CN110387402B - SERS-fluorescence dual-mode probe based on DNA strand displacement and preparation method and application thereof - Google Patents
SERS-fluorescence dual-mode probe based on DNA strand displacement and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a SERS-fluorescence dual-mode probe based on a DNA strand displacement principle, which is DNA modified by a ferroferric oxide magnetic composite material with a certain particle size 1‑2 Double strand and another DNA 3 The single chain complementary pairing is prepared; the DNA 1 And DNA 3 Containing the same aptamer, the DNA 2 End-modified with a fluorophore FAM6, said DNA 3 The end is modified with a fluorophore Cy5. The invention also discloses a preparation method and application of the SERS-fluorescence dual-mode probe based on the DNA strand displacement principle. The SERS-fluorescence dual-mode probe based on the DNA strand displacement principle can detect the biomarker in the superficial living cells simultaneously by using two signals of Raman and fluorescence, and has the advantages of convenient and efficient detection, strong specificity, high sensitivity and high accuracy.
Description
Technical Field
The invention relates to the technical field of biological analysis chemistry, in particular to a SERS-fluorescence dual-mode probe based on DNA strand displacement, and a preparation method and application thereof.
Background
The quality of life level of people is significantly reduced due to the higher incidence of cancer. Even if the incidence rate of partial tumors is low, the malignant degree is high, the metastasis occurs early, and the death rate is high, so that early detection, diagnosis and treatment of cancers are important. The current common cancer prognosis method is to detect some abnormal biological factors in the focus area, including enzymes, proteins, DNA, RNA, etc., and use them as important biomarkers for early cell deterioration of cancer. Although many fluorescence detection methods are currently available, the operation is complicated, signals are unstable, and the position and change of the intracellular active substances cannot be accurately observed. The Raman spectrum is a fingerprint spectrum capable of marking specific chemical groups, particularly a surface enhanced Raman spectrum (surface enhanced Raman spectroscopy, SERS) technology, has the advantages of high sensitivity, good light stability, simplicity in operation and nondestructive detection, solves the problem of low sensitivity of the common Raman spectrum, and is convenient for identifying target molecules specifically. Therefore, based on the DNA strand displacement principle, the DNA with a secondary structure is bonded with noble metal nano particles, a fluorescence technology is effectively combined with an SERS technology, a novel dual-mode probe with high specificity, good stability and weak aqueous solution background is developed, and the novel dual-mode probe is used for direct quantitative analysis and imaging analysis of superficial lesion cells and tissues, so that an SERS-fluorescence accurate diagnosis analysis method for early diseases such as melanin tumors is constructed, and has important research significance.
Disclosure of Invention
The present invention aims to solve the above-mentioned drawbacks and disadvantages of the prior art, and a primary object of the present invention is to provide a SERS-fluorescence dual-mode probe based on DNA strand displacement, and a second object of the present invention is to provide a method for preparing the probe, and another object of the present invention is to provide an application of the probe.
In order to achieve the above purpose, the invention adopts the following technical scheme:
SERS-fluorescence dual-mode probe based on DNA strand displacement and DNA modified by ferroferric oxide magnetic composite material with certain particle size 1-2 Double strand and another DNA 3 The single chain complementary pairing is prepared; the DNA 1 And DNA 3 Containing the same aptamer, the DNA 2 End-modified with a fluorophore FAM6, said DNA 3 The end is modified with a fluorophore Cy5.
Further, the ferroferric oxide magnetic composite material is Fe 3 O 4 @Au。
Still further, the Fe 3 O 4 The particle size of the @ Au is 50-150 nm, and the length of the DNA strand is 20-50 base.
The invention also provides a preparation method of the SERS-fluorescence dual-mode probe based on DNA strand displacement, which comprises the following steps:
1)Fe 3 O 4 is prepared by the steps of (1);
2)Fe 3 O 4 preparation of @ Au: 2a) Fe (Fe) 3 O 4 -preparation of DA; 2b) Preparing gold seeds; 2c) Preparing a growth solution;
2d) Adsorbing; 2e) Growing;
3) Preparation of SERS-fluorescence dual-mode probe: 3a) DNA (deoxyribonucleic acid) 1-2 Double strand and DNA 3 Preparing a single chain; 3b) DNA (deoxyribonucleic acid) 1-2 Double-chain modified Fe 3 O 4 @au magnetic composite material.
