CN113237855B - Cancer cell uptake monitoring method of biosensor based on quantum dots - Google Patents
Cancer cell uptake monitoring method of biosensor based on quantum dots Download PDFInfo
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- CN113237855B CN113237855B CN202110477581.8A CN202110477581A CN113237855B CN 113237855 B CN113237855 B CN 113237855B CN 202110477581 A CN202110477581 A CN 202110477581A CN 113237855 B CN113237855 B CN 113237855B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N2021/6417—Spectrofluorimetric devices
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a biosensor preparation and cancer cell uptake monitoring method based on quantum dots. The method specifically comprises the following steps: a) Carrying out specific targeted modification on the quantum dots; b) Doping the treated quantum dots into a hydrogel precursor solution in a chemical bonding mode; c) And manufacturing the hydrogel biosensor based on the quantum dots by using a die. The fluorescence intensity of the specific targeted modified quantum dot has different change rates in different stages of cancer cell uptake (adsorption, "endocytosis") and good linearity in each stage, so that different stages of the cancer cell uptake process can be monitored and distinguished in real time; meanwhile, the manufactured hydrogel biosensor has good mechanical properties, and can be suitable for monitoring under complex environments such as stretching, bending, extrusion and the like; in addition, the method can also realize the analysis and detection of different cells and other biochemical indexes by carrying out different targeted modification on the quantum dots.
Description
Technical Field
The invention relates to the technical field of biological sensing, in particular to a method for preparing a biological sensor based on quantum dots and monitoring cancer cell uptake.
Background
Malignant tumors with rapid progress, easy recurrence and low clinical cure rate have great harm to patients. Active and effective cancer diagnosis has become a research hotspot in the field of cancer therapy. Surgery is a traditional method of cancer treatment. Unfortunately, the prognosis of surgery is not ideal. Because cancer cells are not, in most cases, completely found and resected by surgery, they often result in recurrence and metastasis.
The development of antitumor drugs makes chemotherapy an effective method for cancer treatment, wherein molecular targeting drugs are the main direction of development of antitumor drugs. Among them, the exploration of the kinetics of drug absorption by cells is a key to promoting the development of new drugs. Therefore, the development of new technologies that can specifically recognize and monitor the real-time uptake process for cancer cells is of great interest for cancer treatment.
The current observation methods for cancer cells mainly comprise the following steps: the first is cell imaging technology; the second is a conventional fiber optic biosensor. The cell imaging technology is high in instrument price, complex in operation and incapable of being monitored in real time; the traditional optical fiber biosensor has the defects of very fragile sensing probe and poor biocompatibility, and cannot meet the requirements of practical application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention discloses a biosensor preparation and cancer cell uptake monitoring method based on quantum dots, which can solve the defects of the prior art.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the biosensor preparation and cancer cell uptake monitoring method based on quantum dots comprises the following steps of
a) Weighing folic acid, acrylic acid, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) with specified mass, pouring into Phosphate Buffer Saline (PBS) with pH equal to about 6, and stirring for specified time under the condition of light shielding;
b) C, pouring the quantum dots into the solution generated in the step a, and continuing to stir for a specified time in a dark place;
c) Dialyzing the solution generated in the step b for a designated time in deionized water under the dark condition;
d) Weighing acrylamide, sodium polyacrylate, the solution generated in the step c, a cross-linking agent and a thermal initiator with specified mass, pouring the mixture into deionized water, and uniformly stirring;
e) Injecting the solution generated in the step d into a tubular rubber mold, and placing a traditional optical fiber with a flat end face cut into one end of the rubber mold;
f) Placing the optical fiber generated in the step e into a drying box, setting a proper temperature, heating and initiating the crosslinking of the hydrogel monomer;
g) And f, pushing out the hydrogel from one end of the rubber mold by external force, and completing the manufacturing of the quantum dot hydrogel biosensor.
In the preferred technical scheme, in the step a, the proportion of folic acid, acrylic acid, EDC and NHS is 0.1-1mol:0.1-1mol:1-2mol:1-2mol, and stirring time is 6-12 h.
In the preferred technical scheme, in the step b, the quantum dot material is at least one of BaS, agInS2, naCl, fe2O3, in2O3, inAs, inN, inP, cdS, cdSe, cdTe, znS, znSe, znTe, gaAs, gaN, gaS, gaSe, inGaAs, mgS, mgSe, mgTe, pbS, pbSe, pbTe, cd (SxSe 1-x), baTiO3, pbZrO3, csPbCl3, csPbBr3, csPbI 3.
