A surface-enhanced raman substrate based on three-dimensional DNA nano-pattern and a preparation method thereof

Abstract

The application discloses a three-dimensional DNA nano-pattern surface-enhanced Raman substrate and a preparation method thereof. The three-dimensional DNA nano-pattern is built by a DNA brick LEGO type pairing method, and metal nanoparticles are closely and orderly inlaid in the hole structure of the DNA nano-pattern. The DNA nano-pattern is built by a plurality of DNA bricks through the LEGO type pairing method, and the structure is stable and firm. When the nanoparticles are inlaid on the pattern surface through base complementary pairing with the DNA pattern, the strong repulsive force between the nanoparticles can be overcome, and the nanoparticles with a larger size can be closely and firmly inlaid on the nano-pattern surface. The three-dimensional hole sizes are different due to the different sequences of the DNA bricks, and the fixing of the nanoparticles with different diameters can be realized. The longitudinal and transverse positions of the preset DNA nano-pattern hole on the pattern surface are adopted to realize the orderly arrangement of the nanoparticles. The SERS substrate with closely contacted and orderly arranged nanoparticles is obtained through the above method. The SERS substrate provided by the application can generate high-intensity and dense and uniform Raman hot spots, and can realize high-sensitivity and high-reproducibility Raman spectrum detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanotechnology, and relates to a surface-enhanced Raman substrate based on a three-dimensional DNA nanograph and a preparation method thereof. BACKGROUND

[0002] Surface-enhanced Raman scattering (SERS) is a sensitive chemical analysis technology, which mainly utilizes the localized surface plasmon resonance of noble metal nanoparticles to greatly enhance the Raman scattering signal of the measured molecules; it has been widely used in the fields of biology, chemistry and medicine, and in the detection technology, SERS is considered as a fast and sensitive universal technology, and its detection limit can reach the single molecule level.

[0003] In order to achieve higher detection limit and sensitivity, SERS substrates with more hot spots have attracted more and more attention. The traditional nanometer array SERS substrate is directly self-assembled in a high-concentration noble metal nanoparticle sol, and in a certain range, the larger the diameter of the nanoparticles, the greater the "hot spot" strength, but the larger the diameter of the nanoparticles, the greater the repulsion between them. Therefore, due to the influence of repulsion, the spacing between the nanoparticles in the traditional nanometer array SERS substrate is large, so that the "hot spot" strength is low. The arrangement of nanoparticles in the traditional SERS substrate is also not uniform, which will also cause the "hot spot" to be uneven, thereby greatly reducing the sensitivity and reproducibility of the Raman detection technology. In recent years, it is still a challenge to obtain a SERS substrate with high and uniform "hot spot" strength. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application proposes a three-dimensional DNA nanograph formed by Lego assembly of DNA bricks, which realizes a surface-enhanced Raman substrate overcoming the strong repulsion between large-diameter gold nanoparticles. The closely and orderly arranged large-diameter gold nanoparticles have high-density, high-strength and uniform distribution of "hot spots", and the SERS substrate solves the problems of high sensitivity and high reproducibility of Raman molecule detection at ultra-low concentration.

[0005] The above technical purpose of the present application is realized by the following technical scheme:

[0006] The three-dimensional DNA nanograph is a stable structure formed by Lego pairing of DNA bricks, and the holes on the surface of the graph can overcome the repulsion between metals when the metal nanoparticles are tightly fixed, so that the metal nanoparticles can be closely and orderly inlaid on the surface of the DNA nanograph, thereby obtaining a SERS substrate with high density and orderly arrangement.

