CN111635752B

CN111635752B - Gold nanorod/DNA/carbon dot nano hybrid material and preparation method and application thereof

Info

Publication number: CN111635752B
Application number: CN202010296389.4A
Authority: CN
Inventors: 周树云; 牛月芳; 谢政; 德罗尔·戴维·菲克斯乐
Original assignee: Technical Institute of Physics and Chemistry of CAS
Current assignee: Technical Institute of Physics and Chemistry of CAS
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2022-09-23
Anticipated expiration: 2040-04-15
Also published as: CN111635752A

Abstract

The invention discloses a gold nanorod/DNA/carbon dot nano hybrid material and a preparation method and application thereof. The gold nanorod/DNA/carbon point nano hybrid material takes a gold nanorod as a core, takes DNA as a medium, and couples carbon points through covalent bonds, so that the carbon points are distributed around the gold nanorod in a satellite emission shape to form a 'core-satellite' structure. The preparation method of the gold nanorod/DNA/carbon dot nano hybrid material is mild and effective. The hybrid material simultaneously realizes the application of noninvasive diffuse reflection imaging and fluorescence lifetime imaging, thereby realizing multi-modal imaging and having good application prospect in the aspect of accurate diagnosis of diseases caused by atherosclerosis. When the DNA is the specific aptamer DNA, the material is expected to realize specific detection and targeted treatment on most cancer diseases such as breast cancer, lung cancer, brain tumor and the like.

Description

Gold nanorod/DNA/carbon dot nano hybrid material and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological imaging. More particularly, relates to a gold nanorod/DNA/carbon dot nano hybrid material, a preparation method and application thereof.

Background

Cancer is a worldwide recognized disease with a very high mortality rate, and early diagnosis helps to improve the survival rate of patients. A single imaging technology cannot meet the requirement of diagnosis, and multi-modal imaging is gradually becoming a research hotspot in the field of biological imaging. The nanometer hybrid material composed of multiple components breaks through the limitation of single component, can acquire multi-directional information through multi-mode imaging to realize more comprehensive diagnosis, and is even expected to realize diagnosis and treatment integration.

Diffuse reflectance imaging (DR) and Fluorescence Lifetime Imaging (FLIM) are two new, safe, easy-to-handle imaging techniques. Among other things, DR can provide important morphologies of biological tissue through processing of optical signals, and does not require high radiation intensity or high penetration depth. FLIM is being used more and more widely for quantitative studies of cellular microenvironments and biomedical applications, where high spatial resolution can provide temporal and spatial information of the fluorescence lifetime variation of fluorescently labeled components in images. The combination of DR and FLIM enables the exploration of optical properties and molecular imaging of biological tissues.

Due to the existence of plasmon, the gold nanorods show unique optical characteristics and are suitable DR imaging contrast agents. However, the gold nanorods are not low in fluorescence quantum efficiency enough to carry out FLIM imaging, and a material with high fluorescence quantum efficiency needs to be introduced. In recent years, carbon dots have been widely used in the field of bio-imaging due to advantages such as excellent fluorescence properties and excellent biocompatibility. The gold nanorods and the carbon dots are combined into the nano hybrid material, different optical properties and excellent biocompatibility of the gold nanorods and the carbon dots are integrated, and the nano hybrid material is a multifunctional imaging material suitable for DR and FLIM. However, the assembly of gold nanorods and carbon dots requires a medium as a bridge for connection. Gold nanorods/silica/carbon dots and gold nanorods/polyethylene glycol/carbon dots hybrid nanomaterial [ Multimodal biomorphic basal on gold nanoparticles and carbon dot nanohybrids as a novel tool for atherospermis detection, Nano Research, 2018, 11, 1262-; gold Rod-Polyethylene Glycol-Carbon Dot Nanohybrids as Phototheranostic Probes, Nanomaterials,2018,8,706 have been developed, but relatively, the thickness of silica is not easily controlled precisely, the rigidity of Polyethylene Glycol chains is insufficient, and the regulation of distance is not easily realized.

