CN115963093A - DNA fluorescent sensor based on UCNPs and preparation method and application thereof - Google Patents
DNA fluorescent sensor based on UCNPs and preparation method and application thereof Download PDFInfo
- Publication number
- CN115963093A CN115963093A CN202211603274.0A CN202211603274A CN115963093A CN 115963093 A CN115963093 A CN 115963093A CN 202211603274 A CN202211603274 A CN 202211603274A CN 115963093 A CN115963093 A CN 115963093A
- Authority
- CN
- China
- Prior art keywords
- ucnps
- hsdna
- anticancer drugs
- concentration
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims description 9
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 58
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 58
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims abstract description 7
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims abstract description 7
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000005642 Oleic acid Substances 0.000 claims abstract description 7
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims abstract description 7
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims abstract description 7
- 239000003446 ligand Substances 0.000 claims abstract description 4
- 238000011068 loading method Methods 0.000 claims abstract description 4
- 238000001514 detection method Methods 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 229910052769 Ytterbium Inorganic materials 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 abstract description 51
- 150000004056 anthraquinones Chemical class 0.000 abstract description 51
- 229940079593 drug Drugs 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 8
- 238000010791 quenching Methods 0.000 abstract description 8
- 230000000171 quenching effect Effects 0.000 abstract description 7
- 230000005284 excitation Effects 0.000 abstract description 6
- 238000012546 transfer Methods 0.000 abstract description 6
- 230000008859 change Effects 0.000 abstract description 5
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 238000002866 fluorescence resonance energy transfer Methods 0.000 abstract description 3
- 239000002086 nanomaterial Substances 0.000 abstract description 3
- 238000004445 quantitative analysis Methods 0.000 abstract description 3
- 229920006395 saturated elastomer Polymers 0.000 abstract 1
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 25
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 21
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 21
- 229960000975 daunorubicin Drugs 0.000 description 21
- 206010028980 Neoplasm Diseases 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000000295 emission spectrum Methods 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 2
- 229910052691 Erbium Inorganic materials 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000013060 biological fluid Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229910052761 rare earth metal Inorganic materials 0.000 description 2
- 150000002910 rare earth metals Chemical class 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 102100040458 2',3'-cyclic-nucleotide 3'-phosphodiesterase Human genes 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 206010065553 Bone marrow failure Diseases 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 102000003915 DNA Topoisomerases Human genes 0.000 description 1
- 108090000323 DNA Topoisomerases Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 102000007537 Type II DNA Topoisomerases Human genes 0.000 description 1
- 108010046308 Type II DNA Topoisomerases Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002927 anti-mitotic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000214 effect on organisms Effects 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 231100001252 long-term toxicity Toxicity 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001945 resonance Rayleigh scattering spectroscopy Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000000790 scattering method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a DNA fluorescence sensor based on UCNPs, belonging to the technical field of sensors. The fluorescence sensor is obtained by removing oleic acid ligands on the surface of up-conversion nano materials (UCNPs) and loading hsDNA. The invention detects the concentration of anthraquinone drugs based on the principle of fluorescence resonance energy transfer, and forms a UCNPs @ hsDNA sensor by loading saturated hsDNA on the surface of UCNPs. The AQ anticancer drugs are combined with base pairs through a double helix structure inserted into DNA, and have wide absorption at 400-600 nm and are just overlapped with an emission peak of UCNPs at 545 nm. Under the excitation of 980nm near-infrared light, energy resonance transfer occurs between UCNPs and AQ anticancer drugs, and the green up-conversion fluorescence quenching of the UCNPs at 545nm is enhanced along with the increase of the concentration of the AQ anticancer drugs. Therefore, the quantitative analysis of the AQ anticancer drugs can be realized according to the change of the fluorescence signal intensity of UCNPs at 545 nm.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a DNA fluorescent sensor based on UCNPs and a preparation method and application thereof.
