CN116218523B - Guanidine-functionalized fluorescent carbon dot and preparation method and application thereof - Google Patents
Guanidine-functionalized fluorescent carbon dot and preparation method and application thereof Download PDFInfo
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- 229910052799 carbon Inorganic materials 0.000 title claims abstract description 82
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 64
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 60
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 60
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000002243 precursor Substances 0.000 claims abstract description 12
- 239000004475 Arginine Substances 0.000 claims abstract description 11
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 11
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims abstract description 9
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims abstract description 6
- 238000001027 hydrothermal synthesis Methods 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 239000003480 eluent Substances 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
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- 239000000203 mixture Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims 4
- 238000009210 therapy by ultrasound Methods 0.000 claims 1
- 238000011068 loading method Methods 0.000 abstract description 7
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine group Chemical group NC(=N)N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 abstract description 6
- 239000002086 nanomaterial Substances 0.000 abstract description 3
- 238000004020 luminiscence type Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 15
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 12
- 230000035945 sensitivity Effects 0.000 description 9
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- 108020004414 DNA Proteins 0.000 description 6
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
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- 241000711573 Coronaviridae Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
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- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 2
- 238000001237 Raman spectrum Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
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- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 1
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- 229960000643 adenine Drugs 0.000 description 1
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- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 229960001149 dopamine hydrochloride Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 125000000524 functional group Chemical group 0.000 description 1
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- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 1
- 238000005087 graphitization Methods 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 238000002173 high-resolution transmission electron microscopy Methods 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
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- 230000003612 virological effect Effects 0.000 description 1
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
The invention relates to the field of fluorescent carbon nano materials, in particular to a guanidine group functionalized fluorescent carbon dot, a preparation method and application thereof. Arginine and o-phenylenediamine are used as precursors, and a carbon point solution can be obtained by a hydrothermal method for 3-9 hours at 150-200 ℃. The carbon dot prepared by the invention has excellent luminescence property and nucleic acid loading capacity, and has wide application prospect in the field of nucleic acid detection. The method can realize rapid and sensitive nucleic acid detection after loading the molecular beacon nucleic acid, and is expected to provide a convenient and effective method for the current nucleic acid detection.
Description
Technical Field
The invention relates to the field of fluorescent carbon nano materials, in particular to a guanidine group functionalized fluorescent carbon dot, a preparation method and application thereof.
Background
The real-time quantitative PCR (qPCR) is based on nucleic acid amplification, dye or fluorescent probe is added into the system, and the quantity of target DNA can be monitored in real time in the amplification process by combining the relationship between the fluorescent signal intensity and the amplification increment. Since the popularity of new coronaviruses, the standard nucleic acid detection technique employed is the RT-qPCR technique, which, despite its powerful detection capabilities, still has some limitations as a diagnostic technique for new coronaviruses, such as the need for relatively long detection procedures (2-4 hours), specialized equipment (thermocyclers, PCR instruments, etc.), laboratory and professional operators, etc. Therefore, it is highly desirable to develop a new coronavirus that is rapid and sensitive to point of care (POCT). Rapid antigen detection is a POCT for specific antigens, which is mainly based on Lateral Flow Immunoassay (LFIA), and utilizes antigen-antibody reaction to provide evidence of early viral infection, and has the advantages of low cost, simple operation method, short detection time (20 minutes or less), and the like. However, the detection result may be affected by other chemical substances with similar structures, so that the sensitivity is 75% -98%, the specificity is 95% -99%, and false positive results are easy to occur. The detection limit of LFIA is 1.12×10 5 to 3.57×10 6 copys/mL due to the effect of viral load, which is only effective in the first week of symptoms, and is prone to false negative results. This relatively low sensitivity of antigen detection presents multiple obstacles to detection and requires further investigation to improve its sensitivity and accuracy.
Carbon Dots (CDs) are zero-dimensional carbon nanomaterials, have many advantages of excellent optical properties, good water solubility, low biotoxicity, wide raw material sources, low preparation cost and the like, and have been applied to the fields of detection, diagnosis, drug delivery and the like.
Disclosure of Invention
In order to solve the problems of long time consumption and low sensitivity of virus or disease detection reported at present, the invention provides a fluorescent carbon dot capable of loading nucleic acid, a preparation method and application of the fluorescent carbon dot in nucleic acid detection.
The invention aims to provide a guanidino-functionalized fluorescent carbon dot.
It is still another object of the present invention to provide a nucleic acid detection application of the above carbon dot material.