DNA has been used for nanoscale self-assembly [ DNA as a power tool for morphology control, spatial position, and dynamic assembly of nanoparticles, Acc. chem. Res.47(2014) 1881-1890 ], and double strands have certain rigidity and can be controlled at high degree of distance. The multifunctional nano hybrid material prepared by using DNA as a framework has important application in aspects of biological imaging, drug delivery, disease treatment, microorganism detection and the like. [ highly effective platinum Nanorods and Upconversion Core-saturated hybridization viruses, Adv. Mater.2016,28, 898-904 ] AS1411-conjugated gold nanospheres and the needle for sparse therapy, Oncotarget 6(2015) 22270-22281 [ fluorescent methods for quick detection of ochratoxin A in flowing and using nitro gene bonded carbon dots and silver nanoparticles, Takara 182 (363) 370 ] gold Nanorods/carbon Nanorods and hybrid materials proposed by the present invention have a promising prospect for achieving accurate diagnosis of atherosclerosis and DR-mediated disease. The detection of breast Cancer, [ Gold-Quantum Dot Core-Specific Assembly for Lighting Up MicroRNA In Vitro and In Vivo, [ New-Generation Cancer-Specific Hybrid materials ] MAGE-A3 Persistent Luminescence characterization for In Situ prediction and characterization, 10.1002/2013741 ] brain tumor, [ target for imaging target and therapeutic target of Cancer ] and the like can be achieved when the DNA is a Specific Aptamer DNA such AS AS1411, MAGE-A3 or GBI-10.

Disclosure of Invention

The first purpose of the invention is to provide a gold nanorod/DNA/carbon dot nano hybrid material.

The second purpose of the invention is to provide a preparation method of the gold nanorod/DNA/carbon dot nano hybrid material.

The third purpose of the invention is to provide the application of the gold nanorod/DNA/carbon dot nano hybrid material in diffuse reflection imaging and fluorescence lifetime imaging.

In order to achieve the purpose, the invention adopts the following technical scheme:

one aspect of the present invention provides a gold nanorod/DNA/carbon dot nanohybrid material, comprising: gold nano-rod,

A double-stranded DNA molecule and a carbon dot; the double-stranded DNA molecule comprises single-stranded DNA1 and complementary single-stranded DNA 2;

the 5 'end of the single-stranded DNA1 is combined with the gold nanorod through a gold-sulfur coordination bond, and the 5' end of the single-stranded DNA2 is combined with a carbon point through a covalent bond.

The gold nanorod/DNA/carbon point nano hybrid material takes the gold nanorod as a core, takes the DNA as a connecting medium (bridge) and leads carbon points to be closely distributed around the gold nanorod in a satellite emission shape to form a 'core-satellite' structure. The structure has high stability and good dispersibility; the gold nanorods are combined with the single-stranded DNA1 through a gold-sulfur coordination bond, and the carbon dots are connected with the single-stranded DNA2 through covalent interaction; the material is used for multi-modal imaging combining fluorescence lifetime imaging and diffuse reflection imaging, and has high sensitivity.

Further, the length-diameter ratio of the gold nanorod is 2-6, the diameter is 10-30nm, the length is 20-100nm, and the longitudinal SPR ultraviolet absorption peak is between 600 and 900 nm.

Furthermore, the 5' end of the single-stranded DNA1 is modified with sulfydryl and forms a gold-sulfur coordination bond with the gold nanorod; the 5' -end of the single-stranded DNA2 is modified with amino or carboxyl;

the particle size of the carbon dots is 1-10nm, and the fluorescence lifetime is 1.0-15.0 ns; the carbon point is modified with a functional group and reacts with an amino group or a carboxyl group at the 5' end of the single-stranded DNA2 to form a covalent bond.

In the gold nanorod/DNA/carbon dot nano hybrid material, the gold nanorod is combined with DNA through a gold-sulfur coordination bond, and the carbon dot is combined with the DNA through covalent action. Compared with physical adsorption, the material has better stability and is not easily decomposed in complex chemical environment in cells.