In the preferred technical scheme, in the step d, the cross-linking agent is at least one of N-N methylene bisacrylamide and polyethylene glycol diacrylate PEGDA, and the thermal initiator is ammonium persulfate.
In the preferred technical scheme, in the step e, the rubber mold is a hollow conduit with an inner diameter of 0.1mm-1mm, and the conventional optical fiber is a communication or sensing optical fiber such as a single-mode optical fiber, a multimode optical fiber and the like.
In the preferred technical scheme, in the step f, the temperature is set to be 40-100 ℃ and the heating time is set to be 30-60 min.
The invention discloses a biosensor preparation and cancer cell uptake monitoring method based on quantum dots, which has the following advantages:
the instinct method has simple manufacturing method and good biocompatibility.
1. The sensor is simple to manufacture, has good biocompatibility and can monitor the ingestion process of cancer cells in real time;
2. the hydrogel biosensor based on the quantum dots, which is prepared by the invention, has good mechanical properties and can be suitable for detection of complex environments such as stretching, bending, extrusion and the like;
3. according to the invention, through carrying out different targeted modification on the quantum dots, detection of other cells and biochemical indexes can be realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
It is evident that the drawings in the following description are only some embodiments of the present invention and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a schematic diagram of a hydrogel biosensor based on quantum dots according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a hydrogel biosensing system based on quantum dots according to an embodiment of the invention;
FIG. 3 is a graph of fluorescence spectrum of a hydrogel biosensor based on quantum dots according to an embodiment of the present invention;
FIG. 4 is a graph showing the tensile property test results of a hydrogel biosensor based on quantum dots according to an embodiment of the present invention;
FIG. 5 is a graph of MCF-7 results of a quantum dot based hydrogel biosensor test according to an embodiment of the present invention;
fig. 6 is a graph of the intensity linear fit results for an embodiment of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments.
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1 to 6, the method for preparing a biosensor and monitoring cancer cell uptake based on quantum dots according to the embodiment of the invention specifically comprises the following steps:
a) Weighing the molar mass ratio of 1:1:1.5:1.5 folic acid, acrylic acid, EDC, NHS are poured into phosphate buffer saline (ph=6.4) and stirred for 12h under the condition of avoiding light;
b) Taking 500 mu l of quantum dots (CdSe/ZnS) with the concentration of 1 mu M, pouring the solution into the solution a), and continuously stirring the solution for 24 hours in a dark place;
c) Dialyzing the solution b) in deionized water for 36h under the dark condition;
d) Weighing the mass ratio of 10:20:0.05:0.004:0.02 of acrylamide, acrylic acid, sodium polyacrylate, N-N methylene bisacrylamide, ammonium persulfate and c) solution, pouring the solution into deionized water, and uniformly stirring;
e) Injecting the solution d) into a hollow tubular rubber mold of ammonium persulfate with the inner diameter of 0.15mm, and placing a traditional multimode optical fiber with a flat end face cut into one end of the rubber mold;
f) Placing the e) into a drying box, setting the temperature to 80 ℃, heating for 30min, and initiating the hydrogel monomer to crosslink;
g) Pushing out the hydrogel in the f) from one end of a rubber mold through external force to prepare the quantum dot hydrogel biosensor;
h) Using an LED with a center wavelength of 475nm as an excitation light source, and using a marine optical spectrometer Pro65E as a signal detection device; the excitation light source, the spectrometer and the prepared hydrogel optical fiber sensor are connected through a wavelength division multiplexer (WDM-475 nm/630 nm) to prepare the hydrogel biosensor based on quantum dots, and the real-time monitoring of the cancer cell uptake process is realized.
Performance test of hydrogel biosensors based on quantum dots:
LED excitation light (center wavelength 475 nm) is LED into the quantum dot hydrogel biosensor through a wavelength division multiplexer WDM, and the corresponding other end is connected with a spectrometer; as can be seen from fig. 3, excitation light is coupled into the quantum dot hydrogel biosensor through WDM, and high-intensity quantum dot radiative fluorescence is detected through a spectrometer at the other end of WDM, with a center wavelength of 631nm.
Mechanical performance test of quantum dot hydrogel biosensor:
the mechanical properties of the quantum dot hydrogel biosensor were tested by stretching the quantum dot hydrogel biosensor, and as can be seen from fig. 4, the elongation of the quantum dot hydrogel biosensor can reach 1650% of the original length, and extremely high toughness is exhibited.
