Application of gold nanoparticles in shellfish blood cell transfection

Abstract

The invention discloses application of gold nanoparticles in shellfish blood cell transfection and a method for efficiently transfecting shellfish blood cells, and the method for efficiently transfecting shellfish blood cells comprises the following steps: S1, preparing gold nanoparticles, and loading biomolecules on the gold nanoparticles by mixing the biomolecules (such as DNA) and a gold nanoparticle solution, a gold nanoparticle suspension is obtained; s2, preparing shellfish blood cell suspension; and S3, adding the biomolecule-loaded gold nanoparticles into the shellfish blood cell suspension, and co-incubating for transfection. According to the high-throughput transfection method based on the gold nanoparticles, the transfection efficiency of shellfish cells can reach 48.3%, the survival rate of the transfected cells reaches up to 74.9%, and a new tool is provided for high-efficiency low-loss gene delivery and editing of shellfish.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, relates to the field of cell biology technology, and specifically relates to the application of gold nanoparticles in shellfish blood cell transfection. Background Art

[0002] The methods of transfecting cells can be divided into three main categories: biological transfection (using engineered viral vectors such as lentivirus and adenovirus to introduce genetic material into recipient cells), chemical transfection (interacting with the cell membrane through liposomes, cationic polymers, etc. to allow endocytosis into the cell), and physical transfection (based on electrofection, microinjection, etc. to mechanically change the physical properties of the local cell membrane and inject genetic material into the cell).

[0003] For shellfish cells, although the difficulty of biotransfection is relatively low, the use of viral vectors will cause shellfish cells to have certain cytotoxic risks. Given the main edible value of shellfish, the application of biotransfection in shellfish cells is very limited. In contrast, physical transfection and chemical transfection methods have a higher priority. Among them, microinjection, as a classic delivery method, was first tried in shellfish cells. Since the diameter of bivalve cells is small, much smaller than that of vertebrates such as mice, it has high requirements for technology and equipment, and the number of fertilized eggs is huge, the natural mortality rate is high, and low-throughput methods based on microinjection are difficult to apply. In addition, electrofection and liposome methods have also been tried in shellfish transfection operations. Electrofection uses electric pulses to process a large number of cells at a high throughput, so it is widely used, but different types of cell membranes are quite different, and the specific electrofection parameters are different. Existing data show that the cell survival rate of shellfish cells after electrofection is extremely low. Among them, the result of electrofection of oyster cells is only 9% at most. As a newer transfection method, liposome delivery is also used in shellfish cells, but its transfection efficiency is low and it is still difficult to meet the delivery needs of shellfish cells. Among them, the highest transfection efficiency in the scallop is only 13.2%. Therefore, the current shellfish transfection method has a low cell survival rate, and the mortality rate of shellfish cells has remained high due to different forms of transfection operations, which greatly limits the in-depth development of shellfish genetics and other work. For shellfish cells, a transfection method with less cell damage and higher transfection efficiency is urgently needed.

[0004] Along with the research of materials science, nanotechnology is gradually applied to biology. Gold nanoparticles (AuNPs) are a type of nano-scale material mainly composed of gold (Au). The particle size is usually between 1-100 nanometers, and can be spherical, rod-shaped, polyhedral, etc. Its optical and catalytic activity can be changed by adjusting the size and shape. The gold element gives it good chemical stability and excellent biocompatibility, which makes gold nanoparticles less prone to chemical reactions such as oxidation, and at the same time has the ability to interact with biological molecules such as proteins and nucleic acids. By modifying the surface of the particles to improve their functions, such as thiol substances, antibodies, etc., the aggregation of particles can be reduced and their stability and targeting effect can be improved.

[0005] These excellent properties of gold nanoparticles make them widely used, and they have broad application prospects in the fields of biological imaging, targeted drug delivery, cancer treatment, biochemical sensors and optical materials. At present, this technology has been successfully applied in cell types such as mammalian cells, yeast, Escherichia coli and stem cells, but has not been reported in aquatic animal cells. Especially for marine invertebrates, the habitat is complex, and cell culture and transfection are challenging. Among them, shellfish, as the largest group of marine invertebrates, have not yet established a stable cell line. Because of its advantages such as large number, easy access and easy operation, blood cells are widely used in shellfish gene manipulation research. Therefore, it is urgent to verify the feasibility of gold nanotransfection technology in shellfish blood cells, and to conduct systematic research on its parameters such as particle size, working concentration and incubation time, and optimize the transfection conditions to achieve the establishment of an efficient transfection method for shellfish blood cells. The implementation of this method will provide important technical support for realizing genetic manipulation of shellfish cells and solving major biological problems such as shellfish development and evolution, and will also provide a reference template for the development of efficient and non-destructive transfection technology for aquatic animal cells. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of gold nanoparticles in shellfish blood cell transfection and a method for efficiently transfecting shellfish blood cells.