Further, step 3 b) the Fe 3 O 4 @Au and DNA 1-2 The molar ratio of the molecules is 1:200-500.
The invention also comprises application of the SERS-fluorescence dual-mode probe based on DNA strand displacement in SERS quantitative detection of intracellular active substances.
Further, the quantitative SERS detection in the application comprises the following steps: after the cells grow to logarithmic phase, inoculating a certain amount of cells onto a sterile quartz plate, sucking out old culture medium after the cells are completely adhered, and adding fully mixed DNA 1-2 Modified Fe 3 O 4 Culture medium of @ Au dual-mode probe; after a period of co-cultivation of the cells, the old medium is aspirated and washed with PBS, and then DNA mixed with the same concentration is added 3 After a period of co-cultivation, the old medium was aspirated, washed with PBS and immediately subjected to SERS imaging.
The invention also comprises the application of the SERS-fluorescence dual-mode probe based on DNA strand displacement in qualitative fluorescence imaging analysis of intracellular active substances.
Further, the in-use fluorescence analysis includes the steps of: after the cells grow to the logarithmic phase, inoculating a certain amount of cells to a fluorescent confocal dish, sucking out the old culture medium after the cells are completely adhered, and adding the DNA 1-2 Modified Fe 3 O 4 Culture medium of @ Au dual-mode probe; after a period of co-cultivation of the cells, the old medium is aspirated and washed with PBS, and then DNA mixed with the same concentration is added 3 After a period of co-cultivation, the old medium was aspirated, washed with PBS, stained with DAPI and immediately subjected to fluorescence imaging after washing with PBS.
The SERS-fluorescence dual-mode probe based on DNA strand displacement is based on the principle of induced DNA strand displacement and is prepared by combining magnetic ferroferric oxide composite gold nanoparticles (Fe 3 O 4 Modified DNA containing specific aptamer on @ Au) 1-2 Double strand (DNA) 1 Containing aptamer, DNA 2 End-modified fluorophore FAM 6), using another complementary DNA containing the same aptamer 3 Single strand, placed by DNA strand in the presence of targetThe exchange and aptamer recognition effect consciously decompetitively competes for DNA in double strand 2 Short chain, complete the displacement process of fluorophores Cy5 and FAM 6. Since fluorescein Cy5 is used as Raman molecule, and Fe 3 O 4 The space distance of the gold nano-particles on the surface of the @ Au is rapidly shortened by the strand displacement, a stronger SERS signal is generated under the irradiation of laser, and the displaced short-chain DNA 2 The fluorescent group FAM6 is contained to obtain a green fluorescent signal, so that a dual-mode probe based on DNA strand displacement is successfully constructed to realize the detection of the dual signal of the specific biomarker in the superficial living cells.
The SERS-fluorescence dual-mode probe synthesized by the invention is characterized by ultraviolet spectrum, scanning electron microscope and X-ray diffraction (XRD) spectrum. Experimental results show that Fe synthesized by adopting the method 3 O 4 The @ Au NPs have stable properties and can be used as potential contrast agents for MRI imaging. Wherein the DNA strand is successfully modified in Fe through Au-S bond 3 O 4 @au NPs surface. The SERS-fluorescence dual-mode probe disclosed by the invention realizes the detection of dual signals by the on-off conversion of SERS-fluorescence dual signals based on the induction type DNA strand displacement principle, so that the detection of biomarkers in superficial living cells has a large application prospect.
The SERS-fluorescence dual-mode probe based on DNA strand displacement can simultaneously carry out quantitative analysis and imaging analysis on specific biomarkers in superficial living cells by adopting two different signals of Raman and fluorescence, is convenient and efficient to detect, has strong specificity, high sensitivity and high accuracy, and is beneficial to improving the identification degree of tumor cells in a cancer focus area.
