CN106610375A - Oxidized mesoporous carbon nanosphere aptasensor, and preparation method and application thereof - Google Patents
Oxidized mesoporous carbon nanosphere aptasensor, and preparation method and application thereof Download PDFInfo
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- CN106610375A CN106610375A CN201510698579.8A CN201510698579A CN106610375A CN 106610375 A CN106610375 A CN 106610375A CN 201510698579 A CN201510698579 A CN 201510698579A CN 106610375 A CN106610375 A CN 106610375A
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Abstract
The invention relates to an oxidized mesoporous carbon nanosphere aptasensor and a preparation method thereof, and application of the aptasensor to cancer detection, belonging to the field of analytical chemistry. According to the invention, an oxidized mesoporous carbon nanosphere (OMCN) is used as a carrier, and the surface of the OMCN is non-covalently connected with a Cy3-fluorescently-labeled single-stranded DNA probe labeled through Phi-Phi interaction, so the oxidized mesoporous carbon nanosphere aptasensor is prepared. The aptasensor based on OMCN has low detection limit and can be applied to detection of MUC1 molecules, tumor cells, in-vitro tumor tissue and in-vivo tumor tissue at different levels so as to ensure true and credible diagnosis. The invention provides the economical, practical and simple preparation method for the aptasensor based on OMCN, and the prepared aptasensor is applicable to multiple in-vivo and in-vitro diagnosis of tumors.
Description
Technical field
The invention belongs to analytical chemistry field, is related to aptamer sensor, and in particular to one kind oxidation mesoporous carbon nanometer
Ball aptamer sensor and preparation method thereof and its application in cancer detection.
Background technology
It is reported that, cancer incidence and fatality rate grow with each passing day, it has also become threaten the major disease of human health,
Therefore, effective diagnosing tumor technology is developed, including detection and imaging, it is very urgent.Current diagnosing tumor
In practice, mainly there is the different biochemistry detection of joint or an imaging technique, such as enzyme linked immunosorbent assay (ELISA),
Hematoxylin-eosin staining method (H＆E dyeing), CT technology (CT), magnetic
Resonance image-forming (MRI) etc., for the diagnosis of different tissues level, practice shows, described checkout and diagnosis skill
Art is complicated, and difficulty is high, and wherein most technologies lack specificity, false positive results easily occur.In recent years,
It is considered as that a kind of sensitivity is high, reliable lesion detection instrument based on the biosensor of fluorescent quenching-recovery,
But most biological sensors are easy to detect tumor markerses such as nucleic acid, polypeptide or the albumen of liquid tissue,
But rarely seen surveying in health check-up for in-vivo tumour tissue, therefore, the fluorescent quenching material of Development of Novel is prepared special
The high biosensor of the opposite sex, is the important research direction of the technical field for multifaceted lesion detection.
Known carbon nanomaterial, including one-dimensional CNT, two dimensional oxidation Graphene have specific function, such as
Interacted by non-covalent π-π between fluorescent labeling single stranded DNA produce excellent fluorescent quenching ability with
And the quantitative fluorescence recovery capability after target spot is exposed to, it has been developed and has been applied to fluorescence " Turn-on " biology
The preparation of sensor；But the main single detection for being still used for extracorporeal liquid tissue at present, wherein main reason is that
CNT or graphene oxide toxicity is larger, easy aggregation, functional group are primarily present in interface edge etc., more
Importantly, a described peacekeeping two-dimensional structure is unfavorable for uniformly being interacted with solid tumor tissue；In comparison,
Three-dimensional carbon nanomaterial, as mesoporous Nano carbon balls (MCN) are potential the one-dimensional, two-dimensional material is overcome
Defect, e.g., has even mesoporous in (1) three-dimensional spherical structure, specific surface area is big, and pore volume is big, easily logical
Cross surface functional group and interaction of biomacromolecules；(2) three-dimensional manometer ball, rotational energy is low, in vivo with
After solid tumor interacts, transducing signal is stable, sensitive；(3) MCN good biocompatibilities, at present
It is explored the pharmaceutical carrier as tumor thermal therapy.Therefore, MCN is a kind of potential can be used for
The fluorescence quencher of " Turn-on " bio-sensing, is expected to be used for many on the tumor tissues varying level of inside and outside
Re-detection.