[0007] The technical solution of the present invention mainly includes the following contents: The present invention provides the application of gold nanoparticles in shellfish blood cell transfection, that is, using gold nanomaterials as carriers to deliver biomolecules into shellfish blood cells, thereby achieving the purpose of improving transfection efficiency and maintaining a high survival rate of blood cells.

[0008] Furthermore, the present invention also provides a method for efficiently transfecting shellfish blood cells mediated by gold nanoparticles, comprising the following steps: S1: preparing a gold nanomaterial loaded with biomolecules; the biomolecules include DNA, and the nucleotide sequence of the DNA is any one of SEQ ID NO: 1 to SEQ ID NO: 4; S2: Preparation of shellfish blood cell suspension; S3: Shellfish blood cell transfection: The DNA-loaded gold nanomaterials are mixed with the blood cell suspension and incubated together.

[0009] Preferably, step S1 is: Preparation of gold nanomaterials loaded with biomolecules: adding tannic acid, sodium citrate dihydrate and potassium carbonate to water, heating, adding chloroauric acid and continuing heating to obtain AuNPs; mixing the biomolecules with the AuNPs in the presence of NaNO3 to obtain a mixed solution A, adding n-butanol to the mixed solution A and rapidly vortexing to obtain a mixed solution B, then adding TBE buffer to the mixed solution B and rapidly vortexing to obtain a lower layer of gold nanomaterials loaded with biomolecules, and storing for later use; the biomolecules include DNA, and the nucleotide sequence of the DNA is any one of SEQ ID NO:1 to SEQ ID NO:4; Preferably, in step S3, after the DNA-loaded gold nanomaterial is mixed with the blood cell suspension, the final concentration of the DNA-loaded gold nanomaterial is 5-10 nM; and the co-incubation time is 3-9 hours.

[0010] Preferably, step S1 is: Preparation of gold nanomaterials loaded with biomolecules: 0.1 mL 2.5 mM tannic acid, 9.7 mL 1 wt% sodium citrate dihydrate solution and 1 mL 150 mM potassium carbonate solution were added to 138.2 mL ultrapure water and heated to 68 °C, 1 mL 1wt% chloroauric acid was added and heated at 68 °C for 15 min to obtain AuNPs; 30 μL 250 nM AuNPs, 15 μL 100 μM biomolecules and 5 μL 200 mM sodium nitrate were added to 50 μL ultrapure water and mixed evenly to obtain a mixed solution A, n-butanol was added to the mixed solution A and vortexed quickly to obtain a mixed solution B, then 0.5×TBE buffer was added to the mixed solution B and vortexed quickly to mix, finally, the two phase solutions were separated by centrifugation to obtain a lower layer of gold nanomaterials loaded with biomolecules, and stored for later use; the biomolecules include DNA, and the nucleotide sequence of the DNA is SEQ ID NO: 1~SEQ ID NO: Any one of 4.

[0011] Preferably, the volume ratio of the n-butanol to the mixed solution A is 7:1.

[0012] Preferably, the volume ratio of the 0.5×TBE buffer to the mixed solution B is 1:4.

[0013] Preferably, the nucleotide sequence of the DNA is as shown in SEQ ID NO:2.

[0014] Preferably, in step S2, the cell concentration in the blood cell suspension is 1×10 6 ~2×10 6 Pieces / mL.

[0015] Preferably, in step S3, the co-incubation temperature is 18-20° C., and the co-incubation time is 6 h.

[0016] Preferably, the shellfish includes Yesso scallop.

[0017] Beneficial effects of the present invention: The present invention uses gold nanomaterials as carriers to carry biomolecules into cells. As a high-throughput transfection method, it has lower toxicity and higher transfection efficiency than commonly used delivery methods (such as liposomes and electroporation). In existing reports, the transfection rate of commonly used commercial liposomes on scallop cells is only 13.2%, and the efficiency of electrotransfection is less than 5%. The transfection method based on the present invention can achieve a transfection efficiency of 48.3% for shellfish cells, and a survival rate of up to 74.9%.