Background
With the development of society and the acceleration of urbanization, the living environment of people is gradually worsened, and the cancer creates the opportunity to attack people. Cancer has now become a leading cause of death in the world. Clinically, the DNA of cancer cells has become the main target of many anticancer drugs, and these drugs can destroy the DNA structure through the interaction with the DNA of cancer cells, influence the expression function of gene regulation, and show the antitumor activity. Among them, anthraquinone (AQ) antitumor antibiotics have a remarkable anti-mitotic ability, generate Reactive Oxygen Species (ROS) and interact with DNA topoisomerase I and II, thereby causing cancer cell necrosis, since it can block all transcription or replication processes by intercalating DNA to form a DNA complex. Doxorubicin (Adriamycin, ADM) and Daunorubicin (DNR) belong to an important class of AQ anticancer drugs. They insert into the double helix of DNA and bind to base pairs, acting by inhibiting DNA and RNA synthesis. ADM and DNR have a broad spectrum of anticancer activity, have a wide range of biochemical effects on organisms, and have strong cytotoxicity. Clinically, ADM is commonly used to treat acute lymphocytic leukemia and acute myeloid leukemia. DNR is an effective anti-oxidant leukemia drug and is used for treating chronic myelocytic leukemia, famous acute myelocytic leukemia, kaposi's sarcoma and acute lymphocytic leukemia. The clinical efficacy of antitumor antibiotics depends on their concentration in humans. When the concentration of the drug reaches a suitable level, the optimal therapeutic effect can be achieved. If the concentration of the drug in the body exceeds the optimum concentration, the drug may cause serious adverse reactions such as cardiotoxicity, myelosuppression and nephrotoxicity. Therefore, monitoring the AQ anticancer drugs in the human body to obtain more accurate therapeutic drug concentrations is beneficial to reducing the toxic and side effects caused by the drugs.
In order to effectively detect AQ anticancer drugs in biological fluids, several conventional detection methods have been developed. Among them, HPLC is the most widely used method for measuring AQ anticancer drugs. In addition, there are reports of electrochemistry, capillary electrophoresis, HPLC-MS and immunoassays. However, these methods have problems that they require the use of expensive, time-consuming and complicated instruments. At present, the spectrum method is relatively few, and a raman scattering method, a resonance rayleigh scattering method, a spectrophotometry method and the like are reported in documents. However, these methods have disadvantages of low sensitivity and poor specificity. In recent years, fluorescent sensors have attracted considerable interest due to their advantages of high sensitivity, low cost, rapid response, and relatively simple operation, and thus many fluorescent sensors have been developed and used in the fields of biology, food, and environment. Some fluorescent sensors based on quantum dots or combined with organic dyes have been developed for the detection of AQ anticancer drugs. These probes are typically designed using changes in their optical properties in the presence of DNR and ADM. Unfortunately, they suffer from several inherent problems, including poor photochemical stability, large emission bandwidths, and high long-term toxicity. In addition, the excitation wavelengths of these sensors are mostly in the ultraviolet and visible range, so that their light penetration depth is low, and autofluorescence intensity hinders further biological applications.
The rare earth doped up-conversion luminescent nano material (UCNPs) has great advantages in biological application due to the unique anti-Stokes luminescent property, and is widely applied to the biological field. Fluorescence resonance energy transfer (LRET) is a non-radiative process, i.e., an energy donor in an excited state transfers energy non-radiatively to an energy acceptor, which releases energy by fluorescence emission or other non-radiative means. The premise of generating fluorescence resonance energy transfer is that the emission spectrum of an energy donor is overlapped with the absorption spectrum of an acceptor, and the space distance between the emission spectrum and the absorption spectrum of the acceptor is within 10nm, so that the LRET technology has the advantages of high sensitivity, high resolution, simplicity, convenience and the like.
The UCNPs have the advantages of good chemical stability, light stability, no autofluorescence and the like, and are a better energy donor for LRET. Based on LRET principle, the application of UCNPs in constructing fluorescence biosensors for drug detection and the like is becoming a research hotspot.
Disclosure of Invention
The invention aims to provide a DNA fluorescent sensor based on UCNPs (nuclear magnetic resonance) and a preparation method and application thereof, which aim to solve the problems in the prior art and realize high-sensitivity detection and high-selectivity detection of AQ anticancer drugs by using the change of the intensity of up-conversion fluorescence.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a preparation method of a DNA fluorescent sensor based on UCNPs, which comprises the following steps: and washing the oleic acid ligand on the surface of the UCNPs, and then loading the hsDNA to obtain the fluorescence sensor.