Compared with the prior art, the invention has the following advantages and effects:
(1) The SERS-fluorescence dual-mode probe has the advantages of stable property, simple preparation, controllable particle size, high biocompatibility and the like, and can be practically applied to biological samples such as cells. Wherein Fe is 3 O 4 The nano-carrier has the advantages of simple preparation, controllable particle size and high biocompatibility, and can be used as a potential contrast agent for MRI imaging.
(2) The invention prepares SERS-fluorescence dual mode based on DNA strand displacement for the first timeThe probe consists of Fe with a certain particle size 3 O 4 Modified DNA containing specific aptamer 1-2 Double strand DNA containing the same aptamer as another strand 3 The single-chain complementary pairing is prepared, and provides technical reference for dual signal detection exploration of biomarkers in superficial living cells.
(3) The detection method of the SERS-fluorescence dual-mode probe based on DNA strand displacement is convenient and efficient, has strong specificity, high sensitivity and high accuracy, is favorable for improving the identification degree of tumor cells in a cancer focus area, and constructs an early SERS-fluorescence accurate diagnosis analysis method for diseases such as melanin tumors and the like.
(4) The target object in the invention is not limited to a certain biomarker used in the invention, and can be selected in various ways according to actual needs.
For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.
Drawings
FIG. 1 is a schematic diagram of the preparation and application principle of the SERS-fluorescence dual-mode probe.
FIG. 2 is a diagram of Fe of a SERS-fluorescence dual-mode probe according to the invention 3 O 4 Performance characterization and condition optimization of @ Au NPs.
FIG. 3 is a diagram of Fe of a SERS-fluorescence dual-mode probe of the present invention 3 O 4 Stability characterization of Au NPs.
FIG. 4 is a quantitative assay of VEGF in superficial living cells A375 using SERS-fluorescent dual-mode probes of the invention.
FIG. 5 is a confocal fluorescence image of VEGF in superficial living cells A375 using SERS-fluorescence dual mode probes of the invention.
FIG. 6 is confocal fluorescence imaging of SERS-fluorescence dual-mode probes of the invention for VEGF of different cell lines A375, heLa, MHCC-97H, HCT.
Detailed Description
All materials, reagents and equipment selected for use in the present invention are well known in the art, but are not limiting of the practice of the invention, and other reagents and equipment known in the art may be suitable for use in the practice of the following embodiments of the invention.
Example 1
A preparation method of a SERS-fluorescence dual-mode probe based on DNA strand displacement is shown in figure 1, and specifically comprises the following steps:
1)Fe 3 O 4 is prepared from the following steps: taking 1-5 g FeCl 2 ·4H 2 O and 1-5 g FeCl 3 ·6H 2 O is dissolved in 20mL of ultrapure water, the solution is filtered by filter paper and diluted by 10 to 30mL, nitrogen is introduced for a plurality of minutes, 10 to 30mL of ammonia water is added dropwise into the solution, and the solution is treated by N 2 Reacting for 30min under protection and aging for 1-3 h. After the reaction is finished, the heat source is removed, the mixture is cooled to room temperature, and after the separation by using a magnet, the mixture is repeatedly washed with water and ethanol for a plurality of times, and the mixture is placed in an oven for drying overnight and is ground into powder for standby.
2)Fe 3 O 4 Preparation of @ Au:
2a)Fe 3 O 4 preparation of DA: weighing 10-30 mg Fe 3 O 4 Dissolving in THF, adding 2mL of dopamine hydrochloride aqueous solution, carrying out ultrasonic homogenization, stirring at room temperature overnight, and centrifuging to remove redundant DA for later use;
2b) Preparation of gold seeds: 30 to 60 mu L of 25mM 1% HAuCl 4 Adding the solution into 30-60 mL of water solution (containing 5-20 mu L of 80% THPC solution and 250 mu L of 2M NaOH solution), and continuously stirring overnight under the dark condition for later use;
2c) Preparation of a growth solution: 1-3 mL 1% HAuCl 4 Adding dissolved 10-30 mg K 2 CO 3 Is left overnight for standby in 100mL of water;
2d) Adsorption: adding 10-20 mL of gold seeds into 5-10 mL of Fe 3 O 4 In DA, the pH is adjusted to 4.0, shaken overnight and the excess Au is removed by magnetic separation;
2e) And (3) growing: adding 80-100 mL of growth solution into 5-10 mL of Fe under stirring 3 O 4 In DA-Au, hydroxylamine hydrochloride is used as reducing agent, added dropwise to the blue color.
