CN110387402A - A kind of SERS- fluorescent dual module probe and its preparation method and application based on DNA chain displacement - Google Patents
A kind of SERS- fluorescent dual module probe and its preparation method and application based on DNA chain displacement Download PDFInfo
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Abstract
The invention discloses a kind of SERS- fluorescent dual module probe based on DNA chain displacement principle, the DNA modified by the Fe 3 O 4 magnetic composite material of certain partial size1‑2Double-strand and another DNA3Single-stranded complementary pairing is made；The DNA1And DNA3Containing identical aptamers, the DNA2It is end modified to have fluorogen FAM6, the DNA3It is end modified to have fluorogen Cy5.The preparation method and application of the invention also discloses the described SERS- fluorescent dual module probe based on DNA chain displacement principle.SERS- fluorescent dual module probe of the present invention based on DNA chain displacement principle can detect simultaneously the biomarker in superficial living cells with two kinds of signals of Raman and fluorescence, and detect convenient and efficient, and high specificity, high sensitivity, accuracy are high.
Description
Technical field
The present invention relates to bioanalytical chemistry technical field more particularly to a kind of SERS- fluorescence based on DNA chain displacement are double
Mould probe and its preparation method and application.
Background technique
Since there are higher disease incidence, and people's quality of life level to be caused to be remarkably decreased for cancer.Even if Partial tumors are fallen ill
Rate is low, but grade malignancy is high, and transfer occurs early, to cause the death rate high, therefore early detection, diagnosis and the treatment of cancer are relatively attached most importance to
It wants.Common cancer pre-judging method is the certain abnormal biotic factors for detecting focal area at present, including enzyme, albumen,
DNA, RNA etc., as the important biomolecule marker of cancer early stage cell deterioration.Although having occurred many fluorescence detections at present
Method, but its is cumbersome, jitter and can not position and variation of the accurate observation to intracellular reactive substance.And Raman
Spectrum is a kind of Fingerprint that can mark specified chemical group, especially Surface enhanced Raman spectroscopy (surface
Enhanced Raman spectroscopy, SERS) technology, there is high sensitivity, good light stability, easy to operate and nothing
The advantage for damaging detection, solves the problems, such as that normal Raman spectroscopy sensitivity is low, identifies target molecule convenient for specificity.Therefore,
It based on DNA chain displacement principle, is bonded using the DNA of secondary structure with noble metal nano particles, by fluorescent technique and SERS technology
It is effectively combined, the novel bimodulus probe that one species specificity of exploitation is high, stability is good, aqueous solution background is weak, and is used for superficial
The analysis of the direct quantitative of sick cell and tissue and imaging analysis, to construct the SERS- of early stage such as melanoma tumors disease
The accurate diagnostic analysis method of fluorescence has important research significance.
Summary of the invention
The shortcomings that the purpose of the present invention is to solve the above-mentioned prior arts and deficiency, primary and foremost purpose of the invention are to provide
A kind of SERS- fluorescent dual module probe based on DNA chain displacement, a second object of the present invention is to provide the preparation sides of the probe
Method, it is a further object of the present invention to provide the applications of the probe.
In order to achieve the above objectives, the present invention adopts the following technical scheme:
A kind of SERS- fluorescent dual module probe based on DNA chain displacement, by the Fe 3 O 4 magnetic composite wood of certain partial size
Expect the DNA of modification1-2Double-strand and another DNA3Single-stranded complementary pairing is made；The DNA1And DNA3Containing identical aptamers, institute
State DNA2It is end modified to have fluorogen FAM6, the DNA3It is end modified to have fluorogen Cy5.
Further, the Fe 3 O 4 magnetic composite material is Fe3O4@Au。
Further, the Fe3O4The partial size of@Au is 50~150nm, and DNA chain length is 20~50base.
The preparation method of the present invention also provides the described SERS- fluorescent dual module probe based on DNA chain displacement, including it is following
Step:
1)Fe3O4Preparation；
2)Fe3O4The preparation of@Au: 2a) Fe3O4The preparation of-DA；2b) the preparation of gold kind；2c) the preparation of growth-promoting media；
2d) adsorb；2e) grow；
3) preparation of SERS- fluorescent dual module probe: 3a) DNA1-2Double-strand and DNA3Single-stranded preparation；3b)DNA1-2Double-strand is repaired
Adorn Fe3O4@Au magnetic composite.
Further, step 3b) Fe3O4@Au and DNA1-2The molar ratio of molecule is 1:200~500.
The invention also includes the described SERS- fluorescent dual module probe based on DNA chain displacement is to intracellular reactive substance
Application in SERS quantitative detection.