Based on the present situation of prior art, present inventor intends providing a kind of new aptamer sensor, makes reality
The tumor multiple diagnostic of existing inside and outside.
The content of the invention
It is an object of the invention to provide a kind of aptamer sensor of efficient and sensible, and in particular to one kind oxidation is mesoporous
Nano carbon balls aptamer sensor and preparation method thereof.Described aptamer sensor is by specifically binding malignant tumor
The MUC1 of apparent height expression, realizes the tumor multiple diagnostic of inside and outside.
In the present invention, a kind of mesoporous Nano carbon balls (OMCN) of oxidation are synthesized, have been interacted by π-π
Fluorescently-labeled ssDNA probe (the P of its surface non-covalent linking Cy30It is fit), prepare aptamer sensor
(OMCN/P0- Cy3), by mark --- the cell table for specifically binding the expression of malignant tumor apparent height
Face MUC1 (MUC1), realizes the bio-sensing lesion detection of " Turn-on ".The new fit sensing
Device not only can MUC1 molecules and MCF-7 Breast Cancer Cell in highly sensitive quantitative liquid, can also realize swelling
The clear specificity imaging of oncocyte, in vitro tissue and solid tumor, realizes the tumor multiple diagnostic of inside and outside.
Specifically, the mesoporous Nano carbon balls aptamer sensor of oxidation of the present invention, to aoxidize mesoporous carbon nanometer
Ball OMCN is carrier, and surface is fluorescently-labeled single-stranded by a kind of Cy3 of π-π interaction non-covalent linking
DNA probe, makes the mesoporous Nano carbon balls aptamer sensor of oxidation.
The method that the present invention prepares the mesoporous Nano carbon balls aptamer sensor of oxidation, comprises the steps：Mesoporous carbon
Nanosphere is processed using strong acid mixture, ultrasound, stirring, centrifugation, and washing is dried, and obtains final product oxidation mesoporous carbon
Nanosphere OMCN.Appropriate OMCN and P0- Cy3 mixes, shaking table shaking, and OMCN/P is obtained0- Cy3 is fitted
Body sensor, stores for future use.
In above-mentioned preparation method, the strong acid mixture is the mixture of sulphuric acid and nitric acid when synthesizing OMCN,
Volume ratio is 2～4:1；
In said method, during synthesis OMCN, ultrasonic power is ordinary ultrasonic and Ultrasonic Cell Disruptor ultrasound；
In said method, during synthesis OMCN, ultrasonic time is 0.5～4h；
In said method, during synthesis OMCN, whipping temp is 30～70 DEG C；
In said method, during synthesis OMCN, mixing time is 0.5～4h；
In said method, OMCN/P is prepared0During-Cy3 aptamer sensors, OMCN and P0The consumption of-Cy3
Than for：30～780mg：100～300nmol；
In said method, OMCN/P is prepared0During-Cy3 aptamer sensors, OMCN and P0The mixing of-Cy3
Mode is shaking table shaking；
In said method, OMCN/P is prepared0During-Cy3 aptamer sensors, OMCN and P0The shaking of-Cy3
Mixing rate is 100～200rpm；
In said method, OMCN/P is prepared0During-Cy3 aptamer sensors, OMCN and P0The shaking of-Cy3
Incorporation time is 10～30min；
In said method, gained OMCN/P0The proper storage temperature of-Cy3 aptamer sensors is 4～25 DEG C.
The present invention uses OMCN/P0The solution system of-Cy3 aptamer sensors detection is Tris-HCl solution, contains
Plasma solutions and solution containing tumor cell.
The present invention uses OMCN/P0The destination object of-Cy3 aptamer sensors detection is MUC1 molecules, swells
Oncocyte, tumor tissue in vitro and live tumor tissue.
The present invention can be characterized by various methods to prepared OMCN, and characterizing method includes：Scanning
Electronic Speculum, transmission electron microscope, small angle X ray scattering (SAXS), X-ray diffraction (XRD) and X-ray light
Electron spectrum (XPS).