3) Preparation of SERS-fluorescence dual-mode probe:
3a)DNA 1-2 double strand and DNA 3 Preparation of single strand: heating all DNA to 95 ℃ for annealing treatment, and slowlyCooled to room temperature, formulated into different concentrations and stored in a refrigerator at 4 ℃. Wherein the DNA 1 And DNA 2 Double strand formation by hybridization annealing, and DNA 3 Then it is a separate anneal. All steps were performed in Tris-HCl (300mM NaCl,5mM MgCl) 2 pH 7.6) buffer.
3b)DNA 1-2 Double-chain modified Fe 3 O 4 @Au：
According to Fe 3 O 4 The mol ratio of the @ Au to the DNA molecules is 1:200-500, and Fe with corresponding dosage is taken out 3 O 4 Aqueous Au solution and DNA 1-2 Mixing the solution, adding sodium chloride to the molar concentration of 0.01-0.05 mol/L, preferably 0.1-0.5 mol/L, and oscillating at room temperature for 18-48 hr to obtain DNA 1-2 Modified Fe 3 O 4 Centrifuging the Au solution to remove excess DNA twice 1-2 The pellet was then resuspended in 5mL of purified water for further use.
Fe prepared by adopting ultraviolet spectrum, scanning electron microscope and XRD spectrum 3 O 4 Characterization and optimization of the @ Au NPs, as shown in FIG. 2, fe can be seen 3 O 4 UV absorption peak of @ Au NPs at 556nm (FIG. 2-A), particle size of 100+ -5 nm (FIG. 2-B), XRD pattern confirmed Fe 3 O 4 Preparation success of @ Au NPs (FIG. 2-C), and Fe 3 O 4 Optimization of DNA immobilization on the surface of the @ Au NPs (FIG. 2-D), demonstrated that when the molar ratio C Fe3O4 :C DNA At 1:200, the DNA immobilization amount was optimal.
Stability experiments were performed on the SERS-fluorescence dual-mode probe prepared as described above, and the results are shown in FIG. 3. As can be seen from fig. 3-a, after the SERS-fluorescent dual-mode probe is added into the buffer solution or the culture medium, the continuous incubation is performed for 0, 12, 24, 36 and 48 hours, and the difference of the SERS signal changes at different time points is small, which indicates that the SERS stability of the SERS-fluorescent dual-mode probe is good; as can be seen from FIG. 3-B, SERS-fluorescence dual mode probes are added mixed with the same concentration of DNA 3 The fluorescence imaging intensity at the time points of 1min, 10min, 20min and 30min is basically unchanged after the incubation for 30min, which indicates that the fluorescence stability of the SERS-fluorescence dual-mode probe is better.
Example 2
The SERS-fluorescence dual-mode probe is used for SERS quantitative detection of VEGF in vitro, as shown in FIG. 4.
The Raman detection in the constructed detection method comprises the following steps: in this example, taking human melanoma cells A375 as an example, after A375 cells grew to log phase, the cells were grown at 1X 10 6 Inoculating 100 μl/mL onto sterile quartz plate, incubating for 24 hr, sucking out old culture medium, adding DNA 1-2 Modified Fe 3 O 4 1mL of culture medium for the @ Au probe, co-cultured with the cells for 24h, the old medium was aspirated and washed 2 times with PBS, then DNA mixed with the same concentration was added 3 Co-culturing for 0, 0.5, 1, 1.5, 2, 2.5 and 3 hours, respectively, and then aspirating the old medium, washing 2 times with PBS, and immediately performing SERS imaging.