It is a further object of the present invention to provide a method for preparing the above guanidino-functionalized fluorescent carbon dot.
The guanidinated fluorescent carbon dot according to the invention is prepared by a process comprising the steps of:
o-phenylenediamine and arginine are used as precursors, the precursors are dissolved in water, a homogeneous solution is formed under the ultrasonic action, and the reaction is carried out for 3 to 9 hours at 150 to 200 ℃ to obtain a brown yellow carbon dot solution;
And eluting and drying the obtained carbon dot solution to obtain carbon dot solid powder.
The guanidino-functionalized fluorescent carbon dot according to the invention, wherein the mass ratio of arginine to o-phenylenediamine is 1:5-5:1.
According to the technical scheme, arginine and phthalic diammine are selected as precursors, through regulating and controlling the reaction sites of the precursor o-phenylenediamine and controlling the connection of arginine guanidyl and amino acid groups, the mass ratio of the precursors in the hydrothermal reaction is controlled between 1:5 and 5:1, the reaction time is 3-9h, the reaction temperature is 150-200 ℃, the guanylated fluorescent carbon dot structure with both guanidyl and alpha-aminocarboxylic acid is synthesized, if the reaction time is too short (less than 3 hours) or the reaction temperature is too low (less than 150 ℃), the designed structure cannot be formed, namely the formed carbon dot cannot be connected with nucleic acid, and if the reaction time is too long (more than 9 hours) or the reaction temperature is too high (more than 200 ℃), the carbon dot is easy to excessively carbonize and cannot be obtained.
According to the preparation method, a mixture of methanol and dichloromethane (the volume ratio of the methanol to the dichloromethane is 1:14-1:7) is used as an eluent, and the purification by adopting a silica gel column chromatography is very important to the purification process. Since the impurities are excessive after the reaction is finished, if the purification is not performed by using a silica gel column, the guanidyl functional fluorescent carbon point capable of loading the nucleic acid medicine cannot be obtained.
According to the technical scheme of the application, the prepared guanidino functional fluorescent carbon dot can load molecular beacon nucleic acid (beacon) through guanidino, wherein the beacon is a hairpin structure, a sequence which can carry out base complementary pairing with target nucleic acid (TARGET DNA) is carried on the fluorescent carbon dot, and a ROX fluorescent group is modified at the 5' end. Molecular beacons (beacons) can arbitrarily alter the sequence therein to achieve detection of other diseases or viruses.
The guanidino functionalized carbon dot prepared by the invention has excellent optical property, good water solubility and nucleic acid loading characteristic. The guanidino functionalized carbon dot prepared by the invention has the capability of loading nucleic acid and has wide application prospect in the field of nucleic acid detection. The fluorescent probe has excellent light-emitting characteristics and nucleic acid loading capacity, so that the target of nucleic acid detection can be realized, and the fluorescent probe is expected to be applied to high-sensitivity and high-specificity rapid nucleic acid detection.
Drawings
FIG. 1 is a graph showing fluorescence spectra of the guanidino-functionalized carbon dots prepared in example 1 under excitation of different wavelengths;
FIG. 2 is a 2-dimensional fluorescence spectrum of the guanidino-functionalized carbon dot prepared in example 1;
FIG. 3 is an ultraviolet absorption spectrum of the guanidino-functionalized carbon dot prepared in example 1;
FIG. 4 is a transmission electron microscope electron micrograph of the guanidino functionalized carbon dots prepared in example 1;
FIG. 5 is a Raman spectrum of the guanidino-functionalized carbon dots prepared in example 1;
FIG. 6 is an X-ray photoelectron spectrum of a guanidino functionalized carbon dot prepared in example 1;
FIG. 7 is an infrared spectrum of the guanidino-functionalized carbon dots prepared in example 1;
FIG. 8 shows the band of Beacon electrophoresis before and after the combination of the guanidino functionalized carbon dots and molecular beacons (Beacon) prepared in example 1, and the gray values of the band;
FIG. 9 is the energy of interaction of the guanidino group on the guanidino functionalized carbon dot prepared in example 1 with four bases of beacon;
FIG. 10 is a diagram showing the specificity for nucleic acid detection after the attachment of beacon with the guanidinium-functionalized carbon dot prepared in example 1;
FIG. 11 is a graph showing the specificity for multi-sample nucleic acid detection after attachment of beacon with guanidinium functionalized carbon dots prepared in example 1;
FIG. 12 is a graph showing the sensitivity for nucleic acid detection after the attachment of beacon with the guanidinium-functionalized carbon dot prepared in example 1;
FIG. 13 is a graph showing the sensitivity for nucleic acid detection after ligation of a second beacon with the guanidinium-functionalized carbon dot prepared in example 1;
FIG. 14 shows the sensitivity for nucleic acid detection after ligation of a third beacon with the guanidino functionalized carbon dot prepared in example 1;
FIG. 15 shows the fluorescence change rate for nucleic acid detection after 3 beacon types are attached using the guanidino-functionalized carbon dots prepared in example 1;
FIG. 16 is a high specificity for nucleic acid detection after attachment of beacon with guanidino functionalized carbon dots prepared in example 1;
FIG. 17 is a graph showing the fluorescence change rate of nucleic acid detection for a mixed sample after the attachment of beacon with the guanidinium-functionalized carbon dot prepared in example 1;
FIGS. 18 and 19 are fluorescence spectra of the guanidino-functionalized carbon dots prepared in example 2 under excitation at different wavelengths.