[0007] The specific implementation steps are as follows:

[0008] Step one, design DNA nano-pattern. Design a pattern containing a hole structure that can inlay gold nanoparticles;

[0009] Step two, prepare DNA bricks. Through computer programming of the designed three-dimensional DNA pattern, calculate the specific base sequence of the DNA bricks required for synthesizing the nano-pattern and synthesize;

[0010] Step three, prepare DNA nano-pattern. By mixing the DNA bricks synthesized in the previous step, and annealing to form the DNA pattern designed in step one;

[0011] Step four, modify the hole surface of the DNA nano-pattern to form a short chain of base sequence A;

[0012] Step five, thiolate the surface of the gold nanoparticles to connect them to the short chain of base sequence T;

[0013] Step six, inlay gold nanoparticles in the three-dimensional DNA nano-pattern to form a surface-enhanced Raman substrate (SERS). Gold nanoparticles are connected through the base sequence complementarity principle and the base sequence in the hole of the DNA nano-pattern. Gold nanoparticles are firmly inlaid on the DNA nano-pattern, forming a SERS substrate that is tightly and orderly arranged in both the longitudinal and lateral directions.

[0014] Compared with the prior art, the present application has the following excellent properties:

[0015] I. The DNA nano-pattern is built by a plurality of DNA bricks through a Lego-type pairing method, and its structure is stable and firm. The hole structure allows the nanoparticles to be firmly inlaid on the surface of the nano-pattern, which can overcome the strong repulsive force between the nanoparticles and achieve tight contact of metal nanoparticles with large diameters, thereby achieving stronger Raman "hot spots".

[0016] II. By pre-setting the longitudinal and lateral positions of the hole in the pattern surface, high-density ordered arrangement of nanoparticles can be achieved.

[0017] III. Due to the high-density ordered arrangement of nanoparticles in the longitudinal and lateral directions, more Raman "hot spots" can be achieved. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 is a schematic diagram of Lego-type pairing synthesis of DNA bricks to form a three-dimensional pattern;

[0019] Figure 2 Figure 2 is a schematic diagram of a DNA nano-pattern formed by DNA bricks with a hole structure on the surface;

[0020] Figure 3 Figure 3 is a DNA nano-pattern with a hole structure modified with a short chain of base A;

[0021] Figure 4 Schematic diagram of short base T modification for gold nanoparticle surface;

[0022] Figure 5 Schematic diagram of SERS substrate for closely arranged large-diameter gold nanoparticles; DETAILED DESCRIPTION

[0023] The technical solutions in the present application will be further described below in combination with the drawings and examples.

[0024] In the present embodiment, what needs to be prepared is a Raman substrate in which gold nanoparticles are closely and orderly inlaid in a three-dimensional DNA nanometer pattern surface in an array form, with 3x4 nanoparticles on the surface, and the size of the gold nanoparticles being 60 nm and the interval between adjacent nanoparticles being 1 nm.

[0025] To realize the above Raman substrate, the following steps are taken:

[0026] Step one, design a three-dimensional DNA nanometer pattern. A 190x250x30 nm DNA nanometer pattern is designed by computer,

[0027] The nanometer pattern surface has 3x4 square holes with a size of 50x50x15 nm as shown in Figure 2 The nanometer pattern is composed of short DNA helix chains spliced with each other, and the chains are spliced with each other like a Lego model as shown in Figure 1 , thereby forming a DNA nanometer pattern. The DNA sequences corresponding to all the short DNA helix chains are designed by computer, and the number of the short DNA helix chains is about 30000.

[0028] Step two, prepare DNA bricks. The required DNA bricks are synthesized by the base sequences designed by computer, which are mainly composed of a short chain of 32 nucleotides (containing four 8-nucleotide regions), and the base sequences of each DNA brick are different, so that each DNA brick can only be Lego-complementarily paired with a specific DNA brick, thereby assembling the three-dimensional nanometer pattern designed by us.

[0029] Step three, prepare the DNA nanometer pattern. All the short DNA chains with specific sequences designed are mixed. Mix at 100 nM in 0.5xTE buffer (5 mM Tris, pH 7.9, 1 mM EDTA) and add 40 mM MgCl2 without carefully adjusting the stoichiometric ratio of the chains. Then anneal in a PCR thermal cycler using a rapid linear cooling step from 80℃ to 60℃ for 1 hour, and then linearly cool the slope from 72 hours from 60℃ to 24℃. According to the length of the second cooling step, change the annealing slope to 168 hours of annealing, to form the hole-type DNA nanometer pattern we need, as shown in Figure 2 .

