CN109709081B - Nano biosensor using fluorescence detection and preparation method and application thereof - Google Patents
Nano biosensor using fluorescence detection and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano biosensor for detecting lactate dehydrogenase activity by using fluorescence, which is characterized in that the nano biosensor is doped Mn which is synthesized by a hydrothermal method and has red light emission2+The cubic phase rare earth up-conversion luminescence nano particle is prepared by the interaction of hydrophilic and hydrophobic water between an oleic acid ligand on the surface of the cubic phase rare earth up-conversion luminescence nano particle and a nile blue derivative (NB-C18PMH-PEG), and has the advantages of water solubility, stable structure, small chemical composition difference and higher sensing signal intensity. The invention also provides a preparation method and application thereof. The hybrid nano biosensor provided by the invention has good stability and biocompatibility, can play a role in assisting cancer cell diagnosis and drug screening in vitro by utilizing the converted fluorescence imaging signals, and meets the requirements of clinical diagnosis and research and development of anti-cancer drugs. The preparation method provided by the invention has the characteristics of simple and efficient preparation process and stable quality.
Description
Technical Field
The invention relates to the technical field of nano biological material manufacturing, in particular to a nano biosensor for detecting lactate dehydrogenase activity by utilizing fluorescence, and a preparation method and application thereof.
Background
Cancer has been one of the leading causes of human death for a long time and has been studied, and the german physiologist Otto Warburg in 1924 proposed that cancer development is due to increased anaerobic glycolysis of intracellular sugars coupled with decreased oxygen consumption, known as the walburg effect. Cancer cells generally energize themselves by enhancing anaerobic glycolysis of glucose, i.e., conversion of glucose to lactate by lactate dehydrogenase. Therefore, lactate dehydrogenase activity in cancer cells is much higher than that in normal cells. While the amide adenine dinucleotide (NAD +) is a coenzyme factor of lactate dehydrogenase in the anaerobic glycolysis process of glucose, and the concentration of NAD + is far higher than that of NAD + in common cells in cancer cells with lactic acid accumulation caused by excessively high metabolic rate. Therefore, the lactate dehydrogenase activity can be reflected by measuring the concentration of NAD +.
In recent years, biosensors have been regarded in various fields as an analytical alternative to the conventional methods. A biosensor is an analytical device, consisting of two basic parts: biological receptors (antibodies, DNA probes, cells, or the like) for specifically detecting substances using the specificity of biomolecular interactions; and a transducer or sensor capable of interpreting and "converting" the biological recognition reaction produced by the receptor into a quantifiable optical or electrical signal. These devices have the greatest advantage of being extremely attractive analytical tools in terms of specificity, high sensitivity, the ability to shorten the reaction time of the analysis, the ability to perform induction in an integrated system, convenience of automation, real-time operability, versatility, and low cost.
In the optical sensing technology, fluorescence imaging becomes a new research hotspot due to the advantages of high sensitivity, high signal intensity, no damage to cells and biological tissues and the like. The rare earth doped up-conversion luminescent nano material attracts people's extensive attention as a new fluorescent material because the luminescence of the material belongs to anti-stokes shift, low-energy light excitation (usually near infrared light 980nm or 808nm) is usually adopted to emit high-energy light (ultraviolet light, visible light and near infrared light), the defects of background fluorescence interference and low signal to noise ratio in biological application can be overcome, and the imaging signal can be widely applied to a series of biological sensing fields such as concentration sensing, temperature sensing and the like.
At present, in order to achieve the purpose of more accurate cancer diagnosis and accelerating the research and development process of related anticancer drugs, it is very necessary to develop a hybrid nano biosensor having the functions of cancer cell diagnosis and anticancer drug screening, which is helpful to promote the diagnosis and treatment of cancer.
The molecular weight of Lactate Dehydrogenase (LDH) is 130-140 KDa, and the Lactate Dehydrogenase (LDH) consists of two subunits: h (for heart) and M (for muscle). They are arranged and combined in different forms to form 5 isozymes containing 4 subunits, namely: LDH1(H4), LDH2(H3M1), LDH3(H2M2), LDH4(HM3), LDH5 (M4). The accurate measurement of the lactate dehydrogenase activity has great clinical significance. The distribution of LDH in the tissue is characterized in that the heart and the kidney mainly use LDH1, and LDH2 times is adopted; the lung is mainly LDH3 and LDH 4; skeletal muscle is mainly LDH 5; liver is mainly LDH5, and LDH4 times. The sequence of LDH content in serum was LDH2> LDH1> LDH3> LDH4> LDH 5. Since LDH is present in almost all somatic cells and is generally highly active in human tissues, the increase in LDH in serum is non-specific for any single tissue or organ. The increase is late in AMI and the peak is late, so the method has little value in early diagnosis. Due to the long half-life (10-163 hours), it is often used for retrospective diagnosis, such as diagnosis and disease monitoring of AMI patients who are admitted late, subacute MI. The clinical incidence of the cases with the serum LDH total activity being increased and the isozyme spectrum being normal (LDH1/LDH2<1) is sequentially as follows; cardiopulmonary disease, malignant tumor, fracture, central nervous system diseases, inflammation, liver cirrhosis, infectious mononucleosis, hypothyroidism, uremia, tissue necrosis, viremia, intestinal obstruction, etc. However, the detection of the lactate dehydrogenase activity by the means in the prior art has the disadvantages of low accuracy, complex operation and high material cost.