Detailed Description
Embodiments of the present invention are described in detail below with reference to the attached drawing figures: the embodiment is implemented on the premise of the technical scheme of the invention, and detailed implementation modes and processes are given, but the protection scope of the invention is not limited to the following embodiment.
According to the technical scheme, arginine and o-phenylenediamine are selected as precursors, and guanidine functional carbon dots are synthesized by regulating the reaction proportion and the reaction time, and the specific method comprises the following steps:
(1) Arginine and o-phenylenediamine are used as precursors, and the precursors are fully dissolved in water by ultrasonic stirring. The solution was then transferred to a stainless steel autoclave lined with polytetrafluoroethylene. And carrying out hydrothermal reaction for 3-9 hours at 150-200 ℃, and then naturally cooling the reaction kettle to room temperature, so as to obtain a carbon dot solution with a brownish yellow appearance.
(2) Collecting the solution after the reaction, filtering with a 0.22 mu m filter membrane, purifying with a mixed solvent of methanol and dichloromethane as an eluent by adopting a silica gel column chromatography, separating and purifying to obtain a carbon dot solution, and drying to obtain carbon dot solid powder. The carbon dot solid powder prepared by the invention has good solubility in water.
According to the technical scheme, a mixture of methanol and dichloromethane (the volume ratio of the methanol to the dichloromethane is 1:14-1:7, and the methane concentration is gradually increased) is used as an eluent, and the purification is performed by adopting a silica gel column chromatography.
Example 1 preparation of guanidino-functionalized carbon dots
0.3G of arginine and 0.15g of o-phenylenediamine are weighed into a beaker respectively, 60mL of secondary water is added, a homogeneous solution is formed under the action of ultrasound, and then the homogeneous solution is transferred to an autoclave lined with polytetrafluoroethylene. And carrying out hydrothermal reaction for 5 hours at 180 ℃ to naturally cool the reaction kettle to room temperature, thereby obtaining a carbon dot solution with a brown yellow appearance. After the reaction is finished, collecting the solution after the reaction, filtering the solution by using a 0.22 mu m filter membrane, taking a mixture of methanol and dichloromethane (the volume ratio of the methanol to the dichloromethane is 1:14-1:7, and the concentration of the methane is gradually increased) as an eluent, purifying the eluent by adopting a silica gel column chromatography, separating and purifying the eluent to obtain a carbon dot solution, and drying the carbon dot solution to obtain carbon dot solid powder.
As an alternative technical solution of this embodiment, the mass ratio of arginine to dopamine hydrochloride is 1:2, 1:3, 1:4, 1:5 or 5:1.
As an alternative to this embodiment, the reaction temperature was 150℃180℃and 200 ℃.
As an alternative technical solution of the embodiment, the volume ratio of the eluent methanol to the dichloromethane is 1:14, 1:10 and 1:7.
The yellow fluorescent carbon dot aqueous solution emits bright yellow fluorescence under a portable ultraviolet lamp (365 nm), and shows the characteristic of intrinsic state fluorescence independent of excitation (fig. 1 and 2), and the emission peak is positioned at 570nm. The yellow light carbon dot characteristic exciton absorption peak is at 415nm (fig. 3), close to the maximum fluorescence excitation wavelength, further illustrating that carbon dot fluorescence results from energy band transitions.