Further, the ultraviolet visible absorption spectrum of the gold nanorod/DNA/carbon dot nano hybrid material has an absorption peak at 500-540nm and a maximum absorption peak at 600-900 nm.

The invention also provides a preparation method of the gold nanorod/DNA/carbon dot nano hybrid material, which comprises the following steps:

1) preparing gold nanorods;

2) reacting the single-stranded DNA1 with the gold nanorods to form gold-sulfur coordination bonds, and preparing a gold nanorod/DNA 1 compound;

3) preparing a carbon dot containing a functional group;

4) reacting the carbon dot containing the functional group with the single-stranded DNA2 to form a covalent bond, and preparing a carbon dot/DNA 2 complex;

5) self-assembling the gold nanorod/DNA 1 compound obtained in the step 2) and the carbon dot/DNA 2 compound obtained in the step 4) in a PBS buffer solution to form the gold nanorod/DNA/carbon dot nano hybrid material.

Further, the 5 'end of the single-stranded DNA1 is modified with a thiol group, and the 5' end of the single-stranded DNA2 is modified with an amino group.

Further, the step 2) specifically comprises: single-stranded DNA1 and gold nanorods were added to the buffer in sequence, and the reaction was carried out at room temperature with shaking.

Preferably, the molar ratio of the single-stranded DNA1 to the gold nanorods is 250:1-1000:1, and the reaction time is 0.5-24 h.

Preferably, the buffer solution of step 2) is not particularly limited in the present invention, as long as it can provide a suitable reaction environment. For example, Tris borate buffer (TBE), Tris acetate buffer (TAE), Tris-EDTA buffer (TE), Tris phosphate buffer (TPE), Phosphate Buffer (PBS), etc. may be used. The above buffers are all buffers commonly used in the art and can be selected arbitrarily. TBE is preferred in the present invention, and more preferably, the buffer comprises 1 XTBE, 500mM sodium chloride (NaCl) and 0.02% Sodium Dodecyl Sulfate (SDS) solution, the pH thereof can be adjusted to 3.0-6.0, and it can be prepared by dissolving 2.16g Tris base, 1.1g boric acid, 0.8mL of 0.5M EDTA (pH 8.0) in 200mL ultrapure water.

In a preferred embodiment, step 2) comprises:

washing CTAB on the surface of the gold nanorod obtained in the step 1) with water, and dispersing the gold nanorod in water to obtain a gold nanorod dispersion liquid; adding DNA1 with one end being modified by sulfydryl into a buffer solution containing TBE, NaCl, HCl and SDS solution, mixing uniformly, then adding gold nanorod dispersion liquid, and fully oscillating at room temperature to obtain the gold nanorod dispersion liquid grafted with DNA 1; then centrifuging and washing to obtain a gold nanorod/DNA 1 compound; wherein the molar ratio of the DNA1 to the gold nanorods is 250:1-1000:1, and the reaction time is 0.5-24 h.

Further, the step 3) specifically comprises: taking organic carboxylic acid or organic amine as a precursor and deionized water as a solvent, preparing a carbon dot crude product by a hydrothermal method, and dialyzing and purifying to obtain the carbon dot.

Preferably, the reaction temperature of the hydrothermal method is 120-200 ℃, and the reaction time is 4-12 h;

preferably, the organic carboxylic acid is one or a combination of more than two of citric acid, lower fatty acid and amino acid; the organic amine is one or the combination of more than two of ethylenediamine, aniline, phenylenediamine, diethanolamine and hydrazine hydrate.

Further, the step 4) specifically includes: adding a coupling agent into the aqueous solution of the carbon dots containing the functional groups for activation, then adding the single-stranded DNA2, and carrying out the reaction by shaking in the dark.

Preferably, the coupling agent is one or a combination of more than two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), Dicyclohexylcarbodiimide (DCC), N-hydroxythiosuccinimide (sulfo-NHS) and N-hydroxysuccinimide (NHS); when the number of the coupling agents is two, the proportion of the two coupling agents is adjusted according to actual needs; still further, the coupling agent is selected from the group consisting of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

The coupling agent adopted by the invention has good catalytic activity in a water system, the reaction condition is mild and controllable, and no pollutant is generated.