Wherein FIG. 4-A is a SERS signal diagram of SERS-fluorescence dual-mode probe for detecting VEGF at different concentrations, showing 1468cm -1 The SERS characteristic adsorption peak of the SERS-fluorescence dual-mode probe is positioned; FIG. 4-B shows that the probe is at 1468cm -1 The linear relationship of SERS intensity shows that the linear relationship between the SERS signal of the probe and the VEGF concentration is good in the concentration range of 0.001-1.0 mu M; FIG. 4-C is a graph showing the change in fluorescence intensity at 490nm and 650nm, respectively, showing that DNA strand displacement processes are evident in vitro; FIG. 4-D shows the Fe concentrations (0, 20, 40, 60, 80, 100. Mu.M) 3 O 4 Cell viability after 24h incubation of the AuSERS-fluorescence dual-mode probe with A375 cells shows that the probe has good biocompatibility in the range of 0-100 mu M.
Example 3
Qualitative fluorescence imaging analysis of SERS-fluorescent dual mode probes is shown in fig. 5.
The fluorescence detection in the constructed detection method comprises the following steps: in this example, taking human melanoma cell A375 as an example, after the cells had grown to the logarithmic phase, the cells were grown at 1X 10 6 Inoculating 100 mu L of the culture medium to a fluorescence confocal dish at a density of 100 mu L/mL, incubating for 24 hours, and sucking out old culture medium after the culture medium is completely adhered to the wallAdding DNA to be mixed fully 1-2 Modified Fe 3 O 4 1mL of culture medium @ Au, co-cultured with cells for 24h, the old medium was aspirated and washed 2 times with PBS, then DNA mixed with the same concentration was added 3 Co-culturing for 0, 1, 10, 20, and 30min, respectively; the old medium was then aspirated, washed 2 times with PBS, and immediately subjected to fluorescence imaging.
The results showed that due to DNA 3 DNA displacement by strand displacement and aptamer recognition 1-2 Short chains in the double strand, thus forming double-stranded DNA 1-3 The red fluorescence of the strand (containing Cy 5) gradually decreases and DNA is displaced 2 The strand green fluorescence (containing FAM 6) was gradually increased and the DNA strand displacement process was substantially completed within 30 min.
Example 4
Different kinds of cancer cells were used to examine the selectivity of SERS-fluorescent dual-mode probes, as shown in fig. 6.
After the cells grew to logarithmic phase, the cells were grown at 1X 10 6 Inoculating 100 mu L of the culture medium to a fluorescence confocal dish at the density of one/mL, incubating for 24 hours until the culture medium is completely adhered, sucking out the old culture medium, adding the DNA fully and uniformly mixed 1-2 Modified Fe 3 O 4 1mL of culture medium @ Au, co-cultured with A375, heLa, MHCC-97H, HCT116 different cell lines for 24h, the old medium was aspirated and washed 2 times with PBS, then DNA mixed with the same concentration was added 3 Co-culturing the basic culture medium of (2) for 30min; old media was then aspirated, washed 2 times with PBS and stained with DAPI for 3min, washed 2 times with PBS, and immediately subjected to fluorescence imaging.
The result shows that the SERS-fluorescence dual-mode probe only has fluorescence response signals to A375 cells containing a target VEGF, and little or no fluorescence signals exist in other cancer cells due to the fact that the VEGF markers are few or absent, so that the SERS-fluorescence dual-mode probe is high in specificity and accuracy. The target object in the invention is not limited to a certain biomarker used in the invention, and can be selected in various ways according to actual needs.
The present invention is not limited to the above-described embodiments, but it is intended that the present invention also includes modifications and variations if they fall within the scope of the claims and the equivalents thereof, if they do not depart from the spirit and scope of the present invention.