Enzymes in the prior art generally have a high degree of specific catalytic activity and thus their enzyme activity can be determined by measuring changes in the concentration of their respective substrates or products. The enzyme activity is usually measured by a physical or chemical method such as a colorimetric method, a calorimetric method, a titration method, a spectrophotometric method, a radiometric method, and an enzyme-coupled assay method, depending on the characteristics of the substrate or the product of the enzyme reaction. Colorimetric methods are commonly used to determine enzyme activity, such as proteases, when the product of an enzymatic reaction can be reacted with a particular chemical reagent to produce a stable colored solution, with the shade of the color produced having a linear relationship with the concentration of the product over a range. When the product of the enzymatic reaction is a gas, the activity of the enzyme, such as amino acid decarboxylase, urease, is usually measured by a gas measurement method. When the product of the enzymatic reaction is a free acidic substance, the activity of the enzyme, such as lipase, is usually determined by titration. When the substrate and product of the enzymatic reaction differ in their light absorption properties, the amount of decrease in the substrate or the amount of increase in the product in the reaction mixture can be directly measured, and the activity of the enzyme, such as a reducing coenzyme, is usually measured spectrophotometrically. While the radiometry method is suitable for the case where a substrate for an enzyme reaction is labeled with a radioisotope, it is common to measure the activity of urease. For some enzymes, there is no suitable assay per se, but the assay can be performed in conjunction with another enzyme reaction, and usually an enzyme-coupled assay method, i.e., a method using a highly specific tool enzyme to allow the enzyme reaction to proceed to a stage where the assay can be directly, continuously, easily and accurately performed, is used. At present, common kits for enzyme activity detection are generally designed and manufactured by using the principle of spectrophotometry, and have some technical limitations.
In the prior art, NaYF modified with oleic acid4The Yb/Er up-conversion nano particle not only has strong luminous intensity, but also has the advantages of easy control of the shape and the size of the particle, simple and reliable preparation, and the NaYF modified by the oleic acid4The conversion of Yb/Er upconversion nanoparticles into water-soluble upconversion nanoparticles is of great significance to various applications in aqueous solutions. Oleic acid modified NaYF4The study of Yb/Er upconversion nanoparticles to water-soluble upconversion nanoparticles has reported that surface oleic acid ligands are oxidized to hydrophilic azelaic acid ligands by old steel and the like (Z.Chen, H.Chen, H.Huetal, J.Am.chem.Soc.,2008,130, 3023-; boyer et al NaYF modified with oleic acid using phosphorylated polyethylene glycol as ligand4Oleic acid exchange on the surface of the up-conversion nanoparticles is carried out to obtain water-soluble NaYF4Upconversion nanoparticles (J-C.Boyer, M-P.Manseau, J.I.Murray, F.C.J.M.vanVeggel, Langmuir,2010,26(2), 1157-.
The invention of Chinese patent application 201310153965.X provides a preparation method of amino acid modified water-soluble rare earth up-conversion nanoparticles, and the method uses oleic acid modified NaYF4Based on Yb/Er up-conversion nano particles, the oleic acid on the surface of the particles is modifiedThe decorations are removed and then modified by amino acid, and the prepared up-conversion nano particle inherits the NaYF modified by oleic acid4The Yb/Er up-conversion nano particle has high luminous intensity, and the surface of the particle is provided with active functional groups for further molecular connection.
However, the hybrid nano biosensor prepared by the existing process has the defects of large difference of main components, limited source of the main components, high cost, complex preparation method, high equipment price and difficulty in realizing stable mass production; the chemical stability and biocompatibility of the finished product are poor, especially the sensing signal intensity is low, and the application of the finished product in the biological field is limited.
Therefore, there is a need to develop a new nano biosensor and a method for preparing the same.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a nano biosensor for detecting the activity of lactate dehydrogenase by using fluorescence, a preparation method and application thereof, so as to overcome the defects of limited component sources, poor stability, large chemical composition difference, low sensing signal intensity and the like of the conventional biosensor and expand the application of the biosensor in the biological field; meanwhile, the preparation process is simplified, the preparation cost is reduced, and the industrialization is easy to realize; the sensor overcomes the technical defects of the existing products, can realize the detection and sensing of the lactate dehydrogenase activity by using fluorescence signals, and the obtained sensor and the preparation process thereof can meet the requirements of cancer cell diagnosis and anticancer drug screening.