The present invention investigates OMCN/P using fluorescence spectrophotometer0The range of linearity of-Cy3 aptamer sensors and detection
Limit.OMCN/P0Respectively (Tris-HCl is molten with the different solutions system containing MUC1 for-Cy3 aptamer sensors
Liquid or the Tris-HCl solution containing 4% blood plasma) or Hank ' the s buffer solution mixing containing MCF-7 tumor cells,
Incubation 10min, using fluorescence spectrophotometer solution fluorescence is detected, investigates the range of linearity and test limit.As a result show,
OMCN/P prepared by the present invention0- Cy3 aptamer sensors are in Tris-HCl solution, containing 4% blood plasma
The range of linearity in Tris-HCl solution and Hank ' the s buffer solution containing MCF-7 tumor cells is respectively 1.06-
10.6 μM, 1.06-10.6 μM and 105–2×106Individual cell/mL, test limit be respectively 67.8nM, 49.5
NM and 9.3 × 104Individual cell/mL.
The present invention further investigates OMCN/P using Infinite M1000Pro microplate reader0Sensing that-Cy3 is fit
The range of linearity and test limit of device.OMCN/P0- Cy3 aptamer sensors respectively from it is different molten containing MUC1
Liquid system (Tris-HCl solution or the Tris-HCl solution containing 4% blood plasma) or containing MCF-7 tumor cells
Hank ' s buffer solution mixes, and is incubated 10min, glimmering using Infinite M1000 Pro microplate reader detection solution
Light, investigates the range of linearity and test limit.As a result show, the OMCN/P prepared by the present invention0Biography that-Cy3 is fit
Sensor is in Tris-HCl solution, the Tris-HCl solution containing 4% blood plasma and the Hank ' s containing MCF-7 tumor cells
The range of linearity in buffer solution is respectively 0.10-2.12 μM, 0.10-2.12 μM and 104–2×105It is individual
Cell/mL, test limit is respectively 7.05nM, 6.52nM and 8500 cell/mL.
The present invention investigates OMCN/P using Laser Scanning Confocal Microscope0Choosing of-Cy3 the aptamer sensors to tumor cell
Select specificity.OMCN/P0- Cy3 aptamer sensors respectively with MCF-7 Human Breast Cancer Cells and breast epithelium
MCF-10A cell incubation 0min, 5min, 30min and 60min, PBS drip washing, Laser Scanning Confocal Microscope
Take pictures observation.As a result show, the red fluorescence on MCF-7 breast cancer cells gradually strengthens over time, and
Breast epithelium MCF-10A cells almost unstressed configuration, shows the OMCN/P prepared by the present invention0- Cy3 is fit
Sensor has extraordinary selection specificly-response to breast cancer cell.
The present invention observes OMCN/P using inverted fluorescence microscope0- Cy3 aptamer sensors are in Ex vivo Tumor group
Knit the response effect of section.Female Balb/c nude mice by subcutaneous is inoculated with MCF-7 cells, treats that tumor volume increases to 200
mm3Can use.Mice with tumor is put to death, and takes tumor, frozen section, partially sliced and OMCN/P0Biography that-Cy3 is fit
Sensor is incubated 1h, PBS drip washing, inverted fluorescence microscope observation section.As a result show, Jing OMCN/P0-Cy3
The tumor biopsy of aptamer sensor process can be observed red fluorescence, and undressed tumor biopsy is not glimmering
Light, shows the OMCN/P prepared by the present invention0- Cy3 aptamer sensors have highly effective to tumor tissue in vitro
Response.
The present invention observes OMCN/P using living imaging system0Sound of-Cy3 the aptamer sensors to in-vivo tumour
Answer effect.Female Balb/c nude mice by subcutaneous is inoculated with MCF-7 cells, treats that tumor volume increases to 200mm3Can use.
