CN103834727A - Preparation method of fluorescence probe and applications of the fluorescence probe - Google Patents
Preparation method of fluorescence probe and applications of the fluorescence probe Download PDFInfo
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Abstract
The invention relates to a fluorescence nano-material, and particularly relates to a preparation method of a fluorescence probe and applications of the fluorescence probe. The fluorescence probe has characteristics of strong fluorescence signals, high stability and good water solubility. The fluorescence probe is prepared by connecting reduced carbon quantum dots capable of emitting blue fluorescence to single-stranded DNA. The reduced carbon quantum dots are prepared from carbon quantum dots, and are prepare by subjecting the carbon quantum dots to surface modification by adopting sodium borohydride as a reductant and adopting a chemical reduction method so as to obtain the reduced carbon quantum dots capable of emitting the blue fluorescence. Detection of the target DNA is achieved by utilization of fluorescence quenching-recovery processes of the fluorescence probe.
Description
Technical field
The present invention relates to a kind of fluorescent nano material, relate in particular to a kind of preparation method and application thereof of fluorescent probe.
Background technology
Fluorescent mark refers to fluorescent substance is attached in the biomolecules that will study, utilizes the special fluorescent signal of fluorescent mark deposits yields, and target biological molecules is carried out to quantitative and qualitative analysis mensuration.In recent years, fluorescent mark technology has been brought into play vital role at aspects such as protein, nucleic acid, cell detection and immunoassays.But, all Shortcomings parts of all kinds of markers that use in biology at present, as larger in fluorescence organic dye molecule general toxicity, bio-compatibility is bad, and photobleaching speed is very fast, unstable, and quenching of fluorescence easily occurs, and fluorescence lifetime is short; Semiconductor-quantum-point has certain bio-toxicity owing to itself containing heavy metal ion, and easily decompose at High Concentration Situation or while being subject to uv irradiating etc.The drawbacks limit of these materials its application in fields such as biomolecules, cell marking, medicine tracking, medical diagnosis on disease and fluorescence senses.Therefore in fluorescent mark technology, excellent in optical properties, and stablize, the exploitation of the marker of toxicological harmless effect is extremely important.
Carbon quantum dot is a kind of novel fluorescence nano material growing up recent years, compares with traditional organic fluorescent dye, has that fluorescence intensity is high, a good light stability, fast light bleaching, and emission wavelength and excitation wavelength such as can regulate and control at the advantage.Meanwhile, compared with the widely used fluorescent nano particles such as quantum dot, have again that toxicity is low, good biocompatibility, molecular weight and a feature such as particle diameter is little, be expected to solve the safety problem of fluorescent material in life studies.There is good application prospect in fields such as cell marking, bio-imaging and biological detection.In recent years, the work that replaces traditional organic fluorescent dye and semiconductor-quantum-point to be applied to living things system as a kind of new fluorescent probe carbon quantum dot has caused scientific worker's very big interest.
Summary of the invention
Main purpose of the present invention is to provide a kind of fluorescent signal strong, and stability is high, the preparation method of the fluorescent probe of good water solubility.
Above-mentioned technical problem of the present invention is implemented by the following technical programs:
A kind of preparation method of fluorescent probe, this probe is to connect single stranded DNA by the reduction carbon quantum dot that can launch blue-fluorescence to obtain, described reduction carbon quantum dot is taking carbon quantum dot as raw material, sodium borohydride is as reductive agent, adopt the method for chemical reduction to carry out surface modification to carbon quantum dot, obtain launching the reduction carbon quantum dot of blue-fluorescence.