Further, SERS quantitative detection is the following steps are included: after cell grows to logarithmic phase in the application, by one
In quantitative cell inoculation to sterile quartz plate, after cell is completely adherent, old culture medium is sucked out and addition mixes well and contains
DNA1-2The Fe of modification3O4The culture medium of@Au bimodulus probe；Old culture medium and clear with PBS is sucked out after co-culturing a period of time in cell
It washes, the DNA for being mixed with same concentrations is then added3Minimal medium, co-culture a period of time after old culture medium is sucked out, use PBS
SERS imaging is carried out after cleaning at once.
The invention also includes the described SERS- fluorescent dual module probe based on DNA chain displacement is to intracellular reactive substance
Application in qualitative fluorescence imaging analysis.
Further, fluorescence analysis includes the following steps: after cell grows to logarithmic phase in the application, will be a certain amount of
Cell inoculation to fluorescence co-focusing ware on, after cell completely it is adherent after, be sucked out old culture medium and be added mix well containing DNA1-2
The Fe of modification3O4The culture medium of@Au bimodulus probe；Cell is sucked out old culture medium after co-culturing a period of time and is cleaned with PBS, so
The DNA for being mixed with same concentrations is added afterwards3Minimal medium, co-culture a period of time after old culture medium is sucked out, cleaned with PBS,
Fluorescence imaging is carried out at once after DAPI is dyed and is cleaned with PBS again.
SERS- fluorescent dual module probe of the present invention based on DNA chain displacement is former based on induction type DNA chain displacement
Reason, passes through the magnetic ferroferric oxide composite gold nano particles (Fe in size tunable3O4@Au) on modification containing special aptamers
DNA1-2Double-strand (DNA1Containing aptamers, DNA2End modified fluorogen FAM6), using another complementation and containing identical aptamers
DNA3It is single-stranded, in the presence of object, competition double-strand is gone by DNA chain displacement and aptamers recognition reaction consciously
Middle DNA2Short chain completes the replacement process of fluorogen Cy5 and FAM6.Since fluorescein Cy5 is as Raman molecular, with Fe3O4@Au
Jenner's grain of rice space length on surface is furthered rapidly by strand displacement effect, and stronger SERS letter is generated under laser irradiation
Number, and the short chain DNA displaced2Green florescent signal is obtained due to FAM6 containing fluorogen, is successfully constructed based on DNA chain displacement
Bimodulus probe is to realize the detection of particular organisms marker dual signal in superficial living cells.
The SERS- fluorescent dual module probe that the present invention synthesizes is through ultraviolet spectra, scanning electron microscope and X-ray diffraction (XRD) map
It is characterized.The experimental results showed that the Fe synthesized using this method3O4@Au NPs with property stablize, be alternatively arranged as MRI at
The potential contrast agent of picture.Wherein, DNA chain is successfully modified by Au-S key in Fe3O4The surface@Au NPs.It is of the present invention
" on-off " of SERS- fluorescent dual module probe, the SERS- fluorescence dual signal based on induction type DNA chain displacement principle is converted to realize
The detection of dual signal, therefore the detection of biomarker has biggish application prospect in superficial living cells.
SERS- fluorescent dual module probe of the present invention based on DNA chain displacement can use two kinds of Raman and fluorescence not
Quantitative analysis and imaging analysis are carried out to particular organisms marker in superficial living cells simultaneously with signal, and detect convenient and efficient,
High specificity, high sensitivity, accuracy are high, are conducive to the identification for improving cancer focal area tumour cell.
The present invention also has the following advantages that compared with the existing technology and effect:
(1) SERS- fluorescent dual module probe of the invention is stablized with property, and preparation is simple, size tunable, and bio-compatible
The advantages that property is high, can be applied in the biological samples such as cell.Wherein Fe3O4As nano-carrier, preparation is simple, grain
Diameter is controllable, and biocompatibility is high, is in addition alternatively arranged as the potential contrast agent of MRI imaging.
(2) present invention prepares the SERS- fluorescent dual module probe based on DNA chain displacement for the first time, by the Fe of certain partial size3O4@Au
Modify the DNA containing special aptamers1-2Double-strand and another DNA equally containing identical aptamers3Single-stranded complementary pairing is made, and is
The dual signal detection of biomarker, which is probed into, in superficial living cells provides Technical Reference.
(3) the present invention is based on its convenient and efficient of the detection method of SERS- fluorescent dual module probe of DNA chain displacement, specificity
By force, high sensitivity, accuracy are high, are conducive to the identification for improving cancer focal area tumour cell, building early stage such as melanin
The accurate diagnostic analysis method of SERS- fluorescence of the diseases such as tumour.