In order to achieve the purpose, the invention adopts the technical scheme that:
a nano biosensor for detecting lactate dehydrogenase activity by fluorescence is characterized in that the nano biosensor is doped Mn with red light emission synthesized by a hydrothermal method2+The cubic phase rare earth up-conversion luminescence nano particle is prepared by the interaction of hydrophilic and hydrophobic phases between an oleic acid ligand on the surface of the cubic phase rare earth up-conversion luminescence nano particle and a nile blue derivative (an expression is NB-C18PMH-PEG), and has the advantages of water solubility, stable structure, small chemical composition difference and higher sensing signal intensity.
A nano biosensor for detecting lactate dehydrogenase activity by using fluorescence is characterized by comprising the following steps:
1) preparing nano particles: synthesis of oil-soluble doped Mn with oleic acid as surface ligand2+Mixing cyclohexane dispersion liquid of the rare earth upconversion luminescent nanoparticles with absolute ethyl alcohol, performing ultrasonic dispersion, centrifuging, taking solid, dispersing into trichloromethane, and coordinating the surface of the rare earth upconversion luminescent nanoparticles with an oleic acid ligand to obtain a first dispersion liquid;
2) preparing a precursor of the ligand: dissolving a polymaleic anhydride/1-octadecene alternating copolymer and methoxypolyethylene diamine in dichloromethane, adding triethylamine and EDC, and stirring at room temperature to obtain a second dispersion liquid;
3) preparing a modified ligand: and (3) carrying out rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in N, N-dimethylformamide, adding Nile blue, EDC and N-hydroxy thiosuccinimide, stirring the mixture at room temperature in a dark place, dialyzing the mixture with deionized water through a dialysis bag, and dispersing the mixture into trichloromethane after freeze drying to obtain a third dispersion liquid, wherein the expression of the third dispersion liquid is as follows: NB-C18 PMH-PEG;
4) integral surface modification: and (3) mixing the first dispersion liquid and the third dispersion liquid, stirring in a dark place under the heating condition of 40-60 ℃, evaporating trichloromethane, and dispersing into deionized water to obtain the hybrid nano biosensor.
In the first dispersion liquid in the step 1), the surface ligand is oil-soluble doped Mn of oleic acid2+The rare earth up-conversion luminescent nanoparticles comprise: NaYbF4:Mn/Er、NaYbF4:Mn/Er/Tm、NaYF4:Yb/Mn/Er、NaYF4:Yb/Mn/Er/Tm。
The application of the nano biosensor for detecting the activity of the lactate dehydrogenase by using fluorescence is characterized in that the hybrid nano biosensor is applied to the detection of the concentration of the lactate dehydrogenase activity marker, namely, the amide adenine dinucleotide, so that the effect of in-vitro auxiliary cancer cell diagnosis is achieved, and the requirement of clinical diagnosis is met.
The application of the nano biosensor for detecting the lactate dehydrogenase activity by using fluorescence is characterized in that the hybrid nano biosensor is applied to the detection of the treatment effect of the anti-cancer drug for inhibiting the lactate dehydrogenase activity, plays a role in drug screening, and meets the requirement of research and development of the anti-cancer drug.
The invention has the beneficial effects that:
(1) the invention provides a nano biosensor for detecting lactate dehydrogenase activity by fluorescence, which focuses on using Mn doping2+The hydrophilic and hydrophobic effects between the rare earth up-conversion luminescent nanoparticle surface ligand and the Nile blue derivative (expression is NB-C18PMH-PEG) modify the surface of the up-conversion luminescent nanoparticle, endow the surface of the up-conversion luminescent nanoparticle with the functions of cancer cell diagnosis and drug screening, and expand the application range of the hybrid nano sensor in the biological field.
(2) The preparation method of the nano biosensor for detecting the activity of the lactate dehydrogenase by using fluorescence provided by the invention adopts a hydrothermal method to obtain the Mn doped with red light emission through controlling special components, proportion and reaction conditions2+The rare earth up-conversion luminescent nanoparticles. The preparation method overcomes the defects of the existing synthesis method, has the advantages of simple preparation process, low equipment investment, mild reaction conditions and easy control, and the prepared nano particles have good uniformity, stable chemical composition and good repeatability, and are easy to realize industrial production.