Preferably, the activation time is 15-30 min.

Preferably, the molar ratio of the carbon dots containing functional groups to the single-stranded DNA2 is 50:1 to 500:1, and the molar ratio of the coupling agent to the carbon dots containing functional groups is 100:1 to 1000: 1.

In a preferred embodiment, step 4) specifically comprises: dispersing the carbon dots obtained in the step 3) in water, sequentially adding a coupling agent, activating for 15-30min in a dark environment, quickly adding the single-stranded DNA2 modified with carboxyl or amino at the 5' end, and oscillating for 12-24h in a dark place; then centrifugating and washing by an ultrafiltration tube to obtain the carbon dot/DNA 2 complex.

In a preferred embodiment, step 5) comprises in particular: adding the gold nanorod/DNA 1 complex obtained in the step 2) into PBS buffer solution containing the carbon dot/DNA 2 complex obtained in the step 4) for self-assembly; then centrifuging and washing to obtain the gold nanorod/DNA/carbon dot nano hybrid material aqueous solution.

Further, the molar ratio of the carbon dots to the gold nanorods in the step 5) is 10:1-50: 1.

The gold nanorod/DNA/carbon dot nano hybrid material prepared by the invention has good water solubility, wherein the gold nanorod is connected with DNA through a gold-sulfur coordination bond, the carbon dot is connected with the DNA through an amide bond, and the two single-stranded

DNAs

1 and 2 are complementarily paired through hydrogen bonds between bases, so that the gold nanorod/DNA/carbon dot nano hybrid material has good stability.

The invention further provides application of the gold nanorod/DNA/carbon dot nano hybrid material in diffuse reflection imaging and fluorescence lifetime imaging. Therefore, multi-mode imaging is realized, and the method has good application prospect in the aspect of accurate diagnosis of diseases caused by atherosclerosis. When the DNA is the specific aptamer DNA, the material is expected to realize specific detection and targeted treatment on most cancer diseases such as breast cancer, lung cancer, brain tumor and the like.

The invention has the following beneficial effects:

(1) the preparation method of the gold nanorod/DNA/carbon dot nano hybrid material provided by the invention is mild and effective, not only ensures the close attachment of carbon dots, but also maintains the surface plasmon resonance effect of the gold nanorod.

(2) The gold nanorod/DNA/carbon dot nano hybrid material provided by the invention has excellent optical properties of the gold nanorod and remarkable fluorescence property of the carbon dot.

(3) The gold nanorod/DNA/carbon dot nano hybrid material skillfully utilizes different optical properties of the gold nanorod and the Carbon Dots (CDs), and effectively proves good response effect of the gold nanorod/DNA/carbon dots in noninvasive multi-modal imaging.

(4) According to the gold nanorod/DNA/carbon dot nano hybrid material provided by the invention, the connection of CDs not only endows the gold nanorods with better fluorescence response in FLIM imaging, but also obtains better diffuse reflection effect than the gold nanorods.

(5) The gold nanorod/DNA/carbon dot nano hybrid material provided by the invention realizes DR and FLIM multi-modal imaging, and has a good application prospect in the aspect of accurate diagnosis of diseases caused by atherosclerosis. When the DNA is the specific aptamer DNA, the material is expected to realize specific detection and targeted treatment on most cancer diseases such as breast cancer, lung cancer, brain tumor and the like.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the TEM image of the gold nanorod/DNA/carbon dot hybrid material in example 1.

FIG. 2 shows UV-visible absorption spectra of Gold Nanorods (GNRs), gold nanorod/DNA complexes (GNR-DNA), and gold nanorods/DNA/carbon dots (GNR-DNA-CDs) in example 1.

FIG. 3 shows fluorescence emission spectra of gold nanorods/DNA/carbon dots in example 1.

FIG. 4 shows the results of diffuse reflectance imaging of blanks (controls), gold nanorods/DNA complexes (GNR-DNA) in comparative example 1, and gold nanorods/DNA/carbon dots (GNR-DNA-CDs) in a phantom like tissue of example 1.