In one possible design, the UCNPs are NaYF4: yb, er.
In one possible design, in particular, the first deoiling was performed in a round bottom flask with oleic acid capped OA-UCNPs: adding HCl solution, adding ether, performing ultrasonic treatment, stirring, and centrifuging; dissolving the precipitate with a small amount of HCl solution after the centrifugation is finished, and transferring the precipitate to a round-bottom flask; repeating the above steps for 2-3 times of deoiling, dispersing the 3 times of deoiled centrifugal product with 5-10mL of acetone, centrifuging, discarding supernatant, and freeze-drying precipitate to obtain Free-UCNPs; mixing Free-UCNPs, tris-HCl buffer solution and hsDNA in solution for reaction, incubating and centrifuging to obtain UCNPs @ hsDNA, namely the fluorescence sensor.
In one possible design, the pH of the HCl is between 3.00 and 4.00.
In one possible design, the final concentration of Free-UCNPs solution is 0.1 mg-mL -1 The final concentration of hsDNA was 20. Mu.g.mL -1 The concentration of Tris-HCl buffer was 10mM.
According to the invention, after the oleic acid ligand on the surface of UCNPs is washed away and the hsDNA is loaded, when the hsDNA is added into a system as an identification element, the phosphate skeleton of the hsDNA has a large amount of negative charges and is attached to the surface of the UCNPs through electrostatic attraction so as to capture the AQ anticancer drugs in the solution, the hydroxyl on the AQ anticancer drugs C9 can interact with N of a base in the hsDNA through a hydrogen bond, thus the distance between the UCNPs and the AQ anticancer drugs is reduced, energy resonance transfer is generated between the UCNPs and the AQ anticancer drugs, and the fluorescence quenching intensity is changed along with the increase of the concentration of the AQ anticancer drugs, so that the detection of the concentration of the AQ anticancer drugs is realized by detecting the fluorescence quenching intensity change of the UCNPs @ hsDNA sensor.
The present invention also provides a UCNPs-based DNA fluorescence sensor prepared by the method of claims 1-5.
The rare earth doped up-conversion luminescent nano material selected by the invention has photochemical stability, long fluorescence life and no background fluorescence due to the unique anti-Stokes luminescent property. The AQ anticancer drug plays a role by inserting into a double helix structure of DNA and combining with base pairs, has wide absorption at 400-600 nm, and is just equal to NaYF 4 The emission peaks of Yb, er UCNPs overlap at 545nm, which all provide conditions for LRET.
The invention also provides an application of the DNA fluorescent sensor based on UCNPs in AQ anticancer drug concentration detection.
The invention also provides a method for detecting the concentration of the AQ anticancer drugs, which comprises the following steps:
and incubating a sample to be detected and a buffer solution prepared by the fluorescence sensor together, and carrying out fluorescence detection.
In one possible design, the buffer solution was prepared using a Tris-HCl buffer solution at a concentration of 10mM and free-UCNPs solution at a final concentration of 0.1 mg. ML -1 The final concentration of hsDNA is 20. Mu.g.mL -1 The final volume of the mixed solution is 500 mu L of UCNPs @ hsDNA solution, and the concentration range of the AQ anticancer drugs is 0-100 mu g/mL -1 。
The detection system of the invention is NaYF under the excitation of 980nm near infrared light 4 Yb, er UCNPs and AQ anticancer drugs are subjected to energy resonance transfer, and NaYF is added along with the increase of the concentration of the AQ anticancer drugs 4 :Yb,Er UGreen up-conversion fluorescence quenching of CNPs at 545nm was enhanced. Thus, it can be based on NaYF 4 The change of the fluorescence signal intensity of Yb and Er UCNPs at 545nm realizes the quantitative analysis of the AQ anticancer drugs.
The invention also provides an application of the DNA fluorescent sensor based on UCNPs and/or the detection method in AQ anticancer drug screening.