(3) The application of the nano biosensor for detecting the lactate dehydrogenase activity by using fluorescence overcomes the limitation of single application of the existing hybrid biosensor, and the fluorescence imaging of the up-conversion luminescent nano particles is fully utilized, and simultaneously the luminescent signal intensity is utilized, so that the nano biosensor has the cancer cell diagnosis function, also has the drug screening function, expands the application of the nano biosensor in the field of biological medicine, and has wide application prospect.
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) photograph of a nanobiocensor for detecting lactate dehydrogenase activity using fluorescence, prepared in example 1 of the present invention;
FIG. 2 is a fluorescence spectrum of a nanobiocensor for detecting lactate dehydrogenase activity by fluorescence according to example 5 of the present invention;
FIG. 3 is a concentration titration graph of the hybrid nano biosensor prepared in example 6 of the present invention for lactate dehydrogenase activity marker NAD +, and the inset is a linear fitting function graph of the intensity of converted emission light at 668nm of the hybrid nano biosensor and the corresponding concentration of NAD +.
Detailed Description
Example 1
The nano biosensor for detecting lactate dehydrogenase activity by fluorescence provided in this example is doped Mn with red light emission synthesized by hydrothermal method2+The cubic phase rare earth up-conversion luminescence nano particle is prepared by the interaction of hydrophilic and hydrophobic phases between an oleic acid ligand on the surface of the cubic phase rare earth up-conversion luminescence nano particle and a nile blue derivative (an expression is NB-C18PMH-PEG), and has the advantages of water solubility, stable structure, small chemical composition difference and higher sensing signal intensity.
The preparation method of the nano biosensor for detecting the activity of the lactate dehydrogenase by using fluorescence is characterized in that Mn is doped and emitted by red light2+Rare earth up-conversion luminescent nano particle NaYbF4Based on Mn/Er, the surface of the Nile blue derivative is functionally modified through the hydrophilic and hydrophobic effects between an oleic acid ligand on the surface and the Nile blue derivative, and the method specifically comprises the following steps:
1) preparing nano particles: synthesis of oil-soluble doped Mn with oleic acid as surface ligand2+Mixing cyclohexane dispersion liquid of the rare earth upconversion luminescent nanoparticles with absolute ethyl alcohol, performing ultrasonic dispersion, centrifuging, taking solid, dispersing into trichloromethane, and coordinating the surface of the rare earth upconversion luminescent nanoparticles with an oleic acid ligand to obtain a first dispersion liquid;
specifically, the method comprises the following steps: oil-soluble doped Mn with prepared 10mL of synthesized surface ligand as oleic acid2+Rare earth up-conversion luminescent nano particle NaYbF4Preparing 10-20 mL of absolute ethyl alcohol in a Mn/Er cyclohexane dispersion liquid, mixing the absolute ethyl alcohol according to a volume ratio of 1: 1-1: 2, and ultrasonically dispersing the absolute ethyl alcohol at room temperatureAnd centrifuging and washing at 8000-10000 revolutions per minute, and dispersing the obtained solid into 10mL of trichloromethane to obtain a first dispersion liquid.
2) Preparing a precursor of the ligand: dissolving a polymaleic anhydride/1-octadecene alternating copolymer and methoxypolyethylene diamine in dichloromethane, adding triethylamine and EDC, and stirring at room temperature to obtain a second dispersion liquid;
specifically, the method comprises the following steps: firstly, 0.1-0.4 g of polymaleic anhydride/1-octadecene alternating copolymer, 1.43-5.72 g of methoxypolyethyleneglycoamine and 0.11-0.44 g of EDC are ultrasonically dissolved in 100-250 mL of dichloromethane, then 0.04-0.16 mL of triethylamine is injected, the materials are uniformly mixed, and the mixture is stirred for 24 hours at room temperature to obtain a second dispersion liquid.
3) Preparing a modified ligand: and (3) carrying out rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in N, N-dimethylformamide, adding Nile blue, EDC and N-hydroxy thiosuccinimide, stirring the mixture at room temperature in a dark place, dialyzing the mixture with deionized water through a dialysis bag, and dispersing the mixture into trichloromethane after freeze drying to obtain a third dispersion liquid, wherein the expression of the third dispersion liquid is as follows: NB-C18 PMH-PEG;
specifically, the method comprises the following steps: and (3) carrying out rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in N, N-dimethylformamide to obtain an N, N-dimethylformamide solution of C18PMH-PEG, dissolving Nile blue, EDC and N-hydroxy thiosuccinimide in the N, N-dimethylformamide, and then uniformly mixing the solution and the N, N-dimethylformamide solution of C18PMH-PEG, and stirring the mixture at room temperature in a dark place for 24 hours. Dialyzing for 48 hours by using deionized water through a dialysis bag with the molecular weight cutoff of 12000-14000, centrifugally washing for three times, freezing and drying, and dispersing the obtained solid into trichloromethane to obtain a third dispersion liquid;
or: and (3) performing rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in 25-100 mL of N, N-dimethylformamide to obtain a C18PMH-PEG N, N-dimethylformamide solution, dissolving 0.1-0.4 g of nile blue, 0.05-0.2 g of EDC and 0.05-0.2 g N-hydroxythiosuccinimide in 25-100 mL of N, N-dimethylformamide, uniformly mixing with the C18PMH-PEG N, N-dimethylformamide solution, and stirring at room temperature in a dark place for 24 hours. Dialyzing for 48 hours by using deionized water through a dialysis bag with the molecular weight cutoff of 12000-14000, centrifugally washing for three times, freezing and drying, and dispersing the obtained solid into 25-100 mL of trichloromethane to obtain a third dispersion liquid.