FIG. 5 shows a histogram of the mean slopes of diffuse reflectance imaging of blanks (Control) and gold nanorods/DNA/carbon dots (GNR-DNA-CDs) in the phantom of tissue-like material in example 1.

FIGS. 6a, 6b, and 6c show the fluorescence lifetime imaging graphs and lifetime value fitting results of the blank (Control), the gold nanorod/DNA complex (GNR-DNA) in comparative example 1, and the gold nanorod/DNA/carbon dots (GNR-DNA-CDs) in the phantom of the tissue-like structure in example 1, respectively.

FIG. 6d, FIG. 6e, and FIG. 6f show the fluorescence lifetime value fitting results of blank (Control), gold nanorod/DNA complex (GNR-DNA) in comparative example 1, and gold nanorod/DNA/carbon dots (GNR-DNA-CDs) in the phantom of tissue-like body in example 1, respectively.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

DNA1, DNA2, DNA1 'and DNA2' were all from Biotechnology engineering (Shanghai) Ltd.

Example 1

The preparation method of the gold nanorod/DNA/carbon dot nano hybrid material comprises the following steps:

1) gold nano-rod

Gold Nanorods [ Babak Nikoobacht, Mostafa A El-Sayed. preparation and Growth Mechanism of Gold Nanoros (NRs) Using Seed-media Growth method. chem. mater.2003,15, 1957-. The diameter of the prepared gold nanorod is about 17nm, the length of the prepared gold nanorod is about 58nm, the length-diameter ratio of the prepared gold nanorod is 3.4, the ultraviolet visible absorption peaks are 510 nm and 735nm, and a transmission electron microscope image of the gold nanorod can be seen in figure 1.

2) Gold nanorod/DNA 1 complex

The DNA1 has a sequence of 5'-SH-AGT CAG TAC AGT CAT TGC GT-3' (shown as a sequence table SEQ ID No. 1), 20 basic groups and SH represents a sulfydryl group.

And (3) centrifuging and washing the gold nanorod dispersion liquid by using deionized water, removing the coated CTAB, and dispersing the precipitate in water again. A buffer solution containing 1 XTBE, 500mM NaCl and 0.02% SDS solution by mass concentration was prepared, the pH of the solution was adjusted to 3.0 with HCl, and the mixture was thoroughly shaken and mixed. Then adding sulfhydryl DNA1 and a gold nanorod solution into the buffer solution in sequence, wherein the molar ratio of the DNA1 to the gold nanorods is 500:1, and continuously oscillating for 12 h. Then, the mixed solution is centrifugally washed for 3 times, and the precipitate is dispersed again to obtain a gold nanorod/DNA 1 complex solution.

3) Carboxyl functional carbon dots

1g of citric acid and 1mL of ethylenediamine were dissolved in 10mL of water, and then transferred to a hydrothermal reactor to carry out hydrothermal reaction at 200 ℃ for 5 hours, thereby obtaining a brownish red mixture containing carboxyl-functionalized carbon dots. And dialyzing the mixed solution of the carboxyl functionalized carbon dots to obtain the carboxyl functionalized carbon dots. The particle size of the obtained carbon dot is 2nm, and the fluorescence emission peak is 400-500 nm.

4) Carbon dot/DNA 2 complex

DNA2 sequence 5' -NH ₂ -ACG CAA TGA CTG TAC TGA CT-3' (shown in SEQ ID No.2 of the sequence Listing), 20 basic groups, NH ₂ Represents an amine group.

150 μ L of carbon dot dispersion (20mg/mL) was added sequentially with coupling agent EDC (100 μ L, 0.4M) and NHS (100 μ L, 0.4M) at a molar ratio of coupling agent to carbon dots of 100:1, and after 30min of activation, 10 μ L of DNA2(150 μ M) was added rapidly and shaken in the dark for 24 h. In order to remove the excess carbon dots and the coupling agent, the resulting mixture was centrifuged and washed by an ultrafiltration tube to obtain a carbon dot/DNA 2 complex.