The invention discloses the following technical effects:
the invention successfully prepares a novel fluorescence sensor of LRET between UCNPs loaded with hsDNA and AQ anticancer drugs, and is used for detecting the concentration of the AQ anticancer drugs. The detection system utilizes the characteristic that AQ anticancer drugs can be embedded into double strands of hsDNA, and uses the hsDNA as a recognition element to capture the AQ anticancer drugs, thereby shortening the distance between UCNPs and the AQ anticancer drugs, realizing LRET between the UCNPs and the AQ anticancer drugs, and being applicable to quantitative detection of the AQ anticancer drugs. The linear range for detecting DNR is 1-100 mu g/mL -1 LOD of 0.5069 mug. Mu.g.mL -1 (ii) a The linear range for detecting ADM is 0.5-100 mug.mL -1 LOD of 0.2390 mug. Mu.g.mL -1 。
The detection method of the invention is based on the fluorescence energy resonance transfer between UCNPs and AQ anticancer drugs to detect the concentration of the AQ anticancer drugs, has high sensitivity and high selectivity, avoids expensive detection cost and complex operation of traditional detection instruments such as ICP-MS and AES, and simplifies the experimental flow of the detection of the concentration of the AQ anticancer drugs.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 (A) is the upconversion emission spectra of NaYF4: yb, er and the UV-Vis absorption spectra of DNR and ADM in example 1; FIG. 1 (B) NaYF in example 1 4 Upconversion of Yb, er UCNPsEmission spectra and UV-Vis absorption spectra of hsDNA + DNR and hsDNA + ADM
FIGS. 2 (A) and (B) fluorescence spectra of UCNPs, UCNPs + DNR, UCNPs @ hsDNA + DNR and UCNPs, UCNPs + ADM, UCNPs @ hsDNA + ADM in example 2, respectively;
FIGS. 3 (A) and (B) are the fluorescence spectra for detecting DNR with different concentrations and the fluorescence spectra for detecting ADM with different concentrations in example 2, respectively, (C) and (D) are the standard working curves for detecting DNR with different concentrations and the standard working curves for detecting ADM with different concentrations, respectively;
FIG. 4 is a strategy diagram of the detection of AQ anticancer drugs based on LRET mechanism in example 2;
FIG. 5 is an alternative diagram of two sensors of example 3.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
The preparation method of the DNA fluorescent sensor based on UCNPs comprises the following steps:
1) Preparation of Free-UCNPs:
the first oil removal was performed in a round bottom flask with oleic acid capped OA-UCNPs (100 mg): adding 10mL of HCl (pH 3.00-4.00), then adding 5mL of diethyl ether, carrying out ultrasonic treatment for 10min, and stirring for 2h.13000rmp centrifugation for 10min; dissolving the precipitate with small amount of HCl (pH 3.00-4.00) after the centrifugation, transferring to round bottom flask, repeating the above steps for 2-3 times of deoiling, dispersing the 3 times of deoiled centrifugation product with 5-10ml of acetone, centrifuging, discarding the supernatant, and freeze-drying the precipitate.
2) Preparation of UCNPs @ hsDNA:
the ligand-free UCNPs were made up to 1 mg/mL with Tris-HCl buffer (10 mM concentration) -1 The solution of (4) is prepared by mixing the hsDNA with a solution of ligand-free UCNPs in a Tris-HCl buffer solution, incubating at 25 ℃ for 20min with shaking at 300rpm, and centrifuging at 8000rpm and 5min for 1 time after the incubation is finished. The final concentration of the hsDNA prepared was 20. Mu.g/mL -1 Final concentration of UCNPs is 0.1 mg. ML -1 The solution of UCNPs @ hsDNA was stored at 4 ℃ until use.
FIG. 1 (A) shows the upconversion emission spectra of NaYF4: yb, er and the UV-Vis absorption spectra of DNR and ADM, when DNR and ADM are respectively embedded into hsDNA, the ultraviolet absorption of hsDNA + DNR and hsDNA + ADM has obvious subtractive effect and the UV-Vis absorption spectra have red shift, as shown in FIG. 1 (B) the upconversion emission spectra of NaYF4: yb, er UCNPs and the UV-Vis absorption spectra of hsDNA + DNR and hsDNA + ADM.