4) Integral surface modification: and adding the third dispersion liquid into the first dispersion liquid, fully stirring and mixing, stirring in a dark place under the heating condition of 40-60 ℃, completely evaporating trichloromethane, adding deionized water, centrifugally washing for three times, and finally dispersing in the deionized water to obtain the hybrid nano biosensor.
Specifically, the method comprises the following steps: mixing 10mL of the first dispersion and 10mL of the third dispersion in equal volume, stirring under heating at 50 ℃ in a dark place, evaporating chloroform, and dispersing into 20mL of deionized water to obtain the doped Mn emitting red light2+Rare earth up-conversion luminescent nano particle NaYbF4Mn/Er based hybrid nano biosensor.
The hybrid nano biosensor is characterized in that Mn doped with red light emission is synthesized by using a hydrothermal method2+Based on the cubic phase rare earth up-conversion luminescence nano particles, the surface oleic acid ligand and the nile blue derivative have good water solubility through the hydrophilic and hydrophobic effects, and the hybridization nano biosensor with stable structure is obtained.
The application of the nano biosensor for detecting the activity of the lactate dehydrogenase by using fluorescence is that the hybrid nano biosensor is applied to the detection of the concentration of the lactate dehydrogenase activity marker, namely, the amide adenine dinucleotide, plays a role in assisting the diagnosis of cancer cells in vitro, and meets the requirement of clinical diagnosis.
The application of the nano biosensor for detecting the lactate dehydrogenase activity by using fluorescence is that the hybrid nano biosensor is applied to the detection of the treatment effect of the anti-cancer drugs for inhibiting the lactate dehydrogenase activity, plays a role in drug screening, and meets the requirement of research and development of the anti-cancer drugs.
FIG. 1 is a TEM photograph of a nano biosensor for detecting lactate dehydrogenase activity by fluorescence, which is prepared in embodiment 1 of the present invention, and it can be seen from the TEM photograph that the structure of the nano particles is stable and the dispersibility is good, which indicates that the method can prepare a nano material with good morphology and good dispersibility, and the particle size distribution of the functionalized hybrid nano biosensor is within the range of 20-40 nm. The small-sized nano particles are easier to be taken up by cells, so the hybrid nano biosensor is more favorable for realizing fluorescence imaging of the cells and has important significance for biological applications such as cancer cell diagnosis, drug screening and the like.
Example 2
This example provides a nano biosensor for detecting lactate dehydrogenase activity using fluorescence and a method for preparing the same, which is substantially the same as example 1, except that the doped Mn emits in red light2+Rare earth up-conversion luminescent nano particle NaYbF4Based on Mn/Er/Tm, comprises the following steps:
(1) oil-soluble doped Mn with prepared 10mL of synthesized surface ligand as oleic acid2+Rare earth up-conversion luminescent nano particle NaYbF4Preparing 10-20 mL of absolute ethyl alcohol, mixing according to the volume ratio of 1: 1-1: 2, performing ultrasonic dispersion at room temperature, performing centrifugal washing at 8000-10000 revolutions per minute, and dispersing the obtained solid into 10mL of trichloromethane to obtain a first dispersion liquid.
(2) Firstly, 0.1-0.4 g of polymaleic anhydride/1-octadecene alternating copolymer, 1.43-5.72 g of methoxypolyethyleneglycoamine and 0.11-0.44 g of EDC are ultrasonically dissolved in 100-250 mL of dichloromethane, then 0.04-0.16 mL of triethylamine is injected, the materials are uniformly mixed, and the mixture is stirred for 24 hours at room temperature to obtain a second dispersion liquid.