(4) object is not limited to certain biomarker used in the present invention in the present invention, and there are many selections, can root
Suitable object is selected according to actual needs.
In order to better understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Detailed description of the invention
Fig. 1 is SERS- fluorescent dual module probe preparation of the present invention and application principle schematic diagram.
Fig. 2 is the Fe of SERS- fluorescent dual module probe of the present invention3O4The performance characterization and condition optimizing of@Au NPs.
Fig. 3 is the Fe of SERS- fluorescent dual module probe of the present invention3O4The stability of@Au NPs characterizes.
Fig. 4 is quantitative detection of the SERS- fluorescent dual module probe of the present invention for VEGF in superficial living cells A375.
Fig. 5 is confocal fluorescent imaging of the SERS- fluorescent dual module probe of the present invention for VEGF in superficial living cells A375.
Fig. 6 is SERS- fluorescent dual module probe of the present invention for A375, HeLa, MHCC-97H, HCT116 difference cell line
The confocal fluorescent of VEGF is imaged.
Specific embodiment
What all material, reagent and the instrument selected in the present invention were all well known in the art, but reality of the invention is not limited
It applies, other some reagents well known in the art and equipment are applied both to the implementation of following implementation of the present invention.
Embodiment 1
A kind of preparation method of the SERS- fluorescent dual module probe based on DNA chain displacement, as shown in Figure 1, specifically including following
Step:
1)Fe3O4Preparation: take 1~5g FeCl2·4H2O and 1~5g FeCl3·6H2O is dissolved in 20mL ultrapure water, filter
Paper filters and dilutes 10~30mL, after being passed through several minutes of nitrogen, above-mentioned solution is added dropwise in 10~30mL ammonium hydroxide, in N2Protection
Lower reaction 30min and 1~3h of aging process.Heat source is removed after reaction to be cooled to room temperature, after being separated using magnet, with water and
Ethyl alcohol cleans for several times repeatedly, is placed in oven dried overnight and grind into powder is spare.
2)Fe3O4The preparation of@Au:
2a)Fe3O4The preparation of-DA: 10~30mg Fe is weighed3O4It is dissolved in THF, 2mL Dopamine hydrochloride aqueous solution is added,
It is ultrasonic uniform, it is stirred overnight at room temperature, it is spare to be centrifuged off extra DA；
2b) the preparation of gold kind: by the 1%HAuCl of 30~60 μ L 25mM4Solution is added to 30~60mL aqueous solution (containing 5
The NaOH solution of the 80%THPC solution of~20 μ L and 250 μ L 2M) in, it is continuously stirring under the conditions of being protected from light overnight, it is spare；
2c) the preparation of growth-promoting media: 1~3mL 1%HAuCl4It is added dissolved with 10~30mg K2CO3100mL water in, place
It is spare overnight；
It 2d) adsorbs: 5~10mL Fe is added in 10~20mL gold kind3O4In-DA, pH to 4.0 is adjusted, overnight, magnetic divides for shaking
The Au extra from removing；
It 2e) grows: 5~10mL Fe is added in 80~100mL growth-promoting media under agitation3O4In-DA-Au, hydrochloric acid is used
Azanol is added dropwise to blue as reducing agent.
3) preparation of SERS- fluorescent dual module probe:
3a)DNA1-2Double-strand and DNA3Single-stranded preparation: being heated to 95 DEG C for all DNA and make annealing treatment, Slow cooling
To room temperature, it is made into various concentration and is stored in 4 DEG C of refrigerators.Wherein, DNA1And DNA2Double-strand is formed to hybridize annealing way, and
DNA3It is then individually annealing.All steps are in Tris-HCl (300mM NaCl, 5mM MgCl2, pH7.6) and it carries out in buffer.
3b)DNA1-2Double-strand modifies Fe3O4@Au:
According to Fe3O4The molar ratio of@Au and DNA molecular is 1:200~500, takes the Fe of phase application amount3O4@Au aqueous solution with
DNA1-2Solution mix, add sodium chloride to its molar concentration be 0.01~0.05mol/L, preferably molar concentration be 0.1~
Then 0.5mol/L vibrates 18~48h under room temperature, DNA is made1-2The Fe of modification3O4@Au solution, centrifuge separation remove twice
Remove extra DNA1-2, then precipitating is resuspended in 5mL pure water, it is spare.