Example 2
The UCNPs @ hsDNA sensor prepared in the embodiment 1 is used for detecting the concentration of AQ anticancer drugs, and the specific detection method comprises the following steps:
taking the final concentration of the solution of the ligand-free UCNPs as 0.1 mg/mL -1 The final concentration of hsDNA is 20. Mu.g.mL -1 The final volume of the mixed solution is 500 μ L of UCNPs @ hsDNA solution, and different concentrations of AQ anticancer drugs (0.5, 1, 10, 20, 30, 40, 50, 80, 100, unit is μ g/mL) -1 ) Shaking and incubating at 25 deg.C and 300rpm for 20min, detecting with a fluorescence spectrophotometer, and detecting fluorescence emission spectrum of the sample in 300-900 nm wavelength range under irradiation of 980nm near infrared exciter. The excitation light source is 980nm exciter with excitation power of 2W/cm 2 And a voltage of 500V.
FIGS. 2 (A) and (B) are fluorescence spectra of UCNPs, UCNPs + DNR, UCNPs @ hsDNA + DNR, UCNPs + ADM, and UCNPs @ hsDNA + ADM, respectively. It can be seen from the figure that when only UCNPs and AQ anticancer drugs exist in the system, the fluorescence intensity of UCNPs at 545nm is basically unchanged. The AQ anticancer drugs quench the green luminescence of UCNPs at 545nm when hsDNA is present in the system.
FIGS. 3 (A) and (B) are fluorescence spectrograms for detecting DNR with different concentrations and fluorescence spectrograms for detecting ADM with different concentrations, respectively, UCNPs @ hsDNA is used as an identification element of AQ anticancer drugs, and the light emission of UCNPs at 545nm is gradually weakened along with the increase of the concentration of the AQ anticancer drugs in the system. Through quenching the fluorescence intensity of UCNPs by AQ anticancer drugs, the regression equation of the standard curve for detecting DNR can be obtained to be y =0.0123c +1.0771, R2=0.9965, and the detection limit is 0.5069 mug. ML -1 (ii) a The regression equation of the standard curve of ADM is detected to be y =0.0217c +1.0612, R2=0.9974, and the detection limit is 0.2390 mug. ML -1 (by 3. Delta./S method), FIGS. 3 (C) and (D) are respectively a standard operation curve for detecting DNR at different concentrations and a standard operation curve for detecting ADM at different concentrations. From this, it can be shown that the fluorescence intensity of DNR-quenched UCNPs is 1 to 100. Mu.g/mL -1 Shows good linear relation, and the fluorescence intensity of ADM quenching UCNPs is between 0.5 and 100 mu g/mL -1 And has a good linear relationship.
FIG. 4 is a schematic diagram of the mechanism of detection of AQ anticancer drugs based on LRET.
When only UCNPs and AQ anticancer drugs exist in the system, the distance is increased due to charge repulsion of the UCNPs and the AQ anticancer drugs, and the LRET condition cannot be reached, so that the green luminescence of the UCNPs at 545nm cannot be quenched. However, when UCNPs are loaded with hsDNA, the distance between the UCNPs and the AQ anticancer drugs is reduced because the AQ anticancer drugs play a role by being embedded on the DNA. Therefore, energy resonance transfer occurs between UCNPs and AQ anticancer drugs under the excitation of 980nm near infrared light of the detection system, and the green up-conversion fluorescence quenching of the UCNPs at 545nm is enhanced along with the increase of the concentration of the AQ anticancer drugs. Therefore, the quantitative analysis of the AQ anticancer drugs can be realized according to the change of the fluorescence signal intensity of UCNPs at 545 nm.
Example 3
Selective experiments of UCNPs-based DNA fluorescence sensors:
under the condition that a detection system is in an optimal reaction condition, different kinds of amino acids and metal ions are selected as possible interference substances in biological fluid, wherein the interference substances comprise Mg 2+ 、K + 、Ca 2+ The concentrations of cysteine (Cys), glycine (Gly), D-fructose (D-Fru) and the like are 20 times of the normal values in normal human body fluid.