Normal mice and mice with tumor difference subcutaneous injection or intratumor injection P0- Cy3 solution and OMCN/P0Sensing that-Cy3 is fit
Device, respectively at 0min, 5min, 15min, 30min and 60min Maestro-2 living imagings system is used
Overall view examines nude mice.As a result show, mice with tumor intratumor injection P0- Cy3 solution, fluorescence signal is very weak, lotus knurl
Mus intratumor injection OMCN/P0- Cy3 aptamer sensors, fluorescence signal first strengthens weaken afterwards as time went on,
Normal sub-cutaneous injections P0- Cy3 solution, fluorescence signal weakens as time went on, normal sub-cutaneous injections
OMCN/P0- Cy3 aptamer sensors, are not detected by fluorescence signal, show prepared by the present invention
OMCN/P0- Cy3 aptamer sensors have very effective response to live tumor tissue.
Table 1 shows OMCN/P0- Cy3 aptamer sensors detect the linear model of MUC1 or MCF-7 cells
Enclose and test limit.
The present invention outstanding advantages and be characterised by, there is provided a kind of efficient and sensible based on OMCN it is fit
Sensor, by the MUC1 for specifically binding the expression of malignant tumor apparent height, is capable of achieving the tumor of inside and outside
Multiple diagnostic.The aptamer sensor based on OMCN prepared by the present invention, with existing based on graphite oxide
The aptamer sensor of alkene compares, and test limit is substantially reduced, and can be applicable to MUC1 molecules, tumor cell,
Detection in tumor tissue in vitro and live tumor tissue varying level, it is ensured that diagnosis is true and reliable.
The invention provides a kind of economical and practical simple aptamer sensor and its preparation side based on OMCN
Method, the aptamer sensor can realize the tumor multiple diagnostic of inside and outside.
Description of the drawings
The physical characterization result of Fig. 1 OMCN.
Wherein, A：Scanning electron microscope (SEM) photograph；
B：Transmission electron microscope picture (insertion figure：The transmission electron microscope picture of single nanosphere)；
C：Small angle X ray scattering figure (insertion figure：X-ray diffractogram)；
D：X-ray photoelectron energy spectrum diagram (insertion figure：Corresponding C 1s figures).
Fig. 2 confocal microscopies investigate OMCN/P0- Cy3 aptamer sensors are to MCF-7 tumor cells
Intake situation,
Wherein, A：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors incubation 0min (200 times of amplifications,
Bar=50 μm)；
B：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors incubation 0min (900 times of amplifications,
Bar=10 μm)；
C：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors are incubated 0min (2.5D tangent planes
Figure)；
D：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors incubation 5min (200 times of amplifications,
Bar=50 μm)；
E：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors incubation 5min (900 times of amplifications,
Bar=10 μm)；
F：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors incubation 5min (2.5D sectional drawings)；
G：MCF-7 cells and OMCN/P0(200 times put-Cy3 aptamer sensors incubation 30min
Greatly, bar=50 μm)；
H：MCF-7 cells and OMCN/P0(900 times put-Cy3 aptamer sensors incubation 30min
Greatly, bar=10 μm)；
I：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors are incubated 30min (2.5D tangent planes
Figure)；
J：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors incubation 60min (200 times of amplifications,
Bar=50 μm)；
K：MCF-7 cells and OMCN/P0(900 times put-Cy3 aptamer sensors incubation 60min
Greatly, bar=10 μm)；
L：MCF-7 cells and OMCN/P0- Cy3 aptamer sensors are incubated 60min (2.5D tangent planes
Figure).
Fig. 3 confocal microscopies investigate OMCN/P0- Cy3 aptamer sensors are to normal breast epithelial
The intake situation of MCF-10A cells,
Wherein, A：MCF-10A cells and OMCN/P0(200 times put-Cy3 aptamer sensors incubation 0min
Greatly, bar=50 μm)；
B：MCF-10A cells and OMCN/P0(900 times put-Cy3 aptamer sensors incubation 0min
Greatly, bar=10 μm)；
C：MCF-10A cells and OMCN/P0(2.5D cuts-Cy3 aptamer sensors incubation 0min
Face figure)；
D：MCF-10A cells and OMCN/P0(200 times put-Cy3 aptamer sensors incubation 5min
Greatly, bar=50 μm)；
E：MCF-10A cells and OMCN/P0(900 times put-Cy3 aptamer sensors incubation 5min
Greatly, bar=10 μm)；
F：MCF-10A cells and OMCN/P0(2.5D cuts-Cy3 aptamer sensors incubation 5min
Face figure)；
G：MCF-10A cells and OMCN/P0- Cy3 aptamer sensors are incubated (200 times of 30min
Amplify, bar=50 μm)；
H：MCF-10A cells and OMCN/P0- Cy3 aptamer sensors are incubated (900 times of 30min
Amplify, bar=10 μm)；
I：MCF-10A cells and OMCN/P0(2.5D cuts-Cy3 aptamer sensors incubation 30min
Face figure)；
J：MCF-10A cells and OMCN/P0- Cy3 aptamer sensors are incubated (200 times of 60min
Amplify, bar=50 μm)；
K：MCF-10A cells and OMCN/P0- Cy3 aptamer sensors are incubated (900 times of 60min
Amplify, bar=10 μm)；
L：MCF-10A cells and OMCN/P0(2.5D cuts-Cy3 aptamer sensors incubation 60min
Face figure).
Fig. 4 inverted fluorescence microscopes observe OMCN/P0- Cy3 aptamer sensors are cut into slices in tumor tissue in vitro
Response effect,
Wherein, A：Tumor tissue in vitro is cut into slices without OMCN/P0The light field figure of-Cy3 aptamer sensors process
(bar=200 μm)；
B：Tumor tissue in vitro is cut into slices without OMCN/P0The fluorogram of-Cy3 aptamer sensors process
(bar=200 μm)；
C：Tumor tissue in vitro section Jing OMCN/P0- Cy3 aptamer sensors process the light field figure of 1h
(bar=200 μm)；
B：Tumor tissue in vitro section Jing OMCN/P0- Cy3 aptamer sensors process the fluorogram of 1h
(bar=200 μm).
Fig. 5 living imaging systems observe OMCN/P0The response effect of-Cy3 aptamer sensors to in-vivo tumour,
Wherein, I groups：Lotus knurl sub-cutaneous injections or intratumor injection P0- Cy3 solution；
II groups：Mice with tumor intratumor injection OMCN/P0- Cy3 aptamer sensors；
III groups：Normal sub-cutaneous injections P0- Cy3 solution；
IV groups：Normal sub-cutaneous injections OMCN/P0- Cy3 aptamer sensors；
A：Nude mice observes result Jing after different disposal 0min using Maestro-2 living imaging systems；
B：Nude mice observes result Jing after different disposal 5min using Maestro-2 living imaging systems；
C：Nude mice observes result Jing after different disposal 15min using Maestro-2 living imaging systems；
D：Nude mice observes result Jing after different disposal 30min using Maestro-2 living imaging systems；
E：Nude mice observes result Jing after different disposal 60min using Maestro-2 living imaging systems.
Specific embodiment
Embodiment 1.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 2 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 2.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 3.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 4 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 4.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 0.5
H, 50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN
(300 μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product
OMCN/P0- Cy3 aptamer sensors, 4 DEG C store for future use.
Embodiment 5.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 4h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 6.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
30 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 7.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
70 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 8.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
30 DEG C of stirring 0.5h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN
(300 μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product
OMCN/P0- Cy3 aptamer sensors, 4 DEG C store for future use.
Embodiment 9.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
30 DEG C of stirring 4h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 10.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ultrasonication 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 11.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(30
μ g/mL) and P0- Cy3 (100nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 12.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(780
μ g/mL) and P0- Cy3 (300nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 13.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 100rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 14.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 200rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 15.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 20min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 16.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 30min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 4 DEG C store for future use.
Embodiment 17.
Using the mixture of sulphuric acid and nitric acid, (volume ratio is 3 to mesoporous Nano carbon balls:1) process, ordinary ultrasonic 2h,
50 DEG C of stirring 2h, centrifugation, washing is dried, and obtains final product the mesoporous Nano carbon balls OMCN of oxidation.OMCN(300
μ g/mL) and P0- Cy3 (200nmol/L) mixes, 160rpm shaking tables shaking 10min, obtains final product OMCN/P0-Cy3
Aptamer sensor, 25 DEG C store for future use.