5) Gold nanorod/DNA/carbon dot nano hybrid material

Adding the gold nanorod-DNA 1 into PBS buffer solution containing carbon dot-DNA 2 for self-assembly, wherein the molar ratio of the carbon dot to the gold nanorod is 20: 1. Shaking, mixing, placing in 60 deg.C water bath for 10min, taking out, placing in 37 deg.C water bath for 2h, taking out, and slowly cooling to room temperature. Then, excess carbon dot-DNA 2 was removed by centrifugation and washing. And dispersing the precipitate in PBS to obtain gold nanorod/DNA/carbon dot dispersion liquid. As shown in FIG. 2, the prepared gold nanorod/DNA/carbon dot has two characteristic absorption peaks of 520 nm and 765nm in an ultraviolet visible spectrum, and as shown in FIG. 3, the fluorescence emission spectrum has an emission peak of 400-500 nm.

And (3) diffuse reflection imaging test: the test was performed using an optical device (NEGOH-OP Technologies, Israel) for measuring the intensity of the reflected light, using the method described in [ Journal of biophotonics,2012,263 and 273 ], when the distance between the light source and the detector was changed from 1mm to 6mm, the intensity of the reflected light was collected and the data was processed using an oscilloscope. The test result is shown in fig. 4, the slope of the diffuse reflection light intensity-distance curve represents the effect of diffuse reflection imaging, and the greater the slope, the more sensitive the imaging and the better the effect. As can be seen from the combination of FIG. 5, the gold nanorod/DNA/carbon dot hybrid material has a larger slope and has better imaging effect and sensitivity.

Fluorescence lifetime imaging test: the scanning confocal microscope used was a PicoQuant MicroTime200, and the light source used was a 406nm picosecond pulsed laser. The test results are shown in fig. 6 a-6 f, and from FLIM imaging graphs, the gold nanorod/DNA/carbon dot hybrid nanomaterial prepared by the invention has a stronger fluorescence response signal, and the corresponding fluorescence lifetime is 6.4 ns.

Example 2

The material preparation method is the same as that of example 1, and only differs from the following method: the diameter of the gold nanorod prepared in the step 1) is about 20nm, the length is about 60nm, the length-diameter ratio is about 3.0, and the ultraviolet visible absorption peaks are 510 nm and 710 nm. The absorption peaks of the gold nanorod/DNA/carbon dot in an ultraviolet visible absorption spectrum are 510 nm and 720nm, and the emission peak of a fluorescence emission spectrum is 400-500 nm.

The diffuse reflection imaging test method is the same as that of the embodiment 1, and the performance of the gold nanorod/DNA/carbon dot nano hybrid material in diffuse reflection imaging is similar to that of the embodiment 1.

The fluorescence lifetime imaging test method is the same as that in the example 1, and the result of the gold nanorod/DNA/carbon dot nano hybrid material in the fluorescence lifetime imaging is similar to that in the example 1.

Example 3

The material preparation method is the same as that of example 1, and only differs from the following method: in steps 2) and 4), "the number of bases of DNA is 20" is replaced with "40".

The sequence of the DNA1 is changed into that of DNA 1': 5'-SH-GTC GCC TGC ATG AGC TGA GAA GTC AGT ACA GTC ATT GCG T-3' (shown in a sequence table SEQ ID No. 3).

The sequence of the DNA2 is changed into that of DNA 2': 5'-NH2-ACG CAA TGA CTG TAC TGA CTT CTC AGC TCA TGC AGG CGA C-3' (shown as sequence table SEQ ID No. 4). .

The absorption peaks of the gold nanorods/DNA/carbon dots in the ultraviolet visible absorption spectrum are 522 nm and 775nm, and the emission peak of the fluorescence emission spectrum is 400-500 nm.

The testing method of diffuse reflection imaging is the same as that of the embodiment 1, and the performance of the gold nanorod/DNA/carbon dot nano hybrid material in diffuse reflection imaging is similar to that of the embodiment 1.