Using ultraviolet spectra, scanning electron microscope and XRD spectrum to the Fe of above-mentioned preparation3O4@Au NPs is characterized and is optimized,
As shown in Figure 2, it can be seen that Fe3O4The ultraviolet absorption peak of@Au NPs (Fig. 2-A) at 556nm, partial size are 100 ± 5nm (figure
2-B), the provable Fe of XRD spectrum3O4@Au NPs is successfully prepared (Fig. 2-C) and Fe3O4The surface@Au NPs DNA supported quantity
Optimize (Fig. 2-D), shows as molar ratio CFe3O4:CDNAWhen for 1:200, DNA supported quantity reaches best.
Stability experiment is carried out to the SERS- fluorescent dual module probe of above-mentioned preparation, as a result as shown in Figure 3.It can from Fig. 3-A
To find out, it is continuously incubated for 0,12,24,36 and 48h after buffer or culture medium is added in SERS- fluorescent dual module probe, in difference
The SERS signal variation at time point is smaller, shows that the SERS stability of SERS- fluorescent dual module probe is preferable；From Fig. 3-B
As can be seen that the DNA for being mixed with same concentrations is added in SERS- fluorescent dual module probe3Minimal medium be incubated for 30min, 1min,
The fluorescence imaging intensity at 10min, 20min and 30min time point is basically unchanged, and shows that the fluorescence of SERS- fluorescent dual module probe is steady
It is qualitative preferable.
Embodiment 2
SERS- fluorescent dual module probe is used for the SERS quantitative detection of external VEGF, as shown in Figure 4.
In constructed detection method Raman detection include the following steps: be with human melanoma cell A375 in the present embodiment
Cell is pressed 1 × 10 after A375 cell grows to logarithmic phase by example6The density of a/mL is inoculated on 100 μ L to sterile quartz plate,
It is incubated for for 24 hours after completely adherent, old culture medium is sucked out, addition is mixed well containing DNA1-2The Fe of modification3O4The culture medium of@Au probe
1mL is co-cultured for 24 hours with cell, and old culture medium is sucked out and is cleaned 2 times with PBS, the DNA for being mixed with same concentrations is then added3Base
Basal culture medium co-cultures 0,0.5,1,1.5,2,2.5 and 3h respectively, old culture medium is then sucked out, and cleans 2 times with PBS, at once into
Row SERS imaging.
Wherein, Fig. 4-A is the SERS signal figure of SERS- fluorescent dual module probe in detecting various concentration VEGF, shows 1468cm-1
Place is the SERS feature adsorption peak of SERS- fluorescent dual module probe；Fig. 4-B is the probe in 1468cm-1The line of the SERS intensity at place
Sexual intercourse illustrates in 0.001~1.0 μM of concentration range, linear relationship between the SERS signal and VEGF concentration of the probe
Well；Fig. 4-C is the variation relation of the fluorescence intensity at 490nm and 650nm respectively, shows to be clearly present DNA chain in vitro
Replacement process；Fig. 4-D is the Fe of various concentration (0,20,40,60,80,100 μM)3O4@AuSERS- fluorescent dual module probe and A375
Cell be incubated for for 24 hours altogether after cell survival rate, show within the scope of 0~100 μM, the probe have good biocompatibility.
Embodiment 3
The qualitative fluorescence imaging analysis of SERS- fluorescent dual module probe, as shown in Figure 5.
In constructed detection method fluorescence detection include the following steps: be with human melanoma cell A375 in the present embodiment
Cell is pressed 1 × 10 after cell grows to logarithmic growth phase by example6The density of a/mL is inoculated with 100 μ L to fluorescence co-focusing ware
On, it is incubated for for 24 hours after completely adherent, old culture medium is sucked out, addition is mixed well containing DNA1-2The Fe of modification3O4The culture medium of@Au
1mL is co-cultured for 24 hours with cell, and old culture medium is sucked out and is cleaned 2 times with PBS, the DNA for being mixed with same concentrations is then added3Base
Basal culture medium co-cultures 0,1,10,20 and 30min respectively；Then old culture medium is sucked out, is cleaned 2 times, is carried out at once glimmering with PBS
Light imaging.
The result shows that due to DNA3DNA is displaced by strand displacement and aptamers recognition reaction1-2Short chain in double-strand, because
This forms double-stranded DNA1-3The red fluorescence of chain gradually weakens (containing Cy5), displaces DNA2Chain green fluorescence (containing FAM6) gradually increases
By force, and in 30min DNA chain displacement process can be basically completed.
Embodiment 4
The selectivity of SERS- fluorescent dual module probe is investigated using different types of cancer cell, as shown in Figure 6.