The transmission electron microscope observed that the eigen-state yellow fluorescent carbon dots were uniformly distributed in size and had an average particle diameter of 4nm (fig. 4). The ratio of I D/IG in the carbon dot raman spectrum was 0.6 (fig. 5), indicating that the degree of graphitization of the carbon dots was very high, consistent with the high crystallinity characterized by high resolution transmission electron microscopy.
The result of the X-ray photoelectron spectroscopy shows that the carbon point mainly consists of three elements of C, N and O, wherein the atomic percentages of C and N are 75.14 percent and 10.14 percent respectively (figure 6). The carbon dot solid infrared spectrum demonstrates the presence of functional groups such as amino groups, carboxyl groups, etc. in the carbon dot (fig. 7).
After Beacon is combined with GelRed in agarose gel, a fluorescent strip can be generated under 254nm excitation, after a gel imager is used for photographing, image J software is used for measuring the gray value of the strip, and the result shows that after carbon points are added into the Beacon, the brightness of the Beacon is weakened, the carbon points interact with the Beacon, movement of the Beacon strip is blocked, when 2 times of carbon points are added into the Beacon, the Beacon strip almost disappears, and the excessive carbon points interact with the Beacon to completely block the movement of the Beacon (figure 8).
And simplifying the structure of the carbon point, and then calculating the binding energy of the carbon point and the beacon. Interactions between amino (-NH 2), carboxyl (-COOH) and guanidino (-CN 3H4) groups in carbon dots and four bases adenine (a), guanine (G), cytosine (C) and thymine (T) were determined using geometric modeling methods, and the geometry was optimized at the b3lyp/6-31G (d) level, and the interaction energies 33 of these models were calculated by computational quantum chemistry from scratch. As shown in the calculation results of FIG. 9, the interaction energy of the guanidine group and the beacon is-1.25 to-7.01 kcal/mol, and the interaction energy of the amino group and the carboxyl group and the beacon are similar (0.58 to-2.21 kcal/mol and 2.55 to-3.07 kcal/mol respectively), which shows that the guanidine group has obvious energy advantage when interacting with the beacon.
After incubation of the carbon dot and beacon binding complex with TARGET DNA for 1min, the fluorescence intensity was increased 6-fold, but when the complex was incubated with MISMATCH DNA, the carbon dot fluorescence did not change significantly, indicating a strong specificity for complex nucleic acid detection (fig. 10).
The carbon spot and beacon binding complex detected 55 samples, 34 of which were positive samples containing TARGET DNA at different concentrations, 21 negative samples containing MISMATCH DNA at different sequences, and the 55 samples were fluorometrically assayed using the complex and analyzed with the detection threshold set at 20% change in fluorescence intensity. As a result, as shown in FIG. 11, the average fluorescence increase rate of the positive samples was 124.75%, and the average fluorescence increase rate of the negative samples was 0.04%. Wherein, the fluorescence increase rate of a sample existing in the negative group is 20.35%, and the negative group is a false positive result. So GCDs-Beacon nucleic acid detection specificity = true negative sample/(true negative sample + false positive sample) ×100% = 95.45%.
The carbon dot and the beacon binding complex are incubated with target nucleic acids with different concentrations, fluorescence changes of the carbon dot are measured and observed, and the result of FIG. 12 shows that the carbon dot and the beacon binding complex have different fluorescence phenomena on the target nucleic acids with different concentrations, and the fluorescence increase phenomenon is still present even when the nucleic acid concentration is as low as 0.01fM (the addition concentration, the final concentration in a detection system is 0.005fM, about 300 copys/mL), which indicates that the carbon dot and the beacon binding complex nucleic acid detection system has stronger sensitivity.
The carbon dots are connected with the beacon molecules (beacon 2 and beacon 3) with different sequences, then the molecules are tested with the corresponding target sequence 2 and the corresponding target sequence 3, as shown in fig. 13 and 14, after the beacon nucleic acid sequence is replaced, the fluorescence intensity change rate of the carbon dots and the beacon binding complex is above 0.5 after the target nucleic acid is detected (fig. 15), and the detection system has similar specificity and sensitivity. Experiments show that the carbon dot and beacon binding complex detection system can be used for detecting nucleic acids of different viruses or diseases by changing the nucleic acid sequence of the beacon.