A preparation method for fluorescent probe, its step comprises:
A. carbon quantum dot is synthetic
Graphite Powder 99 joins in the mixing solutions of the vitriol oil and concentrated nitric acid, ultrasonic 1～3 hour, 60～100 DEG C of heated and stirred reflux 22～26 hours, after reaction finishes, be cooled to the dilution of room temperature adding distil water, obtain dark brown solution sodium carbonate and regulate pH value to neutral, obtain brown solution, the supernatant liquor of brown solution is removed small molecular weight impurity for 2～4 days with dialysis tubing dialysis, finally by the mode of rotary evaporation, the mixing liquid in reactor is concentrated to evaporate to dryness, obtains carbon quantum dot;
B. reduce the synthetic of carbon quantum dot
Take and above-mentioned prepare carbon quantum dot and excessive sodium borohydride solids is dissolved in tetrahydrofuran solution, 60～80 DEG C of heated and stirred reflux 6～10 hours, and after reaction finishes, in solution, unreacted sodium borohydride dialysis is removed, and makes reduction carbon quantum dot;
C. the preparation of fluorescent probe
Taking the above-mentioned reduction carbon quantum dot making is dissolved in the phosphate buffer solution of pH=7.4, adjust pH to 5.0, it is carboxylated to make to reduce carbon quantum dot surface, then add coupling agent, under room temperature, stir 25～40 minutes, add afterwards single stranded DNA to stir 20～28 hours, the mixture obtaining is through filtering with microporous membrane, on film, the substance dissolves of gained, in phosphate buffer soln, obtains fluorescent probe solution.
As preferably, the described vitriol oil and the volume ratio of concentrated nitric acid are 3 ︰ 1.
As preferably, in described steps A ultrasonic 2 hours, 80 DEG C of heated and stirred refluxed 24 hours, and the molecular weight cut-off MWCO of dialysis tubing is 1000～3000.
As preferably, in described step B, 70 DEG C of heated and stirred reflux 8 hours.
As preferably, in described step C, coupling agent is one or both in 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide.1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride be abbreviated as EDC, N-hydroxy-succinamide be abbreviated as NHS.
As preferably, in described step C, add after coupling agent, under room temperature, stir 30 minutes, add afterwards single stranded DNA to stir 24 hours.
The invention still further relates to this fluorescent probe in the application detecting in target dna, based on fluorescent probe and graphene oxide resonance energy transfer principle, using fluorescent probe as electron donor, graphene oxide is as electron acceptor(EA), fluorescent probe is adsorbed onto the surface of graphene oxide by the effect of π – pi accumulation, now fluorescence is by quencher; When after the target dna adding with single stranded DNA complementary pairing, because formation has the double-stranded DNA of rigid structure, make carbon quantum dot and graphene oxide apart from increase, carbon quantum dot is released from graphene oxide surface, now fluorescence is restored, thereby has realized the mensuration to target dna, and in finite concentration, the concentration of relative intensity of fluorescence and target dna is linear, detection by quantitative that therefore can realize target DNA.
As preferably, the detection method that fluorescent probe detects target dna is: get graphene oxide and join in probe solution, ultrasonic 3～6 minutes, leave standstill after 3～6 minutes fluorescence by significantly quencher; Then add target dna, add subsequently PBS buffered soln dilution, ultrasonic 3～6 minutes, leave standstill to add after 20～30 minutes and in quartz colorimetric utensil, carry out fluorometric analysis.
As preferably, the concentration of described graphene oxide is 16 μ g mL
-1.
Beneficial effect of the present invention is: in order further to improve the fluorescence property of carbon quantum dot, use sodium borohydride as reductive agent, adopt the method for simple chemical reduction to carry out surface modification to carbon quantum dot, prepare the reduction carbon quantum dot that can launch blue-fluorescence that performance is more excellent, its fluorescence quantum yield is up to 20.4%, far away higher than 1.7% of original carbon quantum dot, and the carbon quantum point grain diameter after modifying remains unchanged substantially, and this reduction carbon quantum dot fluorescence signal is strong, stability is high, good water solubility.After connecting single stranded DNA, reduction carbon quantum dot forms fluorescent probe, detection by quantitative that can realize target DNA based on fluorescent probe and graphene oxide resonance energy transfer.Detection linearity range is 0.0～46.0nM, and lowest detectable limit reaches 75.0pM.That this method has is highly sensitive, selectivity strong, need the advantages such as the few and method of sample amount is easy.
Brief description of the drawings
Fig. 1 is the preparation of fluorescent probe and the schematic diagram that detects target dna.
Fig. 2 is TEM picture (A) and the high-resolution-ration transmission electric-lens photo (B) of reduction carbon quantum dot.