After cell grows to logarithmic growth phase, cell is pressed 1 × 106The density of a/mL is inoculated with 100 μ L and is copolymerized to fluorescence
On burnt ware, it is incubated for for 24 hours after completely adherent, old culture medium is sucked out, addition is mixed well containing DNA1-2The Fe of modification3O4The training of@Au
Base 1mL is supported, is co-cultured for 24 hours with A375, HeLa, MHCC-97H, HCT116 difference cell line, old culture medium and clear with PBS is sucked out
It washes 2 times, the DNA for being mixed with same concentrations is then added3Minimal medium, co-culture 30min；Then old culture medium is sucked out, uses
PBS is cleaned 2 times, and dyes 3min with DAPI, is cleaned 2 times with PBS, and carries out fluorescence imaging at once.
The result shows that SERS- fluorescent dual module probe only believes the A375 cell containing object VEGF with fluorescence response
Number, and illustrate of the invention since few or there is no VEGF markers almost without fluorescence signal in other cancer cells
SERS- fluorescent dual module probe specificity is strong, accuracy is high.Object is not limited to certain life used in the present invention in the present invention
Object marker, there are many selections, can select suitable object according to actual needs.
The invention is not limited to above embodiment, if not departing from the present invention to various changes or deformation of the invention
Spirit and scope, if these changes and deformation belong within the scope of claim and equivalent technologies of the invention, then this hair
It is bright to be also intended to encompass these changes and change.
Claims (9)
1. a kind of SERS- fluorescent dual module probe based on DNA chain displacement, which is characterized in that the probe is by certain partial size
The DNA of Fe 3 O 4 magnetic composite material modification1-2Double-strand and another DNA3Single-stranded complementary pairing is made；The DNA1With
DNA3Containing identical aptamers, the DNA2It is end modified to have fluorogen FAM6, the DNA3It is end modified to have fluorogen Cy5.
2. the SERS- fluorescent dual module probe according to claim 1 based on DNA chain displacement, which is characterized in that four oxygen
Change three-iron magnetic composite is Fe3O4@Au。
3. the SERS- fluorescent dual module probe according to claim 2 based on DNA chain displacement, which is characterized in that described
Fe3O4The partial size of@Au is 50~150nm, and DNA chain length is 20~50base.
4. the preparation method of the SERS- fluorescent dual module probe as claimed in claim 3 based on DNA chain displacement, which is characterized in that packet
Include following steps:
1)Fe3O4Preparation；
2)Fe3O4The preparation of@Au: 2a) Fe3O4The preparation of-DA；2b) the preparation of gold kind；2c) the preparation of growth-promoting media；
2d) adsorb；2e) grow；
3) preparation of SERS- fluorescent dual module probe: 3a) DNA1-2Double-strand and DNA3Single-stranded preparation；3b)DNA1-2Double-strand modification
Fe3O4@Au magnetic composite.
5. the preparation method of the SERS- fluorescent dual module probe according to claim 4 based on DNA chain displacement, feature exist
In the step 3b) Fe3O4@Au and DNA1-2The molar ratio of molecule is 1:200~500.
6. the SERS- fluorescent dual module probe described in claim 1-3 based on DNA chain displacement is in the SERS to intracellular reactive substance
Application in quantitative detection.
7. application according to claim 6, which is characterized in that SERS quantitative detection in the application the following steps are included:
After cell grows to logarithmic phase, by a certain amount of cell inoculation to sterile quartz plate, after cell is completely adherent, it is sucked out old
Culture medium and be added mix well containing Fe3O4The DNA of@Au modification1-2The culture medium of bimodulus probe；After cell co-cultures a period of time
Old culture medium is sucked out and is cleaned with PBS, the DNA for being mixed with same concentrations is then added3Minimal medium, co-culture a period of time
After old culture medium is sucked out, SERS imaging is carried out after being cleaned with PBS at once.
8. the SERS- fluorescent dual module probe described in claim 1-3 based on DNA chain displacement is to the qualitative of intracellular reactive substance
Application in fluorescence imaging analysis.
9. application according to claim 8, which is characterized in that fluorescence analysis includes the following steps: to thin in the application
After intracellular growth to logarithmic phase, by a certain amount of cell inoculation to fluorescence co-focusing ware, after cell is completely adherent, old training is sucked out
It supports base and is added and mix well containing Fe3O4The DNA of@Au modification1-2The culture medium of bimodulus probe；Cell is inhaled after co-culturing a period of time
It old culture medium and is cleaned out with PBS, the DNA for being mixed with same concentrations is then added3Minimal medium, co-culture a period of time after
Old culture medium is sucked out, is cleaned with PBS, then carries out fluorescence imaging at once after DAPI is dyed and is cleaned with PBS.
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| CN110387402B (en) | 2023-06-06 |
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