The nucleic acid sequence of the target nucleic acid was changed by one base (mismatched nucleic acid 1) or 4 bases (mismatched nucleic acid 4), and then reacted with the carbon dot and the beacon binding complex, respectively, as shown in FIG. 16, when the target nucleic acid was present in the carbon dot and the beacon binding complex system, the fluorescence intensity change rate was 0.24, one base in the target nucleic acid was changed, the fluorescence intensity change rate was reduced to 0.13, and after changing 4 bases, the change rate was only 0.03, which was the same as that of the completely mismatched nucleic acid. The results show that when a nucleic acid sequence very similar to the target nucleic acid exists in the system, the carbon dots do not show positive results, and false positives in the detection process are avoided.
The total concentration of nucleic acids in the system is kept consistent, after mixing nucleic acids with different sequences, the carbon dot and beacon binding complex can still detect trace target nucleic acid contained in the nucleic acid, 0.25 fM target nucleic acid is 0.01fM target nucleic acid which is 0.25 times of that contained in the nucleic acid, namely 0.0025fM target nucleic acid is added into the system, as shown in FIG. 17, so that when a plurality of nucleic acids exist in the system, the target nucleic acid contained in the nucleic acid can still be detected.
Example 2
According to the technical scheme of the invention, the mass ratio of the hydrothermal reaction precursors is controlled to be 1:5-5:1, the reaction time is 3-9h, the reaction temperature is 150-200 ℃, the guanylated fluorescent carbon dot structure with both guanidyl and alpha-aminocarboxylic acid is synthesized, and compared with the embodiment 1, other reflecting conditions are the same, if the reaction time is too short (less than 3 hours) or the reaction temperature is too low (less than 150 ℃), the designed structure cannot be formed, namely the formed carbon dot cannot be connected with nucleic acid, as shown in fig. 18, the reaction is carried out for 2h at 150 ℃, and the carbon dot has weak fluorescence and has a hetero peak. FIG. 19 shows that after 5h of reaction at 120℃the target nucleic acid could not be detected by the carbon dot and beacon complex. If the reaction time is too long (9 hours or more) or the reaction temperature is too high (200 ℃ or more), excessive carbonization is likely to occur, and the inside of the reaction vessel is a black non-fluorescent solution and contains large black residues, so that carbon dots cannot be obtained.
The above embodiments are only for explaining the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (4)
1. Use of a guanidinium-functionalized fluorescent carbon dot for the preparation of a nucleic acid detection reagent, characterized in that the guanidinium-functionalized fluorescent carbon dot is prepared by a method comprising the steps of:
Arginine and o-phenylenediamine are used as precursors, ultrasonic treatment is carried out to dissolve the precursors in water to form a homogeneous solution, then the solution is transferred into a reaction kettle, hydrothermal reaction is carried out for 3-9 hours at 150-200 ℃, and then the reaction kettle is naturally cooled to room temperature, thus directly obtaining a carbon dot solution;
and filtering, eluting and drying the obtained carbon dot solution to obtain the solid powder of the guanidyl functional fluorescent carbon dots.
2. The use of the guanidinium-functionalized fluorescent carbon dot according to claim 1 for preparing a nucleic acid detection reagent, wherein the solid powder of the guanidinium-functionalized fluorescent carbon dot is obtained by separating and purifying the mixture of methanol and methylene chloride as an eluent by a silica gel column chromatography.
3. The use of the guanidino-functionalized fluorescent carbon dot according to claim 2 for preparing a nucleic acid detection reagent, wherein the volume ratio of methanol to dichloromethane is 1:14 to 1:7.
4. The use of a guanidino-functional fluorescent carbon dot according to claim 1 for preparing a nucleic acid detection reagent, wherein the arginine and o-phenylenediamine are in a mass ratio of 5:1 to 1:5.
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CN116281953A (en) * | 2023-03-02 | 2023-06-23 | 北京师范大学 | Base functionalized tumor targeting carbon dot and preparation method and application thereof |
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CN108659833A (en) * | 2018-05-25 | 2018-10-16 | 山西大学 | A kind of yellow fluorescence carbon dots and its preparation method and application |
CN111807349A (en) * | 2020-08-31 | 2020-10-23 | 河南师范大学 | Preparation method of photoluminescence wavelength-stable red light carbon dots |
CN114410300A (en) * | 2022-01-30 | 2022-04-29 | 安徽大学 | Fluorescent probe and preparation method and biological application thereof |
CN114854407A (en) * | 2022-05-27 | 2022-08-05 | 昆明学院 | Preparation method of L-arginine-based carbon dots and application of L-arginine-based carbon dots in lemon yellow detection |
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