Fig. 3 is the TEM picture of graphene oxide.
Fig. 4 is the photo under fluorogram and the ultraviolet lamp of reduction carbon quantum dot.
When Fig. 5 is the fluorescent probe detection graphene oxide in embodiment 1, fluorescence intensity is with the change in concentration figure of graphene oxide.
When Fig. 6 is the fluorescent probe detection graphene oxide in embodiment 1, the linear graph of relative intensity of fluorescence and graphene oxide concentration.
After Fig. 7 is the oxidized Graphene quencher of the fluorescent probe in embodiment 1, fluorescence intensity is with target dna change in concentration figure.
After Fig. 8 is the oxidized Graphene quencher of the fluorescent probe in embodiment 1, the linear graph of relative intensity of fluorescence and target dna.
Embodiment
Below by embodiment, by reference to the accompanying drawings, technical scheme of the present invention is described in further detail:
Embodiment 1: a kind of preparation method of fluorescent probe, this probe is to connect single stranded DNA by the reduction carbon quantum dot that can launch blue-fluorescence to obtain, described reduction carbon quantum dot is taking carbon quantum dot as raw material, sodium borohydride is as reductive agent, adopt the method for chemical reduction to carry out surface modification to carbon quantum dot, obtain launching the reduction carbon quantum dot of blue-fluorescence.Its concrete steps are:
A. carbon quantum dot is synthetic
0.3g Graphite Powder 99 joins in the 180mL vitriol oil and 60mL concentrated nitric acid mixing solutions, ultrasonic 2 hours, 80 DEG C of heated and stirred reflux 24 hours, after reaction finishes, be cooled to room temperature adding distil water and be diluted to 800mL, gained dark brown solution regulates pH value to neutral with sodium carbonate, obtain brown solution, the supernatant liquor of brown solution is removed small molecular weight impurity for 3 days with dialysis tubing dialysis, the molecular weight cut-off MWCO=1000 of dialysis tubing, finally by the mode of rotary evaporation, the mixing liquid in reactor is concentrated to evaporate to dryness, obtain carbon quantum dot;
B. reduce the synthetic of carbon quantum dot:
Take the above-mentioned carbon quantum dot 1.613g preparing, be dissolved in 20mL tetrahydrofuran solution with the excessive sodium borohydride solids of 1.0g, 70 DEG C of heated and stirred reflux 8 hours, after reaction finishes, in solution, unreacted sodium borohydride dialysis is removed, and makes reduction carbon quantum dot.As shown in the TEM picture (A) in Fig. 2, result is known, and reduction carbon quantum dot presents monodispersed evenly spherical, and particle diameter is mainly distributed as 5 ± 3nm.As shown in the high-resolution-ration transmission electric-lens photo (B) in Fig. 2, reduction carbon quantum dot has obvious lattice fringe structure, and the interfringe distance of adjacent crystalline form is
(002) diffraction surfaces data of data and graphite approach.As shown in Figure 4, under the excitation wavelength of 281nm, the transmitting site of the fluorescence intensity maximum of reduction carbon quantum dot is at 443nm place, and the color under ultraviolet lamp is the blueness becoming clear, and the quantum yield in water is 20.4%.
C. the preparation of probe:
Getting the reduction carbon quantum dot 0.01g making is dissolved in the phosphate buffer solution of 10mL pH=7.4, adjust pH to 5.0 to make to reduce carbon quantum dot surface carboxylated, add under coupling agent 0.8g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 0.8g N-hydroxy-succinamide (NHS) room temperature and stir 30 minutes, add afterwards 10 μ L single stranded DNAs (cDNA) to stir 24 hours, the mixture obtaining is through filtering with microporous membrane, retain the insolubles on film, again be dispersed in phosphate buffer solution with distilled water flushing insolubles and by it, obtain probe solution.As shown in Figure 1, carbon quantum dot is through sodium borohydride reduction, and the reduction carbon quantum dot obtaining connects single stranded DNA (cDNA) and forms probe the quenching of fluorescence-recovery process for detection of target dna (tDNA).