Example 4

The material preparation method is the same as that of example 1, and only differs from the following method: in the step 2), "the molar ratio of the DNA to the gold nanorods is 500: 1" is replaced by "1000: 1". The absorption peaks of the gold nanorod/DNA/carbon dot in the ultraviolet visible absorption spectrum are 522 nm and 768nm, and the emission peak of the fluorescence emission spectrum is 400-500 nm.

The fluorescence lifetime imaging test method is the same as that in example 1, and the result of the gold nanorod/DNA/carbon dot nano hybrid material in fluorescence lifetime imaging is similar to that in example 1.

Example 5

The material preparation method is the same as that of example 1, and only differs from the following method: in the step 2), "the reaction time of DNA and gold nanorods is 12 h" is changed to "5 h". The absorption peaks of the gold nanorod/DNA/carbon dot in an ultraviolet visible absorption spectrum are 515 nm and 760nm, and the emission peak of a fluorescence emission spectrum is 400-500 nm.

Example 6

The material preparation method is the same as that of example 1, and only differs from the following method: in the step 3), "the reaction time for preparing the carbon dots is 5 h" is changed to "12 h". The size of the carbon dots is 2-5nm, and the fluorescence emission peak is 400-500 nm. The absorption peaks of the gold nanorod/DNA/carbon dot in the ultraviolet visible absorption spectrum are 520 nm and 767nm, and the emission peak of the fluorescence emission spectrum is 400-500 nm.

Example 7

The material preparation method is the same as that of example 1, and only differs from the following method: in the step 4), "the reaction time of carbon points and DNA 24 h" is changed to "12 h". The absorption peaks of the gold nanorod/DNA/carbon dot in the ultraviolet visible absorption spectrum are 516 nm and 763nm, and the emission peak of the fluorescence emission spectrum is 400-500 nm.

Example 8

The material preparation method is the same as that of example 1, and only differs from the following method: in step 4) "the molar ratio of the coupling agent to the carbon dots is 100: 1" is changed to "500: 1". The absorption peaks of the gold nanorod/DNA/carbon dot in the ultraviolet visible absorption spectrum are 519 and 764nm, and the emission peak of the fluorescence emission spectrum is 400-500 nm.

Example 9

The material preparation method is the same as that of example 1, and only differs from the following method: in the step 5), "the molar ratio of the carbon points to the gold nanorods is 20: 1" is changed into "40: 1". The absorption peaks of the gold nanorod/DNA/carbon dot in the ultraviolet visible absorption spectrum are 525 nm and 770nm, and the emission peak of the fluorescence emission spectrum is 400-500 nm.

Comparative example 1

Preparation of gold nanorods/DNA was the same as in example 1, steps 1) and 2).

The diffuse reflection imaging test in example 1 was repeated except that "the dispersion of gold nanorods/DNA/carbon dot nano-hybrid material" was changed to "gold nanorods/DNA dispersion". The combination of FIG. 4 shows that the slope of gold nanorod/DNA is lower than that of gold nanorod/DNA/carbon dot, and the diffuse reflection imaging effect is poor.

The fluorescence lifetime imaging test in example 1 was repeated except that "the dispersion of gold nanorods/DNA/carbon dot hybrid nanomaterial" was changed to "gold nanorod/DNA dispersion". The combination of FIG. 5 shows that the imaging effect of the gold nanorods/DNA is similar to that of the blank group, no obvious fluorescence corresponding signal exists, and the fluorescence lifetime imaging effect is poor.

Comparative example 2

Preparation of gold nanorods/DNA/carbon dots As in example 1, except that the precursor for preparing the carbon dots in step 3) was changed from "citric acid and ethylenediamine" to "ethylenediamine". As the prepared carbon dots only have amino groups and do not have carboxyl groups, the prepared carbon dots can not be covalently connected with amino-modified DNA2, and a stable gold nanorod/DNA/carbon dot nano hybrid material can not be formed.

The diffuse reflection imaging test in the example 1 is repeated, and the result shows that the slope is similar to the slope of the gold nanorod/DNA, lower than the slope of the gold nanorod/DNA/carbon dot, and the diffuse reflection imaging effect is poorer.