Under the above conditions, the interfering substances do not interfere with the detection of the AQ anticancer drugs, so that the detection system has good recognition specificity for the AQ anticancer drugs, and the results are shown in the selectivity graph of two sensors in fig. 5.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (10)
1. A preparation method of a DNA fluorescent sensor based on UCNPs is characterized by comprising the following steps: and washing the oleic acid ligand on the surface of the UCNPs, and then loading the hsDNA to obtain the fluorescence sensor.
2. The method of claim 1, wherein the UCNPs are NaYF4 Yb, er.
3. The method for preparing a UCNPs-based DNA fluorescence sensor according to claim 1 or 2, wherein the OA-UCNPs capped with oleic acid are taken in a round bottom flask for first deoiling: adding HCl solution, adding ether, performing ultrasonic treatment, stirring, and centrifuging; dissolving the precipitate with a small amount of HCl solution after the centrifugation is finished, and transferring the precipitate into a round-bottom flask; repeating the above steps for 2-3 times of deoiling, dispersing the 3 times of deoiled centrifugal product with 5-10ml of acetone, centrifuging, discarding supernatant, and lyophilizing to obtain Free-UCNPs; mixing Free-UCNPs, tris-HCl buffer solution and hsDNA in solution for reaction, incubating and centrifuging to obtain UCNPs @ hsDNA, namely the fluorescence sensor.
4. The method of claim 3, wherein the HCl has a pH of 3.00 to 4.00.
5. The method of claim 3, wherein the Free-UCNPs solution is at a final concentration of 0.1 mg-mL -1 The final concentration of hsDNA is 20. Mu.g.mL -1 The concentration of Tris-HCl buffer was 10mM.
6. A UCNPs-based DNA fluorescence sensor, wherein the UCNPs-based DNA fluorescence sensor is prepared by the method of claims 1 to 5.
7. Use of the UCNPs-based DNA fluorescence sensor according to claim 6 for the detection of AQ anticancer drug concentration.
8. The method for detecting the concentration of the AQ anticancer drugs is characterized by comprising the following steps of:
incubating a sample to be detected with a buffer solution prepared from the fluorescence sensor of claim 6, and performing fluorescence detection.
9. The detection method according to claim 8, wherein the buffer solution is prepared using a Tris-HCl buffer solution at a concentration of 10mM and a free-UCNPs solution at a final concentration of 0.1 mg-mL -1 The final concentration of hsDNA is 20. Mu.g.mL -1 The final volume of the mixed solution is 500 mu L of UCNPs @ hsDNA solution, and the concentration range of the AQ anticancer drugs is 0-100 mu g/mL -1 。
10. Use of the UCNPs-based DNA fluorescence sensor of claim 6 and/or the detection method of any one of claims 8 to 9 for screening AQ anticancer drugs as anticancer drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211603274.0A CN115963093A (en) | 2022-12-13 | 2022-12-13 | DNA fluorescent sensor based on UCNPs and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211603274.0A CN115963093A (en) | 2022-12-13 | 2022-12-13 | DNA fluorescent sensor based on UCNPs and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115963093A true CN115963093A (en) | 2023-04-14 |
Family
ID=87360967
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211603274.0A Pending CN115963093A (en) | 2022-12-13 | 2022-12-13 | DNA fluorescent sensor based on UCNPs and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115963093A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5001051A (en) * | 1986-12-12 | 1991-03-19 | Regents Of The University Of California | Dose critical in-vivo detection of anti-cancer drug levels in blood |
| CN111053733A (en) * | 2019-12-02 | 2020-04-24 | 天津大学 | Up-conversion DNA nano gel, preparation method and application |
| CN111175266A (en) * | 2020-01-19 | 2020-05-19 | 厦门大学 | Construction method and detection method of near-infrared fluorescence biosensor |
-
2022