Fluorescent probe is in the application detecting in target dna, and using fluorescent probe as electron donor, graphene oxide is as electron acceptor(EA), and fluorescent probe is adsorbed onto the surface of graphene oxide by the effect of π – pi accumulation, and now fluorescence is by quencher; When after the target dna adding with single stranded DNA complementary pairing, because formation has the double-stranded DNA of rigid structure, make carbon quantum dot and graphene oxide apart from increase, carbon quantum dot is released from graphene oxide surface, now fluorescence is restored, and realizes the mensuration to target dna.
The detection method that fluorescent probe detects target dna is: first, get the above-mentioned 3mL probe solution preparing, add the graphene oxide (0.0,0.2,0.5,0.8 of 50 μ L different concns, 1.2,1.6,2.2,3.0,4.0,6.0,8.0,10.0,12.0,16.0,20.0,28.0 μ g mL
-1), finally add PBS buffered soln to be diluted to 5mL, ultrasonic 5 minutes, leave standstill to add after 25 minutes and in quartz colorimetric utensil, carry out fluorometric analysis, record data.As shown in Figure 5, along with the increase of graphene oxide concentration, the Fluorescence quenching effect of probe strengthens.Fig. 6 shows Fluorescence quenching effect and graphene oxide concentration (0.2～3.0 μ g mL
-1) present good linear relationship, when graphene oxide concentration exceedes 16 μ g mL
-1time, the Fluorescence quenching effect of probe presents slowly growth continuously.During for detection target dna, can obtain best recovery effects, adopting concentration is 16 μ gmL
-1graphene oxide as quencher.Fig. 3 has shown pattern, the composition of the graphene oxide of preparation, and graphene oxide prepared by TEM caption is sheet structure.
Get 16 μ g mL
-1graphene oxide 50 μ L join in 3mL probe solution, ultrasonic 5 minutes, leave standstill after 5 minutes fluorescence by significantly quencher; Then add the target dna (0,6.7,13.3,20,27,33 of different concns, 40,46,60,75,92,100,117,133nM), add subsequently PBS buffered soln to be diluted to 5mL, ultrasonic 5 minutes, leave standstill to add after 25 minutes and in quartz colorimetric utensil, carry out fluorometric analysis, record data.As shown in Figure 7, along with the increase of target dna concentration, the fluorescence intensity of probe increases greatly, is recovered by the fluorescence probe of quencher.Fig. 8 shows after the oxidized Graphene quencher of fluorescent probe, and relative intensity of fluorescence and target dna (6.7～46 μ M) present good linear relationship, and lowest detectable limit reaches 75.0pM.According to this linear relationship, by detecting the relative intensity of fluorescence of target dna to be measured, concentration that just can detection by quantitative target dna.
Embodiment 2: a kind of preparation method of fluorescent probe, this probe is to connect single stranded DNA by the reduction carbon quantum dot that can launch blue-fluorescence to obtain, described reduction carbon quantum dot is taking carbon quantum dot as raw material, sodium borohydride is as reductive agent, adopt the method for chemical reduction to carry out surface modification to carbon quantum dot, obtain launching the reduction carbon quantum dot of blue-fluorescence.Its concrete steps are:
A. carbon quantum dot is synthetic
0.1g Graphite Powder 99 joins in the 120mL vitriol oil and 40mL concentrated nitric acid mixing solutions, ultrasonic 1 hour, 60 DEG C of heated and stirred reflux 22 hours, after reaction finishes, be cooled to room temperature adding distil water and be diluted to 600mL, gained dark brown solution regulates pH value to neutral with sodium carbonate, obtain brown solution, the supernatant liquor of brown solution is removed small molecular weight impurity for 2 days with dialysis tubing dialysis, the molecular weight cut-off MWCO=2000 of dialysis tubing, finally by the mode of rotary evaporation, the mixing liquid in reactor is concentrated to evaporate to dryness, obtain carbon quantum dot;
B. reduce the synthetic of carbon quantum dot:
Take the above-mentioned carbon quantum dot 1.213g preparing, be dissolved in 20mL tetrahydrofuran solution with the excessive sodium borohydride solids of 1.0g, 60 DEG C of heated and stirred reflux 6 hours, after reaction finishes, in solution, unreacted sodium borohydride dialysis is removed, and makes the carbon quantum dot of reduction.TEM picture is with embodiment 1, and reduction carbon quantum dot presents monodispersed evenly spherical, and particle diameter is mainly distributed as 5 ± 3nm.The demonstration of high-resolution-ration transmission electric-lens photo, reduction carbon quantum dot has obvious lattice fringe structure, and the interfringe distance of adjacent crystalline form is
(002) diffraction surfaces data of data and graphite approach.Quantum yield in water is 21%.