[0018] The present invention co-incubates gold nanomaterials with shellfish blood cells, allowing the gold nanomaterials to enter the cells through endocytosis and deliver the biomolecules they carry into the cells. The method uses gold nanomaterials with low cell damage as carriers to carry biomolecules into cells, thereby improving cell survival rate and transfection efficiency and simplifying the operation procedure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the process of transfecting cells with gold nanomaterials loaded with biomolecules.

[0020] Figure 2 The following are fluorescence micrographs of blood cells of the scallop transfected with AuNP-DNA-FAM and FAM-DNA alone under white light and 495 nm excitation light at 20× magnification. Figure 2A is a fluorescence micrograph of untransfected Yesso scallop blood cells under white light, B is a fluorescence micrograph of untransfected Yesso scallop blood cells under excitation light of 495 nm, C is a fluorescence micrograph of Yesso scallop blood cells transfected with FAM-DNA (i.e., DNA modified with a FAM fluorescent tag) under white light, D is a fluorescence micrograph of Yesso scallop blood cells transfected with FAM-DNA under excitation light of 495 nm, E is a fluorescence micrograph of Yesso scallop blood cells transfected with gold nanomaterials under white light, F is a fluorescence micrograph of Yesso scallop blood cells transfected with gold nanomaterials under excitation light of 495 nm, and G is a superposition of a photograph of Yesso scallop blood cells transfected with gold nanomaterials under the green fluorescence channel and a photograph under bright field.

[0021] Figure 3 This is a fluorescence micrograph of cells treated with Incucyte Annexin V Red cell viability indicator under white light and 20× field of view under excitation light with a wavelength of 594 nm. Figure 3 A is a fluorescence micrograph of untransfected Yesso scallop blood cells under white light, B is a fluorescence micrograph of untransfected Yesso scallop blood cells under excitation light of 594 nm, C is a fluorescence micrograph of Yesso scallop blood cells transfected with 5 nM gold nanomaterials under white light, and D is a fluorescence micrograph of Yesso scallop blood cells transfected with 5 nM gold nanomaterials under excitation light of 594 nm.

[0022] Figure 4 It is a line graph of cell survival rate after 5 nM-AuNP transfection.

[0023] Figure 5 It is a line chart of transfection efficiency after transfection using different gold nanoparticle working concentrations.

[0024] Figure 6 It is a bar graph showing the transfection efficiency and cell survival rate after using different transfection conditions.

[0025] Figure 7 to Figure 16 The following are fluorescence micrographs of blood cells of the scallop transfected with different lengths of payload sequences under excitation light at wavelengths of 495 nm and 360 nm. Figure 7 This is a fluorescence micrograph of the control group under 495 nm excitation light. Figure 8 is a fluorescence micrograph of the control group under excitation light of wavelength 360 nm; Fig. 9 This is a fluorescence micrograph of group "20" under the excitation light of wavelength 495 nm. Fig.10 This is a fluorescence micrograph of group “20” under excitation light of wavelength 360 nm; Fig.11This is a fluorescence micrograph of group "60" under the excitation light of wavelength 495 nm. Fig.12 This is a fluorescence micrograph of group “60” under excitation light of wavelength 360 nm; Fig.13 This is a fluorescence micrograph of the "60-F" group under 495 nm excitation light. Fig.14 This is a fluorescence micrograph of the “60-F” group under excitation light of wavelength 360 nm; Fig.15 This is a fluorescence micrograph of the "100" group under the excitation light of wavelength 495 nm. Fig.16 This is a fluorescence micrograph of group “100” under excitation light of wavelength 360 nm.

[0026] Fig.17 It is a statistical graph of transfection efficiency of loading sequences of different lengths. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0028] Example 1 Preparation of DNA-AuNPs The preparation process of DNA-loaded gold nanomaterials is as follows: Add 0.1 mL of 2.5 mM tannic acid, 9.7 mL of 1 wt% sodium citrate dihydrate solution, and 1 mL of 150 mM K2CO3 (potassium carbonate) solution to 138.2 mL of ultrapure water and heat to 68 °C. Add 1 mL of 1 wt% HAuCl4 (chloroauric acid) and heat at 68 °C for 15 min to obtain AuNPs, which are then stored at 4 °C.