- 2022-12-13 CN CN202211603274.0A patent/CN115963093A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5001051A (en) * | 1986-12-12 | 1991-03-19 | Regents Of The University Of California | Dose critical in-vivo detection of anti-cancer drug levels in blood |
| CN111053733A (en) * | 2019-12-02 | 2020-04-24 | 天津大学 | Up-conversion DNA nano gel, preparation method and application |
| CN111175266A (en) * | 2020-01-19 | 2020-05-19 | 厦门大学 | Construction method and detection method of near-infrared fluorescence biosensor |
Non-Patent Citations (2)
| Title |
|---|
| PINGPING LI 等: "A sensitive sensor for anthraquinone anticancer drugs and hsDNA based on CdTe/CdS quantum dots fluorescence reversible control", COLLOIDS AND SURFACES A: PHYSICOCHEMICAL AND ENGINEERING ASPECTS, vol. 392, no. 1, pages 7 - 14 * |
| 黄丽姣: "基于上转换纳米材料的生物传感新方法", 湖南大学硕士学位论文 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Carbon-based dots for electrochemiluminescence sensing | |
| Ye et al. | Formation of N, S-codoped fluorescent carbon dots from biomass and their application for the selective detection of mercury and iron ion | |
| Dehghani et al. | Aptamer-based biosensors and nanosensors for the detection of vascular endothelial growth factor (VEGF): A review | |
| Zhang et al. | Application of multiplexed aptasensors in food contaminants detection | |
| Wu et al. | Ratiometric fluorescence, electrochemiluminescence, and photoelectrochemical chemo/biosensing based on semiconductor quantum dots | |
| Wang et al. | Nitrogen-doped graphene quantum dots@ SiO2 nanoparticles as electrochemiluminescence and fluorescence signal indicators for magnetically controlled aptasensor with dual detection channels | |
| Gorris et al. | Tuning the dual emission of photon‐upconverting nanoparticles for ratiometric multiplexed encoding | |
| Li et al. | DNA-templated silver nanocluster as a label-free fluorescent probe for the highly sensitive and selective detection of mercury ions | |
| Hu et al. | Nucleic acid-functionalized nanomaterials for bioimaging applications | |
| Zhang et al. | Engineering DNA on the surface of upconversion nanoparticles for bioanalysis and therapeutics | |
| KR100914455B1 (en) | Kits and methods for biological detection using quantum dots | |
| Du et al. | Quantum dot: Lightning invisible foodborne pathogens | |
| Chen et al. | Near-infrared to near-infrared upconverting NaYF4: Yb 3+, Tm 3+ nanoparticles-aptamer-Au nanorods light resonance energy transfer system for the detection of mercuric (II) ions in solution | |
| Leung et al. | A highly selective G-quadruplex-based luminescent switch-on probe for the detection of nanomolar strontium (II) ions in sea water | |
| Lin et al. | DNA functionalized gold nanoparticles for bioanalysis | |
| Gholivand et al. | DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives | |
| Yang et al. | Highly sensitive and selective detection of silver (I) in aqueous solution with silver (I)-specific DNA and Sybr green I | |
| Liu et al. | Construction of a molecular beacon based on two-photon excited fluorescence resonance energy transfer with quantum dot as donor | |
| Zhang et al. | Plasmonic modulation of the upconversion luminescence based on gold nanorods for designing a new strategy of sensing MicroRNAs | |
| Xie et al. | Stem-loop DNA-assisted silicon nanowires-based biochemical sensors with ultra-high sensitivity, specificity, and multiplexing capability | |
| Harvey et al. | Ratiometric detection of enzyme turnover and flavin reduction using rare-earth upconverting phosphors | |
| Shi et al. | Upconversion core/shell nanoparticles with lowered surface quenching for fluorescence detection of Hg 2+ ions | |
| Alexaki et al. | A DNA sensor based on upconversion nanoparticles and two-dimensional dichalcogenide materials | |
| Nasirian et al. | miRNA-21 rapid diagnosis by one-pot synthesis of highly luminescent red emissive silver nanoclusters/DNA | |
| Guo et al. | Exogenous interference and autofluorescence-free ratiometric aptasensor for detection of OTA based on dual-colored persistent luminescence nanoparticles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |