proposes a controllable assembly method of gold nanoparticles-DNA probe based on protein-assisted freezing acceleration. 300 μL of AuNPs solution 1 is mixed with 5 μL of protein solution 2 and 5 μL of thiol-modified DNA solution 3 in a centrifuge tube, and the molar ratio of protein to AuNPs is 200:1 or more. Then, the mixture is placed in a low-temperature condition and frozen to complete freezing, and then the mixture is taken out and thawed to liquid. After thawing, the unattached protein and DNA are removed by centrifugation, and the precipitate is redissolved to obtain the assembled AuNPs-DNA probe 4.

[0036] Specifically, the gold nanoparticles AuNPs solution is: 100 mL of 0.01% chloroauric acid solution and 0.645 mL of 1% trisodium citrate solution are mixed and added to a conical flask. Then, 3 mL of freshly prepared 0.1 M sodium borohydride solution is added, stirred for 30 minutes, and then the resulting solution is left to stand for 2 hours to obtain a gold nanoparticle solution with a diameter of 4 nanometers.

[0037] Specifically, the protein solution is: glucose oxidase (GOx) solution, and the GOx stock solution is dissolved in 1×PBS buffer to a concentration of 10 mg / mL.

[0038] Specifically, the thiol-modified DNA solution is: the sequence of the DNA is: 5'-SH-TTTTTTTTTTCAGACCGAGACAAGTGCAATGCTTTTTTTTTTTTTT-3', and the DNA powder is dissolved in TE buffer to different concentrations.

[0039] Specifically, the low-temperature condition is: the mixture is frozen in a -20°C refrigerator for 2 hours and then taken out;

[0040] Specifically, the centrifugation condition is: the mixture is transferred to an ultrafiltration device (pore size: 100 kDa) and centrifuged at 4000 rpm for two times. Each time 5 minutes for purification.

[0041] The above synthesis method, the actual picture of the obtained probe is as Figure 4 As shown, the assembled probe still maintains the original red color after freezing, which indicates the successful synthesis of the probe and proves the feasibility of the method.

[0042] Example 4:

[0043] Verification of controllable assembly of AuNPs-DNA: To quantify the amount of DNA assembled on the surface of AuNPs, fluorescently labeled DNA (DNA-FAM) was used to characterize. First, a series of concentrations of DNA-FAM were prepared and the fluorescence intensity was recorded to establish the relationship between fluorescence intensity and DNA-FAM concentration. Then dithiothreitol solution (10 μL, 2 M) was added to the assembled probe, followed by incubation at 4 °C for 12 hours to replace the DNA-FAM assembled on the surface of AuNPs. After centrifugation (14000 rpm, 30 minutes), the supernatant was collected and subjected to fluorescence measurement. The quantification of DNA-FAM was performed by using the relationship between fluorescence intensity and DNA-FAM concentration that had been established. The results, as shown in Fig. 2, indicated that the amount of DNA-FAM assembled on the surface of AuNPs gradually increased with the increase of the amount of added DNA-FAM, verifying that the method of the present application can achieve controllable assembly of AuNPs-DNA. Figure 5

[0044] It should be noted that the content and specific embodiments of the present application are intended to demonstrate the practical application of the technical solutions provided by the present application, and should not be interpreted as a limitation on the scope of protection of the present application. Any modifications and changes made to the present application within the spirit and protection scope of the claims fall within the scope of protection of the present application.​

Claims

1. A method for the controlled assembly of gold nanoparticle-DNA probes based on protein-assisted cryo-acceleration, characterized in that, A gold nanoparticle solution was mixed with a sufficient amount of protein solution and a thiol-modified DNA solution to obtain a mixture. The gold nanoparticle solution contained gold nanoparticles with a diameter of 15 nm synthesized by the trisodium citrate-chloroauric acid reduction method or gold nanoparticles with a diameter of 4 nm synthesized by the sodium borohydride-chloroauric acid reduction method. The protein was horseradish peroxidase, glucose oxidase, or bovine serum albumin. The mixture was then frozen until completely frozen, and then removed and thawed into a liquid state. A sufficient amount of protein formed a "protein crown" on the surface of the gold nanoparticles, which protected the gold nanoparticles from dispersion during freezing by steric hindrance. Based on the "confinement effect" of the ice crystals formed during freezing, the thiol-modified DNA was accelerated to attach to the surface of the gold nanoparticles through Au-S bonds. The required DNA density on the surface of the gold nanoparticles was controlled by the amount of DNA added. After thawing, the unattached protein and DNA were removed by centrifugation, and the precipitate was redissolved to obtain the assembled gold nanoparticle-DNA probe.

2. The method for controllable assembly of gold nanoparticle-DNA probes based on protein-assisted cryo-acceleration according to claim 1, characterized in that, The molar ratio of protein to gold nanoparticles is 200:1.

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