[0030] Step four, modification of DNA nano-pattern. A short chain of about 10 bases A is formed in the hole of DNA nano-pattern by modification of the chain, to realize the connection of the short chain of thio-base A on the surface of gold nanoparticles, as shown in Figure 3 .

[0031] Step five, modification of gold nanoparticles. The thio-DNA sequence and colloidal gold solution are mixed and stirred at room temperature, and the connection of gold nanoparticles and thio-DNA sequence is realized through the sulfuration process. The thio-DNA sequence used is: 5'-TGACTCAATGACTCGTTTTTTTTTT-3'-phosphate-(CH2)3-SH), as shown in Figure 4 .

[0032] Step six, realization of the fixation of nanoparticles on the surface of DNA pattern. Gold nanoparticles are connected with the base sequence in the hole of DNA nano-pattern through the principle of base sequence complementarity. One gold nanoparticle is fixed in one nanopore hole, and the nanoparticle is stably inlaid on the surface of DNA nano-pattern, forming a SERS substrate that is tightly and orderly arranged in both longitudinal and transverse directions, as shown in Figure 5 .

[0033] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should all be covered in the scope of the claims of the present application.

Claims

1. A surface-enhanced Raman substrate based on three-dimensional structure DNA nano-pattern, characterized in that: The structure is composed of three-dimensional DNA nano-pattern and metal nanoparticles; the three-dimensional DNA nano-pattern is built by DNA brick Lego pairing method; the metal nanoparticles are closely and orderly inlaid on the surface of the DNA nano-pattern; the surface of the DNA nano-pattern can form holes of any size to realize the inlay of nanoparticles of different diameters; the DNA brick is formed by folding 180° symmetrically on both sides of the DNA helix chain in the middle; the hole type three-dimensional nano-pattern built by the DNA brick is stable, and when the metal nanoparticles are closely fixed, the repulsive force between the metals can be overcome, and the minimum spacing is 1 nm.

2. The surface-enhanced Raman substrate based on three-dimensional DNA nanopatterns as described in claim 1, characterized in that: The DNA nano-pattern can be any pattern, which is built by a plurality of DNA bricks through Lego pairing method, and the structure is stable and firm.

3. The surface-enhanced Raman substrate based on three-dimensional DNA nanopatterns as described in claim 1, characterized in that: The metal nanoparticles are arranged into any close pattern according to the hole distribution on the DNA pattern.

4. The surface-enhanced Raman substrate based on three-dimensional DNA nanopatterns as described in claim 1, characterized in that: The particle size of the metal nanoparticles ranges from 2 nm to 60 nm.

5. The preparation method of the surface enhanced Raman substrate based on the three-dimensional structure DNA nano-pattern, according to claim 1, characterized in that: The method comprises the following steps: Step one, design the DNA nano-pattern, design the pattern containing the hole structure capable of inlaying the gold nanoparticles; Step two, prepare the DNA brick, compile the designed three-dimensional DNA pattern by computer, calculate the specific base sequence of the DNA brick required for synthesizing the nano-pattern and synthesize; Step three, prepare the DNA nano-pattern, mix the DNA bricks synthesized in the previous step, and anneal to form the DNA pattern designed in step one; Step four, modify the surface of the hole of the DNA nano-pattern to form a short chain of base sequence A; Step five, thiolate the surface of the gold nanoparticles to connect with the short chain of base sequence T; Step six, inlay the gold nanoparticles in the three-dimensional DNA nano-pattern to form the surface enhanced Raman substrate (SERS), the gold nanoparticles are connected with the base sequence in the hole of the DNA nano-pattern through the base sequence complementary principle, the gold nanoparticles are stably inlaid in the hole of the DNA nano-pattern, and the SERS substrate is closely and orderly arranged in the longitudinal and transverse directions.

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