Preparation method of Raman composite substrate material with biological recognition activity

Abstract

The invention relates to the field of biological recognition, in particular to a preparation method of a Raman composite substrate material with biological recognition activity, which comprises the following steps: S1, preparing a GO dispersion liquid; s2, gold nanospheres are prepared, and a gold nanosphere solution with OD 0.5 is prepared after purification; s3, respectively preparing a sodium chloride solution, a chloroauric acid solution and a single-stranded DNA solution; s4, mixing and reacting the GO dispersion liquid, the single-stranded DNA solution, NaCl, HAuCl4 and the gold nanosphere solution for 1 hour; s5, adding an NH2OH solution into the mixture prepared in the step S4, and reacting for 3 hours; and S6, centrifuging the reaction product obtained in the step S5, removing the supernatant, washing twice with ultrapure water, and re-suspending in ultrapure water to obtain the Au / DNA / GO composite nanomaterial. According to the invention, the compounding of the GO and the gold nanomaterial and the fixation of the DNA sequence are realized through the gold ion reduction regulated by DNA, the biological recognition activity of the fixed DNA sequence is maintained, and the prepared Raman composite substrate material has a better Raman signal enhancement effect.

Description

Technical Field

[0001] The invention relates to the field of biometric identification, and in particular to a method for preparing a Raman composite substrate material with biometric identification activity. Background Art

[0002] The biological recognition activity of Raman substrate materials is generally obtained by modifying aptamers or antibodies. However, this kind of stable surface modification generally requires additional reactions to form covalent bonds, which is cumbersome.

[0003] To this end, the present invention designs an Au / DNA / GO composite nanomaterial, which regulates the reduction of gold ions on the surface of graphene oxide (GO) and gold spheres through short-chain nucleic acid sequences (oligoDNA), thereby achieving material composite, morphology regulation and biorecognition activity. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a Raman composite substrate material with biological recognition activity to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing a Raman composite substrate material with biological recognition activity comprises the following steps:

[0007] S1, preparing GO dispersion;

[0008] S2, preparing gold nanospheres, and obtaining a gold nanosphere solution with an OD of 0.5 after purification;

[0009] S3, prepare sodium chloride solution, chloroauric acid solution, and single-stranded DNA solution respectively;

[0010] S4, mixing GO dispersion, single-stranded DNA solution, NaCl, HAuCl4, and gold nanosphere solution for reaction for 1 h;

[0011] S5. Add NH2OH solution to the mixture prepared in step S4 and react for 3 hours;

[0012] S6. Centrifuge the reaction product obtained in step S5, remove the supernatant, wash it twice with ultrapure water, and resuspend it in ultrapure water to obtain Au / DNA / GO composite nanomaterials.

[0013] Preferably, the S1 comprises the following steps:

[0014] S1.1, dissolve GO in water;

[0015] S1.2. Ultrasonic dispersion of the solution obtained in step S1.1 for 15-30 min to obtain a GO dispersion.

[0016] Preferably, S2 comprises the following steps:

[0017] S2.1, preparing a gold nanosphere solution using a trisodium citrate method, wherein the particle size of the gold nanosphere is 20-50 nm;

[0018] S2.2. Purify the solution obtained in step S2.1 to obtain a gold nanosphere solution with an OD of 0.5.

[0019] Preferably, the concentration of the GO dispersion in step S4 is 40 μg / mL, the concentration of the single-stranded DNA is 80 nM, the particle size of the NaCl crystal is 2.5 nM, and the mass fraction of HAuCl4 is 0.02%.

[0020] Preferably, the concentration of GO in S4 is 30-60 μg / mL, the concentration of single-stranded DNA is 60-100 nM, the crystal particle size of NaCl is 2.0-3.0 nM, the concentration of HAuCl4 is 0.02-0.05%, the gold nanospheres have an OD of 0.005, and a particle size of 20-50 nm.

[0021] Preferably, in S5, the pH value of the NH2OH solution is 4.0-6.0.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention synthesizes the composite material through only one-step reaction without additional modification.

[0024] 2. In the present invention, the composite of GO and gold nanomaterials and the fixation of DNA sequences are achieved through the reduction of gold ions regulated by DNA. The fixed DNA sequences maintain biological recognition activity, and the prepared Raman composite substrate material has a good Raman signal enhancement effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a flow chart of the method of the present invention;

[0026] Figure 2 The Raman spectra of rhodamine without substrate material and with AU / DNA / GO composite nanomaterial as substrate at the same concentration;

[0027] Figure 3 The Raman spectra of Au / DNA / GO composite nanomaterials to five different concentrations of rhodamine;

[0028] Figure 4 The bar graph is a comparison of the recognition ability of Au / DNA / GO composite nanomaterials to fluorescently labeled complementary DNA chains;

[0029] Figure 5These are the scanning electron microscopy results of Au / DNA / GO composite nanomaterials at two magnifications. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "top / bottom" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific position, be constructed and operated in a specific position, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0032] Embodiment 1:

[0033] See also Figure 1 The present invention provides a technical solution: a method for preparing a Raman composite substrate material with biological recognition activity, comprising the following steps:

[0034] S1. Prepare GO dispersion, specifically comprising the steps of:

[0035] S1.1, dissolve GO in water;

[0036] S1.2. Ultrasonic dispersion of the solution obtained in step S1.1 for 15-30 min to obtain a GO dispersion.