C. the preparation of probe:
Getting the reduction carbon quantum dot 0.01g making is dissolved in the phosphate buffer solution of 10mL pH=7.4, adjust pH to 5.0 to make to reduce carbon quantum dot surface carboxylated, add under coupling agent 1.5g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) room temperature and stir 25 minutes, add afterwards 15 μ L single stranded DNAs (cDNA) to stir 20 hours, the mixture obtaining is through filtering with microporous membrane, retain the insolubles on film, again be dispersed in phosphate buffer solution with distilled water flushing insolubles and by it, obtain probe solution.As shown in Figure 1, carbon quantum dot is through sodium borohydride reduction, and the reduction carbon quantum dot obtaining connects single stranded DNA (cDNA) and forms probe the quenching of fluorescence-recovery process for detection of target dna (tDNA).
Fluorescent probe is in the application detecting in target dna, and using fluorescent probe as electron donor, graphene oxide is as electron acceptor(EA), and fluorescent probe is adsorbed onto the surface of graphene oxide by the effect of π – pi accumulation, and now fluorescence is by quencher; When after the target dna adding with single stranded DNA complementary pairing, because formation has the double-stranded DNA of rigid structure, make carbon quantum dot and graphene oxide apart from increase, carbon quantum dot is released from graphene oxide surface, now fluorescence is restored, and realizes the mensuration to target dna.
The detection method that fluorescent probe detects target dna is: first, get the above-mentioned 3mL probe solution preparing, add the graphene oxide (0.0,0.2,0.5,0.8 of 50 μ L different concns, 1.2,1.6,2.2,3.0,4.0,6.0,8.0,10.0,12.0,16.0,20.0,28.0 μ g mL
-1), finally add PBS buffered soln to be diluted to 5mL, ultrasonic 5 minutes, leave standstill to add after 25 minutes and in quartz colorimetric utensil, carry out fluorometric analysis, record data.As shown in Figure 5, along with the increase of graphene oxide concentration, the Fluorescence quenching effect of probe strengthens.Fig. 6 shows Fluorescence quenching effect and graphene oxide concentration (0.2～3.0 μ g mL
-1) present good linear relationship, when graphene oxide concentration exceedes 16 μ g mL
-1time, the Fluorescence quenching effect of probe presents slowly growth continuously.During for detection target dna, can obtain best recovery effects, adopting concentration is 16 μ gmL
-1graphene oxide as quencher.
Get 16 μ g mL
-1graphene oxide 50 μ L join in 3mL probe solution, ultrasonic 3 minutes, leave standstill after 3 minutes fluorescence by significantly quencher; Then add the target dna (0,6.7,13.3,20,27,33 of different concns, 40,46,60,75,92,100,117,133nM), add subsequently PBS buffered soln to be diluted to 5mL, ultrasonic 3 minutes, leave standstill to add after 20 minutes and in quartz colorimetric utensil, carry out fluorometric analysis, record data.Result shows with embodiment 1, and along with the increase of target dna concentration, the fluorescence intensity of probe increases greatly, is recovered by the fluorescence probe of quencher.The fluorescence recovery Effects of fluorescent probe and target dna (6.7～46 μ M) present good linear relationship, and lowest detectable limit reaches 75.0pM.According to this linear relationship, by detecting the relative intensity of fluorescence of target dna to be measured, concentration that just can detection by quantitative target dna.