(3) And (3) performing rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in 25-100 mL of N, N-dimethylformamide to obtain a C18PMH-PEG N, N-dimethylformamide solution, dissolving 0.1-0.4 g of nile blue, 0.05-0.2 g of EDC and 0.05-0.2 g N-hydroxythiosuccinimide in 25-100 mL of N, N-dimethylformamide, uniformly mixing with the C18PMH-PEG N, N-dimethylformamide solution, and stirring at room temperature in a dark place for 24 hours. Dialyzing for 48 hours by using deionized water through a dialysis bag with the molecular weight cutoff of 12000-14000, centrifugally washing for three times, freeze-drying, dispersing the obtained solid into 25-100 mL of trichloromethane to obtain a third dispersion liquid, and preparing the nile blue derivative, wherein the expression of the third dispersion liquid is as follows: NB-C18 PMH-PEG;
(4) mixing 10mL of the first dispersion and 10mL of the third dispersion in equal volume, stirring under heating at 50 ℃ in a dark place, evaporating chloroform, and dispersing into 20mL of deionized water to obtain the doped Mn emitting red light2+Rare earth up-conversion luminescent nano particle NaYbF4Mn/Er/Tm based hybrid nano biosensor.
Example 3
This example provides a nano biosensor for detecting lactate dehydrogenase activity using fluorescence and a method for preparing the same, which is substantially the same as examples 1 and 2, except that the doped Mn emits in red light2+Rare earth up-conversion luminescent nano particle NaYF4Based on Yb/Mn/Er, comprises the following steps:
(1) oil-soluble doped Mn with prepared 10mL of synthesized surface ligand as oleic acid2+Rare earth up-conversion luminescent nano particle NaYF4Preparing 10-20 mL of absolute ethyl alcohol in a Yb/Mn/Er cyclohexane dispersion solution, mixing the mixture according to a volume ratio of 1: 1-1: 2, performing ultrasonic dispersion at room temperature, performing centrifugal washing at 8000-10000 revolutions per minute, and dispersing the obtained solid into 10mL of trichloromethane to obtain a first dispersion solution.
(2) Firstly, 0.1-0.4 g of polymaleic anhydride/1-octadecene alternating copolymer, 1.43-5.72 g of methoxypolyethyleneglycoamine and 0.11-0.44 g of EDC are ultrasonically dissolved in 100-250 mL of dichloromethane, then 0.04-0.16 mL of triethylamine is injected, the materials are uniformly mixed, and the mixture is stirred for 24 hours at room temperature to obtain a second dispersion liquid.
(3) And (3) performing rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in 25-100 mL of N, N-dimethylformamide to obtain a C18PMH-PEG N, N-dimethylformamide solution, dissolving 0.1-0.4 g of nile blue, 0.05-0.2 g of EDC and 0.05-0.2 g N-hydroxythiosuccinimide in 25-100 mL of N, N-dimethylformamide, uniformly mixing with the C18PMH-PEG N, N-dimethylformamide solution, and stirring at room temperature in a dark place for 24 hours. Dialyzing for 48 hours by using deionized water through a dialysis bag with the molecular weight cutoff of 12000-14000, centrifugally washing for three times, freeze-drying, and dispersing the obtained solid into 25-100 mL of trichloromethane to obtain a third dispersion liquid;
(4) mixing 10mL of the first dispersion and 10mL of the third dispersion in equal volume, stirring under heating at 50 ℃ in a dark place, evaporating chloroform, and dispersing into 20mL of deionized water to obtain the doped Mn emitting red light2+Rare earth up-conversion luminescent nano particle NaYF4Hybrid nano biosensor based on Yb/Mn/Er.
Example 4
This example provides a nano biosensor for detecting lactate dehydrogenase activity using fluorescence and a method for preparing the same, which is substantially the same as examples 1-3, except that Mn is doped in a red light emission2+Rare earth up-conversion luminescent nano particle NaYF4Based on Yb/Mn/Er/Tm, comprising the following steps:
(1) oil-soluble doped Mn with prepared 10mL of synthesized surface ligand as oleic acid2+Rare earth up-conversion luminescent nano particle NaYF4Preparing 10-20 mL of absolute ethyl alcohol, mixing the mixture according to the volume ratio of 1: 1-1: 2, performing ultrasonic dispersion at room temperature, performing centrifugal washing at 8000-10000 revolutions per minute, and dispersing the obtained solid into 10mL of trichloromethane to obtain a first dispersion liquid.
(2) Firstly, 0.1-0.4 g of polymaleic anhydride/1-octadecene alternating copolymer, 1.43-5.72 g of methoxypolyethyleneglycoamine and 0.11-0.44 g of EDC are ultrasonically dissolved in 100-250 mL of dichloromethane, then 0.04-0.16 mL of triethylamine is injected, the materials are uniformly mixed, and the mixture is stirred for 24 hours at room temperature to obtain a second dispersion liquid.