Claims (6)
1. A SERS-fluorescence dual-mode probe based on DNA strand displacement is characterized in that the probe is DNA modified by ferroferric oxide magnetic composite material with a certain particle size 1-2 Double strand and another DNA 3 The single chain complementary pairing is prepared; the ferroferric oxide magnetic composite material is Fe 3 O 4 @au; the Fe is 3 O 4 The particle size of the@Au is 50-150 nm; the DNA 1 And DNA 3 Containing the same aptamer, the DNA 2 End-modified with a fluorophore FAM6, said DNA 3 The tail end is modified with a fluorophore Cy5; the preparation method of the SERS-fluorescence dual-mode probe based on DNA strand displacement comprises the following steps:
1)Fe 3 O 4 is prepared by the steps of (1);
2)Fe 3 O 4 preparation of @ Au:
2a)Fe 3 O 4 preparation of DA: weighing 10-30 mg Fe 3 O 4 Dissolving in THF, adding 2mL of dopamine hydrochloride aqueous solution, carrying out ultrasonic homogenization, stirring at room temperature overnight, and centrifuging to remove redundant DA for later use;
2b) Preparation of gold seeds: 30 to 60 mu L of 25mM 1% HAuCl 4 Adding the solution into 30-60 mL of water solution (containing 5-20 mu L of 80% THPC solution and 250 mu L of 2M NaOH solution), and continuously stirring overnight under the dark condition for later use; 2c) Preparation of a growth solution: 1-3 mL 1% HAuCl 4 Adding dissolved 10-30 mg K 2 CO 3 Is left overnight for standby in 100mL of water;
2d) Adsorption: adding 10-20 mL of gold seeds into 5-10 mL of Fe 3 O 4 In DA, the pH is adjusted to 4.0, shaken overnight and the excess Au is removed by magnetic separation;
2e) And (3) growing: adding 80-100 mL of growth solution into 5-10 mL of Fe under stirring 3 O 4 In DA-Au, hydroxylamine hydrochloride is used as a reducing agent, and is added dropwiseBlue color is entered;
obtaining Fe 3 O 4 @Au, the Fe 3 O 4 The particle size of the @ Au is 100+/5 nm;
3) Preparation of SERS-fluorescence dual-mode probe:
3a)DNA 1-2 double strand and DNA 3 Preparing a single chain;
3b)DNA 1-2 double-chain modified Fe 3 O 4 Magnetic composite material of @ Au, the Fe 3 O 4 @Au and DNA 1-2 The molar ratio of the molecules was 1:200.
2. The DNA strand displacement-based SERS-fluorescent dual-mode probe according to claim 1, wherein the length of the DNA strand is 20 to 50base.
3. Use of a DNA strand displacement based SERS-fluorescent dual mode probe according to claims 1-2 for quantitative SERS detection of intracellular active substances.
4. The use according to claim 3, wherein the quantitative SERS detection in the use comprises the steps of: after the cells grow to logarithmic phase, inoculating a certain amount of cells onto a sterile quartz plate, sucking out old culture medium after the cells are completely adhered, and adding Fe 3 O 4 Modified DNA @ Au 1-2 Culture medium of dual-mode probe; after a period of co-cultivation of the cells, the old medium is aspirated and washed with PBS, and then DNA mixed with the same concentration is added 3 After a period of co-cultivation, the old medium was aspirated, washed with PBS and immediately subjected to SERS imaging.
5. Use of a SERS-fluorescent dual mode probe based on DNA strand displacement according to claims 1-2 for qualitative fluorescent imaging analysis of intracellular active substances.
6. The use according to claim 5, wherein the in-use fluorescence analysis comprises the steps of: after the cells grow to the logarithmic phase, a certain amount ofInoculating the cells on a fluorescent confocal dish, sucking out the old culture medium after the cells are completely adhered, and adding the Fe-containing material which is fully and uniformly mixed 3 O 4 Modified DNA @ Au 1-2 Culture medium of dual-mode probe; after a period of co-cultivation of the cells, the old medium is aspirated and washed with PBS, and then DNA mixed with the same concentration is added 3 After a period of co-cultivation, the old medium was aspirated, washed with PBS, stained with DAPI and immediately subjected to fluorescence imaging after washing with PBS.
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