Sensing characteristic test of quantum dot hydrogel biosensor for real-time monitoring of cancer cell uptake process:
inserting the quantum dot hydrogel biosensor into PBS solution containing human breast cancer cells (MCF-7), introducing excitation light into the quantum dot hydrogel biosensor, and receiving a feedback signal through a spectrometer; the results of the feedback fluorescence spectrum test are shown in fig. 5, and the fluorescence intensity is enhanced with time. Fig. 6 shows the result of linear fitting of fluorescence intensity, and the fluorescence intensity of the quantum dot hydrogel biosensor of the present invention has good linearity of response to different stages of the uptake process (adsorption, "endocytosis") of breast cancer cells, and can identify cancer cells according to the linearity, and the occurrence of slope change in the graph can be applied to monitoring the progress of the "endocytosis" effect.
The fluorescence intensity of the specific targeted modified quantum dot has different change rates in different stages of cancer cell uptake (adsorption, "endocytosis") and good linearity in each stage, so that different stages of the cancer cell uptake process can be monitored and distinguished in real time; meanwhile, the manufactured hydrogel biosensor has good mechanical properties, and can be suitable for monitoring under complex environments such as stretching, bending, extrusion and the like; in addition, the method can realize analysis and detection of different cells and other biochemical indexes by carrying out different targeted modification on the quantum dots, and has wide application prospects in the research fields of biosensing, disease diagnosis, pharmacological analysis and the like.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. The method for monitoring the cancer cell uptake of the biosensor based on the quantum dots is characterized by comprising the following steps of: comprises the following steps
Step a), weighing folic acid, acrylic acid, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) with specified mass, pouring the folic acid, the acrylic acid, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and the N-hydroxysuccinimide into Phosphate Buffer Saline (PBS) with pH of about 6, and stirring the mixture for specified time under the condition of light shielding;
in the step a, the proportion of folic acid, acrylic acid, EDC and NHS is 0.1-1mol:0.1-1mol:1-2mol:1-2mol, and stirring time is 6-12 h;
step b), pouring the quantum dots into the solution generated in the step a, and continuing to stir for a specified time in a dark place;
step c), dialyzing the solution generated in the step b for a specified time in deionized water under the dark condition;
step d), weighing acrylamide, sodium polyacrylate, the solution generated in the step c, the cross-linking agent and the thermal initiator with specified mass, and pouring the solution into deionized water to be stirred uniformly;
step e), injecting the solution generated in the step d into a tubular rubber mold, and placing a traditional optical fiber with a flat end face cut into one end of the rubber mold;
step f) placing the optical fiber generated in the step e into a drying box, setting a proper temperature, heating, and initiating the crosslinking of the hydrogel monomer;
step g), pushing out the hydrogel in the step f from one end of the rubber mold through external force, and completing the manufacturing of the quantum dot hydrogel biosensor;
step h) using an LED with a center wavelength of 475nm as an excitation light source and using a spectrometer as a signal detection device; the excitation light source, the spectrometer and the prepared hydrogel optical fiber sensor are connected through the wavelength division multiplexer to prepare the hydrogel biosensor based on the quantum dots, and the real-time monitoring of the cancer cell uptake process is realized.
2. The method for monitoring the uptake of cancer cells by a quantum dot-based biosensor according to claim 1, wherein: in the step b, the quantum dot material is at least one of BaS, agInS2, fe2O3, in2O3, inAs, inN, inP, cdS, cdSe, cdTe, znS, znSe, znTe, gaAs, gaN, gaS, gaSe, inGaAs, mgS, mgSe, mgTe, pbS, pbSe, pbTe, cd (SxSe 1-x), baTiO3, pbZrO3, csPbCl3, csPbBr3, csPbI 3.
3. The method for monitoring the uptake of cancer cells by a quantum dot-based biosensor according to claim 1, wherein: in the step d, the cross-linking agent is at least one of N-N methylene bisacrylamide and polyethylene glycol diacrylate PEGDA, and the thermal initiator is ammonium persulfate.
4. The method for monitoring the uptake of cancer cells by a quantum dot-based biosensor according to claim 1, wherein: in the step e, the rubber mold is a hollow conduit with an inner diameter of 0.1mm-1mm, and the traditional optical fiber is any one of a single-mode optical fiber and a multimode optical fiber.
5. The method for monitoring the uptake of cancer cells by a quantum dot-based biosensor according to claim 1, wherein: in the step f, the temperature is set to be 40-100 ℃ and the heating time is set to be 30-60 min.
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