[0037] S2, preparing gold nanospheres, and obtaining a gold nanosphere solution with an OD of 0.5 after purification, specifically comprising the steps of:

[0038] S2.1, preparing a gold nanosphere solution using a trisodium citrate method, wherein the particle size of the gold nanosphere is 20-50 nm;

[0039] S2.2, purify the solution obtained in step S2.1 to obtain a gold nanosphere solution with an OD of 0.5

[0040] S3, prepare sodium chloride solution, chloroauric acid solution, and single-stranded DNA solution respectively;

[0041] S4. Mix the GO dispersion, single-stranded DNA solution, NaCl, HAuCl4, and gold nanosphere solution for reaction for 1 hour, wherein the concentration of the GO dispersion is 40 μg / mL, the concentration of the single-stranded DNA is 80 nM, the NaCl crystal particle size is 2.5 nM, and the mass fraction of HAuCl4 is 0.02%.

[0042] S5. Add NH2OH solution to the mixture prepared in step S4 and react for 3 hours until the pH value is 4.0;

[0043] S6. Centrifuge the reaction product obtained in step S5, remove the supernatant, wash it twice with ultrapure water, and resuspend it in ultrapure water to obtain Au / DNA / GO composite nanomaterials.

[0044] Embodiment 2:

[0045] The difference from Example 1 is that: the concentration of GO in step S4 is 60 μg / mL, the concentration of single-stranded DNA is 100 nM, the crystal particle size of NaCl is 3.0 nM, the concentration of HAuCl4 is 0.05%, the OD of gold nanospheres is 0.005, and the particle size is 20-50 nm;

[0046] The pH value of the NH2OH solution in step S5 is 6.0.

[0047] In some other embodiments, the reduced gold ions can be replaced by silver ions or platinum ions.

[0048] like Figure 2 As shown, at the same rhodamine concentration (concentration: 1×10 -4 mol / L), the Raman spectrum without substrate material (grey curve) is compared with the Raman spectrum with Au / DNA / GO composite nanomaterial as substrate (black curve), which shows that the Raman enhancement effect of the substrate material prepared by the present invention is better.

[0049] like Figure 3 As shown, at different rhodamine concentrations (concentration: 1×10 -4 , 0.5×10 -4 , 1×10 -5 , 0.5×10 -5 , 1×10 -6 mol / L), which is in order from top to bottom in the figure. It can be seen that as the concentration of the target increases, the effect of increasing the Raman signal intensity becomes more obvious.

[0050] refer to Figure 4, using fluorescently labeled complementary DNA sequences, to explore the biorecognition ability of DNA in composite materials. First, use an ELISA instrument to detect the fluorescence value of fluorescently labeled DNA; secondly, use a composite material containing a non-complementary DNA sequence to react, and take the supernatant after centrifugation to detect the fluorescence value (the composite material does not bind to the fluorescently labeled non-complementary sequence, and the fluorescent part is in the supernatant after centrifugation, and the fluorescence value in the supernatant remains basically unchanged); finally, use a composite material containing a complementary DNA sequence to react, and take the supernatant after centrifugation to detect the fluorescence value (if the DNA in the composite material has biorecognition activity, it can be combined with the fluorescently labeled complementary sequence, and the fluorescent part is in the precipitate after centrifugation, and the fluorescence value in the supernatant decreases). The DNA sequence used in this embodiment is a poly A sequence or a poly T sequence.

[0051] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0052] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a Raman composite substrate material with biological recognition activity, characterized in that: The method comprises the following steps: S1, preparing GO dispersion; S2, preparing gold nanospheres, and obtaining a gold nanosphere solution with an OD of 0.5 after purification; S3, prepare sodium chloride solution, chloroauric acid solution, and single-stranded DNA solution respectively; S4, mixing GO dispersion, single-stranded DNA solution, NaCl, HAuCl4 and gold nanosphere solution for reaction for 1 h; S5. Add NH2OH solution to the mixture prepared in step S4 and react for 3 hours; S6. Centrifuge the reaction product obtained in step S5, remove the supernatant, wash it twice with ultrapure water, and resuspend it in ultrapure water to obtain Au / DNA / GO composite nanomaterials.

2. The method for preparing a Raman composite substrate material with biological recognition activity according to claim 1, characterized in that: The S1 comprises the following steps: S1.1, dissolve GO in water; S1.

2. Ultrasonic dispersion of the solution obtained in step S1.1 for 15-30 min to obtain a GO dispersion.

3. The method for preparing a Raman composite substrate material with biological recognition activity according to claim 1, characterized in that: The S2 comprises the following steps: S2.1, preparing a gold nanosphere solution using a trisodium citrate method, wherein the particle size of the gold nanosphere is 20-50 nm; S2.

2. Purify the solution obtained in step S2.1 to obtain a gold nanosphere solution with an OD of 0.

5.

4. The method for preparing a Raman composite substrate material with biological recognition activity according to claim 1, characterized in that: The concentration of the GO dispersion in step S4 is 40 μg / mL, the concentration of the single-stranded DNA is 80 nM, the particle size of the NaCl crystal is 2.5 nM, and the mass fraction of HAuCl4 is 0.02%.

5. The method for preparing a Raman composite substrate material with biological recognition activity according to claim 1, characterized in that: The concentration of GO in the S4 is 30-60 μg / mL, the concentration of single-stranded DNA is 60-100 nM, the crystal particle size of NaCl is 2.0-3.0 nM, the concentration of HAuCl4 is 0.02-0.05%, the OD of gold nanospheres is 0.005, and the particle size is 20-50 nm.

6. The method for preparing a Raman composite substrate material with biological recognition activity according to claim 1, characterized in that: In the S5, the pH value of the NH2OH solution is 4.0-6.0.

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