[0029] Add 30 μL 250 nM AuNPs, 15 μL 100 μM DNA and 5 μL 200 mM NaNO3 (sodium nitrate) to 50 μL ultrapure water and mix well. Add n-butanol to the mixed solution (the volume ratio of n-butanol to the mixed solution is 7:1) and vortex quickly to mix. Subsequently, add 0.5×TBE buffer (pH 7.4) to the above solution (the volume ratio of TBE to the above solution is 1:4) and vortex quickly to mix. Finally, separate the two phases of the solution by centrifugation at 4000 rpm for several seconds, and remove the lower layer of DNA-AuNPs. The prepared DNA-AuNPs are stored at 4 °C.

[0030] The complete flow chart of the gold nanocluster delivery system is shown in Figure 1 As shown, after the gold nanomaterials carry biological molecules into the cells, they release the carried molecules outside the nucleus and perform their functions according to the modification of the molecules.

[0031] Example 2 Preparation of Scallop Blood Cell Suspension After removing the scallop, use a paper towel to absorb the excess seawater on the shell, and place it in a clean bench for ultraviolet irradiation for 30 minutes. Take a syringe, insert the injection needle into the smooth muscle and striated muscle of the scallop to extract the hemolymph mixture, and store it in a 1.5 mL centrifuge tube pre-cooled on ice.

[0032] The hemolymph mixture stored on ice was taken out and placed in a clean bench. At the same time, the L-15 basal medium was taken out from the 4°C refrigerator. A 15 mL centrifuge tube was prepared and the hemolymph mixture obtained from the Yesso scallop was mixed with the L-15 basal medium at a volume ratio of 1:2-5 to dilute the blood cell density to 1×10 6 ~2×10 6 Because the hemolymph mixture of the scallop contains a variety of nutrients that maintain the activity of blood cells, it is not necessary to add fetal bovine serum to the L-15 basic medium used for mixing.

[0033] Experimental Example 1 Analysis of DNA-AuNPs delivery to DNA The FAM fluorescent probe was modified on DNA and loaded onto gold nanomaterials to indicate the delivery ability of gold nanomaterials to nucleic acid biomolecules. The constructed material was co-incubated with the blood cells of the Yesso scallop cultured overnight at 18-20 ℃ for 6 hours. After 6 hours of co-incubation, the fresh L-15 complete medium supplemented with 10% fetal bovine serum was replaced and cultured in an incubator at 18-20 ℃ for 18 hours. During the culture process, 0.5 μL of Incucyte Annexin VRed cell activity indicator was added to the culture environment to indicate dead cells. The co-incubated cells were observed under the green fluorescence channel, red fluorescence channel and bright field channel of the Incucyte®S3 live cell analyzer respectively. In order to eliminate the influence of the autofluorescence of the blood cells of the Yesso scallop, the green fluorescence intensity of the untreated blood cells of the Yesso scallop was counted, and the highest intensity of the green fluorescence was set as the threshold. The number of cells with fluorescence intensity above the threshold in the observation results after co-incubation was counted, and the number of cells and the number of red fluorescence under the bright field were counted at the same time. In the statistical results, the ratio of the number of green fluorescence without red fluorescence overlap to the total number of cells was taken as the transfection efficiency, and the ratio of the number of cells without red fluorescence to the total number of cells was taken as the cell survival rate.

[0034] The cell transfection effect is shown in the figure Figure 2 As shown in the figure, after setting the threshold of green fluorescence, the transfected cells were observed and it can be observed that Figure 2 In (B), no green fluorescent spots were observed, which means that the green fluorescence shown in the subsequent fluorescence observation does not contain the autofluorescence of the blood cells of the scallop; Figure 2In (D), after the blood cells of the scallop were transfected with DNA modified with FAM fluorescent label alone, no obvious green fluorescence with cell outlines was observed; Figure 2 In (F), green fluorescence can be clearly observed; Figure 2 In (G), the image observed by the green fluorescence channel is superimposed with the observation image of all cells under the bright field, and the identified blood cells of the scallop are circled with a yellow outline, and the identified cells that excite green fluorescence are covered with a purple block. This indicates that the DNA modified with the FAM fluorescent label loaded with gold nanomaterials is released in the cells and is excited to emit green fluorescence, while the DNA modified with the FAM fluorescent label not loaded with gold nanomaterials is difficult to enter the cells and cannot be enriched in the cells and excited to emit obvious fluorescence. This shows that the present invention is feasible for transfecting blood cells using gold nanoparticles.