Embodiment 18.
The OMCN prepared by Philips XL-30 scanning electron microscopic observations embodiment 2, as a result shows the OMCN
Spherical in rounding with homogeneous size, particle diameter asks for an interview accompanying drawing 1A in 100nm or so.
Embodiment 19.
The OMCN prepared by JEOL-2100F transmission electron microscope observings embodiment 2, as a result shows the OMCN
Good dispersion, with order mesoporous, aperture is about 3nm, asks for an interview accompanying drawing 1B.
Embodiment 20.
OMCN prepared by embodiment 2 carries out small angle X ray scattering (SAXS) test and X-ray is more
Brilliant diffraction (XRD) analysis, as a result shows that the OMCN has body-centered cubic Im3m of high-sequential mesoporous
Structure, its skeleton has graphitized carbon region, asks for an interview accompanying drawing 1C.
Embodiment 21.
OMCN prepared by embodiment 2 carries out x-ray photoelectron energy-spectrum scanning, as a result shows the OMCN
It is elementary composition for 74.6at%C and 24.8at%O, its structure composition be graphitized carbon (C=C/C-C) and
The carbon (C-O/C=O) of oxidation, asks for an interview accompanying drawing 1D.
Embodiment 22.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors are molten with the Tris-HCl containing MUC1
Liquid mixes, and is incubated 10min, and using fluorescence spectrophotometer solution fluorescence is detected, investigates the range of linearity, as a result shows
(as shown in table 1), prepared OMCN/P0- Cy3 aptamer sensors are linear in Tris-HCl solution
Scope is 1.06-10.6 μM,
Table 1
Embodiment 23.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors with containing MUC1 containing 4% blood plasma
Tris-HCl solution mixes, and is incubated 10min, and using fluorescence spectrophotometer solution fluorescence is detected, investigates the range of linearity.
As a result (as shown in table 1) is shown, prepared OMCN/P0- Cy3 aptamer sensors are containing 4% blood plasma
The range of linearity in Tris-HCl solution is 1.06-10.6 μM.
Embodiment 24.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors and the Hank ' s containing MCF-7 tumor cells
Buffer solution mixes, and is incubated 10min, and using fluorescence spectrophotometer solution fluorescence is detected, investigates the range of linearity, knot
Fruit shows (as shown in table 1), prepared OMCN/P0- Cy3 aptamer sensors are thin in tumor containing MCF-7
The range of linearity in Hank ' the s buffer solution of born of the same parents is 105–2×106Individual cell/mL.
Embodiment 25.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors are molten with the Tris-HCl containing MUC1
Liquid mixes, and is incubated 10min, and using Infinite M1000Pro microplate reader solution fluorescence is detected, investigates linear
Scope, as a result shows (as shown in table 1), prepared OMCN/P0- Cy3 aptamer sensors are in Tris-HCl
The range of linearity in solution is 0.10-2.12 μM.
Embodiment 26.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors with containing MUC1 containing 4% blood plasma
Tris-HCl solution mixes, and is incubated 10min, and using Infinite M1000Pro microplate reader solution fluorescence is detected,
The range of linearity is investigated, as a result (as shown in table 1) is shown, prepared OMCN/P0- Cy3 aptamer sensors
The range of linearity in the Tris-HCl solution containing 4% blood plasma is 0.10-2.12 μM.
Embodiment 27.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors and the Hank ' s containing MCF-7 tumor cells
Buffer solution mixes, and is incubated 10min, and using Infinite M1000Pro microplate reader solution fluorescence is detected, examines
The range of linearity is examined, as a result (as shown in table 1) is shown, prepared OMCN/P0- Cy3 aptamer sensors exist
The range of linearity in Hank ' s buffer solution containing MCF-7 tumor cells is 104–2×105Individual cell/mL.
Embodiment 28.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors are molten with the Tris-HCl containing MUC1
Liquid mixes, and is incubated 10min, and using fluorescence spectrophotometer solution fluorescence is detected, investigates test limit, as a result shows (such as
Shown in table 1), prepared OMCN/P0Detection of-Cy3 the aptamer sensors in Tris-HCl solution is limited to
67.8nM。
Embodiment 29.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors with containing MUC1 containing 4% blood plasma
Tris-HCl solution mixes, and is incubated 10min, and using fluorescence spectrophotometer solution fluorescence is detected, investigates test limit,
As a result (as shown in table 1) is shown, prepared OMCN/P0- Cy3 aptamer sensors are containing 4% blood plasma
Detection in Tris-HCl solution is limited to 49.5nM.
Embodiment 30.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors and the Hank ' s containing MCF-7 tumor cells
Buffer solution mixes, and is incubated 10min, and using fluorescence spectrophotometer solution fluorescence is detected, investigates test limit, as a result
Show (as shown in table 1), prepared OMCN/P0- Cy3 aptamer sensors are in tumor cell containing MCF-7
Hank ' s buffer solution in detection be limited to 9.3 × 104Individual cell/mL.
Embodiment 31.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors are molten with the Tris-HCl containing MUC1
Liquid mixes, and is incubated 10min, and using Infinite M1000Pro microplate reader solution fluorescence is detected, investigates detection
Limit, as a result shows (as shown in table 1), prepared OMCN/P0- Cy3 aptamer sensors are in Tris-HCl
Detection in solution is limited to 7.05nM1.
Embodiment 32.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors with containing MUC1 containing 4% blood plasma
Tris-HCl solution mixes, and is incubated 10min, and using Infinite M1000Pro microplate reader solution fluorescence is detected,
Test limit is investigated, as a result (as shown in table 1) is shown, prepared OMCN/P0- Cy3 aptamer sensors exist
Detection in Tris-HCl solution containing 4% blood plasma is limited to 6.52nM.
Embodiment 33.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors and the Hank ' s containing MCF-7 tumor cells
Buffer solution mixes, and is incubated 10min, and using Infinite M1000Pro microplate reader solution fluorescence is detected, examines
Test limit is examined, as a result (as shown in table 1) is shown, prepared OMCN/P0- Cy3 aptamer sensors are containing
Detection in Hank ' the s buffer solution of MCF-7 tumor cells is limited to 8500 cell/mL.
Embodiment 34.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors and MCF-7 Human Breast Cancer Cells incubation 0
Min, 5min, 30min and 60min, PBS drip washing, Laser Scanning Confocal Microscope is taken pictures observation, is as a result shown,
Red fluorescence on MCF-7 breast cancer cells gradually strengthens over time, shows prepared
OMCN/P0- Cy3 aptamer sensors have extraordinary response to breast cancer cell, ask for an interview accompanying drawing 2.
Embodiment 35.
OMCN/P prepared by embodiment 20- Cy3 aptamer sensors are incubated with breast epithelium MCF-10A cells
Educate 0min, 5min, 30min and 60min, PBS drip washing, Laser Scanning Confocal Microscope is taken pictures observation.As a result show
Show that breast epithelium MCF-10A cells almost unstressed configuration shows prepared OMCN/P0Biography that-Cy3 is fit
Sensor, without response, asks for an interview accompanying drawing 3 to breast epithelium MCF-10A cells.
Embodiment 36.
Female Balb/c nude mice by subcutaneous is inoculated with MCF-7 cells, treats that tumor volume increases to 200mm3Can use.Lotus
Tumor Mus are put to death, and take tumor, frozen section, the partially sliced OMCN/P prepared with embodiment 20- Cy3 is fit
Sensor is incubated 1h, and as a result PBS drip washing, inverted fluorescence microscope observation section shows, Jing OMCN/P0-Cy3
The tumor biopsy of aptamer sensor process can be observed red fluorescence, and undressed tumor biopsy is not glimmering
Light, shows the OMCN/P prepared by the present invention0- Cy3 aptamer sensors have extraordinary to tumor tissue in vitro
Response, asks for an interview accompanying drawing 4.
Embodiment 37.
Female Balb/c nude mice by subcutaneous is inoculated with MCF-7 cells, treats that tumor volume increases to 200mm3Can use.Just
Often Mus and mice with tumor distinguish subcutaneous injection or intratumor injection P0Prepared by-Cy3 solution and embodiment 2
OMCN/P0- Cy3 aptamer sensors, make respectively at 0min, 5min, 15min, 30min and 60min
Nude mice is observed with Maestro-2 living imaging systems, is as a result shown, mice with tumor intratumor injection P0- Cy3 solution,
Fluorescence signal is very weak, mice with tumor intratumor injection OMCN/P0- Cy3 aptamer sensors, fluorescence signal with when
Between extend first to strengthen and weaken afterwards, normal sub-cutaneous injections P0- Cy3 solution, fluorescence signal subtracts as time went on
It is weak, normal sub-cutaneous injections OMCN/P0- Cy3 aptamer sensors, are not detected by fluorescence signal, show this
Bright prepared OMCN/P0- Cy3 aptamer sensors have extraordinary response to live tumor tissue, ask for an interview attached
Fig. 5.
Claims (10)
1. it is a kind of to aoxidize mesoporous Nano carbon balls aptamer sensor, it is characterised in that to aoxidize mesoporous Nano carbon balls
OMCN is carrier, and surface passes through the fluorescently-labeled single stranded DNA of π-π interaction non-covalent linking Cy3
Probe, makes the mesoporous Nano carbon balls aptamer sensor of oxidation.
2. the preparation method of the mesoporous Nano carbon balls aptamer sensor of oxidation described in claim 1, it is characterised in that
It includes：Mesoporous Nano carbon balls are processed using strong acid mixture, ultrasound, stirring, centrifugation, washing,
It is dried, mesoporous Nano carbon balls OMCN must be aoxidized；Appropriate OMCN and P0- Cy3 mixes, shaking table shaking,
Obtain OMCN/P0- Cy3 aptamer sensors, store for future use.
3. the preparation method of the mesoporous Nano carbon balls aptamer sensor of oxidation as described in claim 2, it is characterised in that：
The mesoporous Nano carbon balls aptamer sensor of described oxidation is OMCN/P0- Cy3 aptamer sensors, wherein,
For sulphuric acid and the mixture of nitric acid, volume ratio is 2～4 to the strong acid mixture adopted in synthesis OMCN:1.
4. the preparation method of the mesoporous Nano carbon balls aptamer sensor of oxidation as described in claim 2, it is characterised in that：
In synthesis OMCN, using ordinary ultrasonic or Ultrasonic Cell Disruptor ultrasound.
5. the preparation method of the mesoporous Nano carbon balls aptamer sensor of oxidation as described in claim 4, it is characterised in that
In synthesis OMCN, ultrasonic time is 0.5～4h, and whipping temp is 30～70 DEG C, and mixing time is
0.5～4h.
6. the preparation method of the mesoporous Nano carbon balls aptamer sensor of oxidation as described in claim 2, it is characterised in that
OMCN and P0- Cy3 mixing amount ratio be：30～780mg：100～300nmol.
7. the preparation method of the mesoporous Nano carbon balls aptamer sensor of oxidation as described in claim 2, it is characterised in that
OMCN and P0The hybrid mode of-Cy3 is shaking table shaking, and shaking mixing rate is 100～200rpm,
Shaking incorporation time is 10～30min.
8. the preparation method of the mesoporous Nano carbon balls aptamer sensor of oxidation as described in claim 2, it is characterised in that
Described OMCN/P0- Cy3 storage temperatures are 4～25 DEG C.
9. the OMCN/P that preparation method described in claim 1-9 is prepared0- Cy3 aptamer sensors, can be used for
The solution system of detection be Tris-HCl solution, containing plasma solutions and solution containing tumor cell.
10. the OMCN/P described in claim 10- Cy3 aptamer sensors are being used to detect MUC1 molecules, tumor
Purposes in cell, tumor tissue in vitro or live tumor tissue destination object.
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Application publication date: 20170503 |