Embodiment 3: a kind of preparation method of fluorescent probe, this probe is to connect single stranded DNA by the reduction carbon quantum dot that can launch blue-fluorescence to obtain, described reduction carbon quantum dot is taking carbon quantum dot as raw material, sodium borohydride is as reductive agent, adopt the method for chemical reduction to carry out surface modification to carbon quantum dot, obtain launching the reduction carbon quantum dot of blue-fluorescence.Its concrete steps are:
A. carbon quantum dot is synthetic
0.55g Graphite Powder 99 joins in the 240mL vitriol oil and 80mL concentrated nitric acid mixing solutions, ultrasonic 3 hours, 100 DEG C of heated and stirred reflux 26 hours, after reaction finishes, be cooled to room temperature adding distil water and be diluted to 1000mL, gained dark brown solution regulates pH value to neutral with sodium carbonate, obtain brown solution, the supernatant liquor of brown solution is removed small molecular weight impurity for 4 days with dialysis tubing dialysis, the molecular weight cut-off MWCO=3000 of dialysis tubing, finally by the mode of rotary evaporation, the mixing liquid in reactor is concentrated to evaporate to dryness, obtain carbon quantum dot;
B. reduce the synthetic of carbon quantum dot:
Take the above-mentioned carbon quantum dot 1.913g preparing, be dissolved in 30mL tetrahydrofuran solution with the excessive sodium borohydride solids of 1.5g, 80 DEG C of heated and stirred reflux 10 hours, after reaction finishes, in solution, unreacted sodium borohydride dialysis is removed, and makes the carbon quantum dot of reduction.TEM Image Display is with embodiment 1, and reduction carbon quantum dot presents monodispersed evenly spherical, and particle diameter is mainly distributed as 5 ± 3nm.The demonstration of high-resolution-ration transmission electric-lens photo, reduction carbon quantum dot has obvious lattice fringe structure, and the interfringe distance of adjacent crystalline form is
(002) diffraction surfaces data of data and graphite approach.Quantum yield in water is 22.5%.
C. the preparation of probe:
Getting the reduction carbon quantum dot 0.01g making is dissolved in the phosphate buffer solution of 10mL pH=7.4, adjust pH to 5.0 to make to reduce carbon quantum dot surface carboxylated, add coupling agent 1.7g N-to clap under imide (NHS) room temperature and stir 40 minutes through base glass, add afterwards 20 μ L single stranded DNAs (cDNA) to stir 20 hours, the mixture obtaining is through filtering with microporous membrane, retain the insolubles on film, be again dispersed in phosphate buffer solution with distilled water flushing insolubles and by it, obtain probe solution.As shown in Figure 1, carbon quantum dot is through sodium borohydride reduction, and the reduction carbon quantum dot obtaining connects single stranded DNA (cDNA) and forms probe the quenching of fluorescence-recovery process for detection of target dna (tDNA).
Fluorescent probe is in the application detecting in target dna, and using fluorescent probe as electron donor, graphene oxide is as electron acceptor(EA), and fluorescent probe is adsorbed onto the surface of graphene oxide by the effect of π – pi accumulation, and now fluorescence is by quencher; When after the target dna adding with single stranded DNA complementary pairing, because formation has the double-stranded DNA of rigid structure, make carbon quantum dot and graphene oxide apart from increase, carbon quantum dot is released from graphene oxide surface, now fluorescence is restored, and realizes the mensuration to target dna.
The detection method that fluorescent probe detects target dna is: first, get the above-mentioned 3mL probe solution preparing, add the graphene oxide (0.0,0.2,0.5,0.8 of 50 μ L different concns, 1.2,1.6,2.2,3.0,4.0,6.0,8.0,10.0,12.0,16.0,20.0,28.0 μ g mL
-1), finally add PBS buffered soln to be diluted to 5mL, ultrasonic 5 minutes, leave standstill to add after 25 minutes and in quartz colorimetric utensil, carry out fluorometric analysis, record data.As shown in Figure 5, along with the increase of graphene oxide concentration, the Fluorescence quenching effect of probe strengthens.Fig. 6 shows Fluorescence quenching effect and graphene oxide concentration (0.2～3.0 μ g mL
-1) present good linear relationship, when graphene oxide concentration exceedes 16 μ g mL
-1time, the Fluorescence quenching effect of probe presents slowly growth continuously.During for detection target dna, can obtain best recovery effects, adopting concentration is 16 μ gmL
-1graphene oxide as quencher.