[0035] Cell death after transfection Figure 3 , Figure 4 As shown, by observing the transfected cells through the red fluorescence channel, it can be observed that Figure 3 (B) Figure 3 There is obvious red fluorescence in (D), which indicates the death of cells in the culture environment. Figure 3 The red fluorescence in (B) indicates that a certain proportion of blood cells die under natural conditions. Figure 3 As can be seen from (D), some cells also died in the gold nanoparticle-treated experimental group (the mortality rate was 25.1% after co-transfection for 6 hours and replacement of the culture medium for further 18 hours), but it was much lower than the mortality rate of electrofection and liposome transfection.

[0036] Experimental Example 2: Concentration Optimization The inoculum concentration was 1.5×10 6 Blood cells of 100 nM / mL were inoculated in a 96-well plate, and two different transfection treatment groups were set up: the working concentrations were 10 nM and 5 nM, respectively. After the gold nanomaterials were co-incubated with the cells for 9 hours, the co-incubated cells were observed under the green fluorescence channel, red fluorescence channel, and bright field channel of the Incucyte® S3 live cell analyzer to determine the transfection effect of the gold nanomaterials on the cells.

[0037] Transfection effect statistics Figure 5 As shown, both 5 nM working concentration and 10 nM working concentration showed a certain transfection efficiency. After taking the average transfection efficiency of multiple groups, the gold nanomaterial with a working concentration of 5 nM could reach a maximum transfection efficiency of 48.3%, and the gold nanomaterial with a working concentration of 10 nM could reach a maximum transfection efficiency of 46%.

[0038] Experimental Example 3: Incubation Time Optimization Blood cells were inoculated in a 96-well plate, and a co-incubation time gradient (3 hours, 6 hours, and 9 hours) was set based on the concentrations of 5 nM and 10 nM. After the gold nanomaterials were co-incubated with the cells, the 96-well plate was removed and replaced with fresh L-15 medium containing 10% fetal bovine serum for continued culture. The total culture time before and after the medium change was 48 hours. The co-incubated cells were observed under the green fluorescence channel, red fluorescence channel, and bright field channel of the Incucyte® S3 live cell analyzer to determine the effect of different co-incubation times on the transfection effect.

[0039] Transfection effect statistics Figure 6 As shown. Figure 6 In the experiment, the working concentrations of 5 nM and 10 nM showed the highest transfection efficiency (48.3% and 51.7%) under the condition of 6 h of co-incubation, but the cell survival rate under the condition of 10 nM (40.3%) was much lower than that under the condition of 5 nM (74.9%), which indicated that the working concentration of 5 nM and the co-incubation time of 6 h were the optimal transfection conditions for gold nanomaterials.

[0040] Experimental Example 4: Load Sequence Optimization Through the primer synthesis service of Sangon Biotech (Shanghai) Co., Ltd., short sequences with lengths of 20 nt, 60 nt, and 100 nt were synthesized. The specific information of the sequences is shown in Table 1. The synthesized short sequences were co-incubated with gold nanomaterials to obtain gold nanomaterials that bind to DNA sequences of different lengths. Blood cells were inoculated in a 96-well plate, and the co-incubation conditions of gold nanomaterials and blood cells were set to a working concentration of 5 nM and a co-incubation time of 6 hours. After the co-incubation, the 96-well plate was removed and replaced with fresh L-15 medium containing 10% fetal bovine serum for a further 9 hours. The cells after co-incubation were observed through the green fluorescence channel and blue fluorescence channel of the Zeiss LSM 900 confocal laser scanning microscope, and the number of fluorescence in the image was counted and calculated to determine the effect of the length of the loaded sequence on the transfection effect.