(3) And (3) performing rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in 25-100 mL of N, N-dimethylformamide to obtain a C18PMH-PEG N, N-dimethylformamide solution, dissolving 0.1-0.4 g of nile blue, 0.05-0.2 g of EDC and 0.05-0.2 g N-hydroxythiosuccinimide in 25-100 mL of N, N-dimethylformamide, uniformly mixing with the C18PMH-PEG N, N-dimethylformamide solution, and stirring at room temperature in a dark place for 24 hours. Dialyzing for 48 hours by using deionized water through a dialysis bag with the molecular weight cutoff of 12000-14000, centrifugally washing for three times, freeze-drying, and dispersing the obtained solid into 25-100 mL of trichloromethane to obtain a third dispersion liquid;
(4) mixing 10mL of the first dispersion and 10mL of the third dispersion in equal volume, stirring under heating at 50 ℃ in a dark place, evaporating chloroform, and dispersing into 20mL of deionized water to obtain the doped Mn emitting red light2+Rare earth up-conversion luminescent nano particle NaYF4Namely a hybrid nano biosensor based on Yb/Mn/Er/Tm.
Example 5
The application of the nano biosensor for detecting the activity of the lactate dehydrogenase by using fluorescence prepared in the embodiment is that the hybrid nano biosensor is applied to the detection of the concentration of the lactate dehydrogenase activity marker, namely, the amide adenine dinucleotide, plays a role in assisting the diagnosis of cancer cells in vitro, and meets the requirement of clinical diagnosis.
The nano biosensor for detecting the lactate dehydrogenase activity by using fluorescence, which is prepared in example 1, is used as a sensor for cancer cell diagnosis and is used for cell fluorescence imaging; the method is particularly used for cell fluorescence imaging, and comprises the following steps:
(1) preparing the hybrid nano biosensor obtained in the example 1, and preparing a culture solution with the concentration of 200-400 mug/mL of the hybrid nano biosensor by using a culture medium;
(2) culturing HeLa cells, A549 cells and Ges-1 cells in the culture solution for 2, 4 and 6 hours;
(3) washing the cells with Phosphate Buffered Saline (PBS) for 5 times, and washing away the nano-materials which are not absorbed by the cells;
(4) and imaging the cultured cells on a confocal microscope, wherein a steady-state 980nm laser with adjustable power of 0-500 mW and continuous wave excitation is used as an excitation light source in the imaging process, and emitted light with the wavelength of 640-680 nm is observed.
FIG. 2 is a fluorescence spectrum of 980nm laser excitation using the hybrid nano biosensor of the present invention in application example 5, which can be observedEmission peak around 665nm, corresponding to Er3+Is/are as follows4F9/2→4I15/2And the transition is carried out, and 980nm is just positioned at an optical window of the biological tissue, which shows that the hybrid nano biosensor still maintains the good up-conversion fluorescence property of the up-conversion nano particles and is very suitable for cell fluorescence imaging.
Example 6
The application of the nano biosensor for detecting the lactate dehydrogenase activity by using fluorescence prepared in the embodiment is to detect the treatment effect of the anti-cancer drug for inhibiting the lactate dehydrogenase activity, so that the effect of drug screening and the research and development requirements of the anti-cancer drug are met.
The nano biosensor for detecting the activity of the lactate dehydrogenase by using fluorescence, which is prepared in example 1, is used as a sensor for screening drugs and is used for cell fluorescence imaging; the method is particularly used for cell fluorescence imaging, and comprises the following steps:
(1) preparing the hybrid nano biosensor obtained in the example 1, and preparing a culture solution with the concentration of 200-400 mug/mL of the hybrid nano biosensor by using a culture medium;
(2) adding an anti-cancer drug for inhibiting the activity of the lactate dehydrogenase into the culture solution to ensure that the concentration of the anti-cancer drug is 1-5 mu g/mL, and then respectively culturing HeLa cells, A549 cells and Ges-1 cells for 2, 4 and 6 hours;
(3) washing the cells with phosphate buffer solution for 5 times, and washing away the nano materials which are not absorbed by the cells;
(4) and imaging the cultured cells on a confocal microscope, wherein a steady-state 980nm laser with adjustable power of 0-500 mW and continuous wave excitation is used as an excitation light source in the imaging process, and emitted light with the wavelength of 640-680 nm is observed.
FIG. 3 is a concentration titration graph of the hybrid nano biosensor prepared in example 6 of the present invention for lactate dehydrogenase activity marker NAD +, and the inset is a linear fitting function graph of the intensity of converted emission light at 668nm of the hybrid nano biosensor and the corresponding concentration of NAD +. The influence of the change of NAD + concentration on the red light emission intensity of the hybrid nano biosensor proves that the hybrid nano biosensor can be used as a sensor for drug screening for drug effect screening of drugs inhibiting lactate dehydrogenase activity.