Get 16 μ g mL
-1graphene oxide 50 μ L join in 3mL probe solution, ultrasonic 6 minutes, leave standstill after 6 minutes fluorescence by significantly quencher; Then add the target dna (0,6.7,13.3,20,27,33 of different concns, 40,46,60,75,92,100,117,133nM), add subsequently PBS buffered soln to be diluted to 5mL, ultrasonic 6 minutes, leave standstill to add after 30 minutes and in quartz colorimetric utensil, carry out fluorometric analysis, record data.Result shows with embodiment 1, and along with the increase of target dna concentration, the fluorescence intensity of probe increases greatly, is recovered by the fluorescence probe of quencher.The fluorescence recovery Effects of fluorescent probe and target dna (6.7～46 μ M) present good linear relationship, and lowest detectable limit reaches 75.0pM.According to this linear relationship, by detecting the relative intensity of fluorescence of target dna to be measured, concentration that just can detection by quantitative target dna.
Specific embodiment described herein is only to the explanation for example of the present invention's spirit.Those skilled in the art can make various amendments or supplement or adopt similar mode to substitute described specific embodiment, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Claims (10)
1. the preparation method of a fluorescent probe, it is characterized in that, this probe is to connect single stranded DNA by the reduction carbon quantum dot that can launch blue-fluorescence to obtain, described reduction carbon quantum dot is taking carbon quantum dot as raw material, sodium borohydride is as reductive agent, adopt the method for chemical reduction to carry out surface modification to carbon quantum dot, obtain launching the reduction carbon quantum dot of blue-fluorescence.
2. the preparation method of fluorescent probe according to claim 1, is characterized in that, its step comprises:
A. carbon quantum dot is synthetic
Graphite Powder 99 joins in the mixing solutions of the vitriol oil and concentrated nitric acid, ultrasonic 1～3 hour, 60～100 DEG C of heated and stirred reflux 22～26 hours, after reaction finishes, be cooled to the dilution of room temperature adding distil water, obtain dark brown solution sodium carbonate and regulate pH value to neutral, obtain brown solution, the supernatant liquor of brown solution is removed small molecular weight impurity for 2～4 days with dialysis tubing dialysis, finally by the mode of rotary evaporation, the mixing liquid in reactor is concentrated to evaporate to dryness, obtains carbon quantum dot;
B. reduce the synthetic of carbon quantum dot
Take and above-mentioned prepare carbon quantum dot and excessive sodium borohydride solids is dissolved in tetrahydrofuran solution, 60～80 DEG C of heated and stirred reflux 6～10 hours, and after reaction finishes, in solution, unreacted sodium borohydride dialysis is removed, and makes reduction carbon quantum dot;
C. the preparation of fluorescent probe
Taking the above-mentioned reduction carbon quantum dot making is dissolved in the phosphate buffer solution of pH=7.4, adjust pH to 5.0, it is carboxylated to make to reduce carbon quantum dot surface, then add coupling agent, under room temperature, stir 25～40 minutes, add afterwards single stranded DNA to stir 20～28 hours, the mixture obtaining is through filtering with microporous membrane, on film, the substance dissolves of gained, in phosphate buffer soln, obtains fluorescent probe solution.
3. the preparation method of fluorescent probe according to claim 2, is characterized in that, the described vitriol oil and the volume ratio of concentrated nitric acid are 3 ︰ 1.
4. the preparation method of fluorescent probe according to claim 2, is characterized in that, in described steps A ultrasonic 2 hours, 80 DEG C of heated and stirred refluxed 24 hours, and the molecular weight cut-off MWCO of dialysis tubing is 1000～3000.
5. the preparation method of fluorescent probe according to claim 2, is characterized in that, in described step B, 70 DEG C of heated and stirred reflux 8 hours.
6. the preparation method of fluorescent probe according to claim 2, is characterized in that, in described step C, coupling agent is one or both in 1-ethyl-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide.