[0041] Table 1 DNA sequences

[0042] The transfection effect of the optimization attempt of the gold nanomaterial loading sequence conditions is as follows Figure 7 to Figure 17 As shown. Figure 7 to Figure 16 The green fluorescence images of the control group and the four experimental groups are shown in ( Figure 7 , Fig. 9 , Fig.11 , Fig.13 , Fig.15 ) and blue fluorescence images ( Figure 8 , Fig.10 , Fig.12, Fig.14 , Fig.16 ), and the fluorescence quantity in each figure was counted, and the statistical values ​​were all greater than 200 and were statistically significant. Statistical results ( Fig.17 ) showed that when the sequence length was 60 bp, it exhibited a transfection efficiency of 48.5% (60 group) and 41.0% (60-F group). The 60 bp loading sequence had the highest transfection efficiency, suggesting that the complex formed with gold nanoparticles is more stable and closer to the optimal size preferred by scallop endocytic vesicles. At the same time, the transfection efficiency of the 60 group was higher than that of the 60-F group, indicating that when constructing the loading sequence, the tail poly (A) sequence commonly used for gold nanomaterial loading can be shortened, which allows more working sequences to be transferred into cells to perform their functions. This discovery provides a direct basis for the length and sequence optimization of the design of gold nanocarriers in scallops, and has practical guiding significance in the design of nucleic acid sequences such as scallop gene editing (such as CRISPR gRNA) or RNA interference (siRNA / shRNA).

[0043] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of gold nanoparticles in transfection of shellfish blood cells.

2. A method for efficiently transfecting shellfish blood cells, characterized in that: The method comprises the following steps: S1: preparing a gold nanomaterial loaded with biomolecules; the biomolecules include DNA, and the nucleotide sequence of the DNA is any one of SEQ ID NO: 1 to SEQ ID NO: 4; S2: Preparation of shellfish blood cell suspension; S3: Shellfish blood cell transfection: The gold nanomaterials loaded with biomolecules are mixed with the shellfish blood cell suspension and incubated together.

3. The method according to claim 2, characterized in that Step S1 is: The method comprises the following steps of: adding tannic acid, sodium citrate dihydrate and potassium carbonate to water, heating the mixture, adding chloroauric acid and continuing to heat the mixture to obtain AuNPs; mixing the biomolecules with the AuNPs in the presence of sodium nitrate to obtain a mixed solution A; adding n-butanol to the mixed solution A and rapidly vortexing the mixed solution to obtain a mixed solution B; subsequently adding TBE buffer to the mixed solution B and rapidly vortexing the mixed solution; and finally, separating the two phases of the solution by centrifugation to obtain a lower layer of the gold nanomaterial loaded with the biomolecules, which is stored for later use; the biomolecules include DNA, and the nucleotide sequence of the DNA is any one of SEQ ID NO:1 to SEQ ID NO:

4.

4. The method according to claim 2, characterized in that: In step S3, after the gold nanomaterial loaded with biomolecules is mixed with the blood cell suspension, the final concentration of the gold nanomaterial loaded with biomolecules is 5-10 nM; and the co-incubation time is 3-9 hours.

5. The method according to claim 2, characterized in that: Step S1 is: Preparation of gold nanomaterials loaded with biomolecules: 0.1 mL of 2.5 mM tannic acid, 9.7 mL of 1 wt% sodium citrate dihydrate solution and 1 mL of 150 mM potassium carbonate solution were added to 138.2 mL of ultrapure water and heated to 68 °C, 1 mL of 1wt% chloroauric acid was added and heated at 68 °C for 15 min to obtain AuNPs; 30 μL of 250 nM AuNPs, 15 μL of 100 μM biomolecules and 5 μL of 200 mM sodium nitrate were added to 50 μL of ultrapure water and mixed evenly to obtain a mixed solution A, n-butanol was added to the mixed solution A and vortexed quickly to obtain a mixed solution B, then 0.5×TBE buffer was added to the mixed solution B and vortexed quickly to mix, finally, the two-phase solution was separated by centrifugation to obtain a lower layer of gold nanomaterials loaded with biomolecules, which were stored for later use; the biomolecules included DNA, and the nucleotide sequence of the DNA was SEQ ID NO: 1 to SEQ ID NO: Any one of 4.

6. The method according to claim 5, characterized in that The volume ratio of the n-butanol to the mixed solution A is 7:1, and the volume ratio of the 0.5×TBE buffer to the mixed solution B is 1:

4.

7. The method according to claim 2, characterized in that The nucleotide sequence of the DNA is shown in SEQ ID NO:

2.

8. The method according to claim 2, characterized in that: In step S2, the cell concentration in the blood cell suspension is 1×10 6 ~2×10 6 Pieces / mL.

9. The method according to claim 2, characterized in that: In step S3, the co-incubation temperature is 18-20° C., and the co-incubation time is 6 hours.

10. The method according to claim 2, characterized in that The shellfish include the Yesso scallop.

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