The fluorescence lifetime imaging test in example 1 was repeated, and the results showed that there was no significant fluorescence response signal and the fluorescence lifetime imaging effect was poor.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

SEQUENCE LISTING

<110> research institute of physical and chemical technology of Chinese academy of sciences

<120> gold nanorod/DNA/carbon dot nano hybrid material, and preparation method and application thereof

<130> JLP20I0224

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Claims

1. A gold nanorod/DNA/carbon dot nano hybrid material for diffuse reflection imaging and fluorescence lifetime imaging is characterized by comprising: gold nanorods, double-stranded DNA molecules, and carbon dots;

the double-stranded DNA molecule comprises single-stranded DNA1 and complementary single-stranded DNA 2;

the 5 'end of the single-stranded DNA1 is combined with the gold nanorod through a gold-sulfur coordination bond, and the 5' end of the single-stranded DNA2 is combined with the carbon point through a covalent bond; the 5' end of the single-stranded DNA1 is modified with sulfydryl and forms a gold-sulfur coordination bond with the gold nanorod; the 5' -end of the single-stranded DNA2 is modified with amino or carboxyl;

2. The gold nanorod/DNA/carbon dot hybrid nanomaterial according to claim 1, wherein the gold nanorod has an aspect ratio of 2-6, a diameter of 10-30nm, and a length of 20-100 nm.

3. The gold nanorod/DNA/carbon dot hybrid nanomaterial according to claim 1, wherein the modified functional group on the carbon dot is a carboxyl group or an amino group.

4. A method for preparing gold nanorod/DNA/carbon dot nano hybrid material according to any one of claims 1 to 3, which is characterized by comprising the following steps:

1) preparing gold nanorods;

3) preparing carbon dots containing functional groups;

5. The method of claim 4, wherein the single-stranded DNA1 is modified at its 5 'terminus with a thiol group and the single-stranded DNA2 is modified at its 5' terminus with an amino group.

6. The method according to claim 4, wherein the step 2) comprises:

single-stranded DNA1 and gold nanorods were added to the buffer in this order, and the reaction was carried out with shaking at room temperature.

7. The preparation method according to claim 4, wherein the molar ratio of the single-stranded DNA1 to the gold nanorods is 250:1-1000:1, and the reaction time is 0.5h-24 h.

8. The method according to claim 4, wherein step 3) comprises:

taking organic carboxylic acid or organic amine as a precursor and deionized water as a solvent, preparing a carbon dot crude product by a hydrothermal method, and dialyzing and purifying to obtain the carbon dot.

9. The method as claimed in claim 8, wherein the hydrothermal method has a reaction temperature of 120 ℃ and 200 ℃ and a reaction time of 4-12 h.

10. The method according to claim 8, wherein the organic carboxylic acid is one or a combination of two or more of citric acid, a lower fatty acid, and an amino acid; the organic amine is one or the combination of more than two of ethylenediamine, aniline, phenylenediamine, diethanolamine and hydrazine hydrate.

11. The preparation method according to claim 4, wherein the step 4) specifically comprises:

adding a coupling agent into the aqueous solution of the carbon dots containing the functional groups for activation, then adding the single-stranded DNA2, and carrying out the reaction by shaking in the dark.

12. The production method according to claim 11, wherein the coupling agent is one or a combination of two or more of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N-hydroxythiosuccinimide, and N-hydroxysuccinimide; the activation time is 15-30 min;

the molar ratio of the carbon point containing the functional group to the single-stranded DNA2 is 50:1-500:1, and the molar ratio of the coupling agent to the carbon point containing the functional group is 100:1-1000: 1.

13. The production method according to claim 4, wherein the molar ratio of the carbon dots to the gold nanorods in step 5) is 10:1 to 50: 1.

14. Use of the gold nanorod/DNA/carbon dot hybrid nanomaterial as defined in any one of claims 1-3 in preparation of diffuse reflection imaging and fluorescence lifetime imaging materials.