7. the preparation method of fluorescent probe according to claim 2, is characterized in that, adds after coupling agent and stir 30 minutes under room temperature in described step C, adds afterwards single stranded DNA to stir 24 hours.
8. a fluorescent probe preparing as any in claim 1～7 is in the application detecting in target dna, it is characterized in that, fluorescent probe is as electron donor, graphene oxide is as electron acceptor(EA), fluorescent probe is adsorbed onto the surface of graphene oxide by the effect of π – pi accumulation, now fluorescence is by quencher; When after the target dna adding with single stranded DNA complementary pairing, because formation has the double-stranded DNA of rigid structure, make carbon quantum dot and graphene oxide apart from increase, carbon quantum dot is released from graphene oxide surface, now fluorescence is restored, thereby realizes the mensuration to target dna.
9. fluorescent probe according to claim 8, in the application detecting in target dna, is characterized in that, detection method is: get graphene oxide and join in probe solution, ultrasonic 3～6 minutes, leave standstill after 3～6 minutes fluorescence by significantly quencher; Then add target dna, add subsequently PBS buffered soln dilution, ultrasonic 3～6 minutes, leave standstill to add after 20～30 minutes and in quartz colorimetric utensil, carry out fluorometric analysis.
10. fluorescent probe according to claim 8, in the application detecting in target dna, is characterized in that, the concentration of described graphene oxide is 16 μ g mL
-1.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104087295A (en) * | 2014-06-25 | 2014-10-08 | 广西师范大学 | Preparation method and application of white-fluorescence-emitting carbon quantum dots |
| CN104215621A (en) * | 2014-06-26 | 2014-12-17 | 广西师范学院 | Method for detecting trivalent chromic ions by using graphene quantum dot probe |
| CN104531148A (en) * | 2014-12-31 | 2015-04-22 | 天津大学 | Preparation method of assembly of carbon quantum dots (CQDS) |
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| CN104087295B (en) * | 2014-06-25 | 2016-01-20 | 广西师范大学 | Launch preparation method and the application of the carbon quantum dot of white fluorescent |
| CN104215621A (en) * | 2014-06-26 | 2014-12-17 | 广西师范学院 | Method for detecting trivalent chromic ions by using graphene quantum dot probe |
| CN105219383A (en) * | 2014-07-02 | 2016-01-06 | 中国人民解放军军事医学科学院毒物药物研究所 | Oligonucleotide functional fluorescence nano-sized carbon point, Preparation Method And The Use |
| CN104531148A (en) * | 2014-12-31 | 2015-04-22 | 天津大学 | Preparation method of assembly of carbon quantum dots (CQDS) |
| CN105424927A (en) * | 2015-11-09 | 2016-03-23 | 山东省海洋生物研究院 | Method for detecting vibrio parahaemolyticus |
| CN105651753A (en) * | 2016-03-01 | 2016-06-08 | 上海应用技术学院 | Graphene oxide fluorescent sensor and preparation method and application thereof |
| CN105651753B (en) * | 2016-03-01 | 2018-07-13 | 上海应用技术学院 | Graphene oxide fluorescent optical sensor, preparation method and applications |
| CN106018373A (en) * | 2016-07-19 | 2016-10-12 | 济南大学 | Three-dimensional metal-enhanced fluorescent/colorimetric dual-mode paper chip and ATP measurement |
| CN106018373B (en) * | 2016-07-19 | 2018-06-08 | 济南大学 | The preparation of 3-dimensional metal enhancing fluorescence/colorimetric bimodulus paper chip and ATP detections |
| CN108949910A (en) * | 2018-06-08 | 2018-12-07 | 武汉博杰生物医学科技有限公司 | A kind of detection method and biosensor of high throughput miRNAs express spectra |
| CN116590008A (en) * | 2023-05-17 | 2023-08-15 | 合肥工业大学 | Background elimination-based ratio fluorescent probe for escherichia coli O157: H7 detection and preparation method thereof |
| CN116590008B (en) * | 2023-05-17 | 2024-03-19 | 合肥工业大学 | Background elimination-based ratio fluorescent probe for escherichia coli O157: H7 detection and preparation method thereof |
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