The invention is characterized in that the invention is based on doping Mn with red light emission2+Rare earth up-conversion luminescence nano-particles and Nile blue derivatives, provides a nano biosensor for detecting the activity of lactate dehydrogenase by fluorescence and a preparation method thereof, and expands the application of the nano biosensor in the field of biological medicine so as to meet the requirements of cancer cell diagnosis and anticancer drug screening. The preparation method provided by the invention has the advantages of simple and efficient process, controllable operation, good repeatability, low investment cost and the like, and is easy to industrialize.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Those skilled in the art can make many possible variations or modifications to the disclosed solution, using the methods and techniques disclosed above, to equivalents thereof without departing from the scope of the invention. Therefore, all equivalent modifications made according to the structure, structure and principle of the present invention should be covered within the protection scope of the present invention without departing from the contents of the technical scheme of the present invention.
Claims (9)
1. A nano biosensor for detecting lactate dehydrogenase activity by fluorescence is characterized in that the nano biosensor is doped Mn with red light emission synthesized by a hydrothermal method2+The hybrid nano biosensor is prepared by the interaction of hydrophilic and hydrophobic water between an oleic acid ligand on the surface of the hybrid nano biosensor and a nile blue derivative on the basis of the cubic phase rare earth up-conversion luminescent nano particles, and has the advantages of water solubility, stable structure, small chemical composition difference and higher sensing signal intensity; wherein the expression of the nile blue derivative is NB-C18 PMH-PEG.
2. The method for preparing a nanobiocensor for detecting lactate dehydrogenase activity according to claim 1, comprising the steps of:
1) preparing nano particles: combination of Chinese herbsOil-soluble doped Mn with oleic acid as surface ligand2+Mixing cyclohexane dispersion liquid of the rare earth upconversion luminescent nanoparticles with absolute ethyl alcohol, performing ultrasonic dispersion, centrifuging, taking solid, dispersing into trichloromethane, and coordinating the surface of the rare earth upconversion luminescent nanoparticles with an oleic acid ligand to obtain a first dispersion liquid;
2) preparing a precursor of the ligand: mixing polymaleic anhydride/1-octadecene alternating copolymer (C18PMH) and methoxypolyethyleneglycol amine (mPEG-NH)2) Dissolving in dichloromethane, adding triethylamine and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC-HCl), and stirring at room temperature to obtain a second dispersion liquid;
3) preparing a modified ligand: and (3) carrying out rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in N, N-dimethylformamide, adding Nile blue, EDC-HCl and N-hydroxy thiosuccinimide, stirring at room temperature in a dark place, dialyzing with deionized water through a dialysis bag, and dispersing into trichloromethane after freeze drying to obtain a third dispersion liquid, wherein the expression of the third dispersion liquid is as follows: NB-C18 PMH-PEG;
4) integral surface modification: and (3) mixing the first dispersion liquid and the third dispersion liquid, stirring in a dark place under the heating condition of 40-60 ℃, evaporating trichloromethane, and dispersing into deionized water to obtain the hybrid nano biosensor.
3. The preparation method according to claim 2, wherein the first dispersion in step 1) comprises the following specific steps:
synthesis of oil-soluble doped Mn with oleic acid as surface ligand2+The method comprises the steps of dispersing rare earth up-conversion luminescent nanoparticles in cyclohexane, preparing anhydrous ethanol with a certain volume, mixing the anhydrous ethanol with the volume ratio of 1: 1-1: 2, performing ultrasonic dispersion at room temperature, performing centrifugal washing at 8000-10000 revolutions per minute, and dispersing the obtained solid into trichloromethane to obtain a first dispersion liquid.
4. The preparation method according to claim 2, wherein the second dispersion in step 2) comprises the following specific steps:
firstly, ultrasonically dissolving the polymaleic anhydride/1-octadecene alternating copolymer, the methoxy polyethylene glycol amine and the EDC-HCl in dichloromethane, then injecting triethylamine, uniformly mixing, and stirring for 24 hours at room temperature to obtain a second dispersion liquid.
5. The preparation method according to claim 2, wherein the third dispersion in step 3) comprises the following specific steps:
firstly, carrying out rotary evaporation on the second dispersion liquid to obtain pink solid powder, dissolving the pink solid powder in N, N-dimethylformamide to obtain an N, N-dimethylformamide solution of C18PMH-PEG, then dissolving Nile blue, EDC-HCl and N-hydroxy thiosuccinimide in the N, N-dimethylformamide, and then uniformly mixing the solution with the N, N-dimethylformamide solution of C18PMH-PEG, and stirring the solution at room temperature in a dark place for 24 hours; and dialyzing for 48 hours by using deionized water through a dialysis bag with the molecular weight cutoff of 12000-14000, centrifugally washing for three times, freeze-drying, and dispersing the obtained solid into trichloromethane to obtain a third dispersion liquid.
6. The preparation method according to claim 2, wherein the specific steps in step 4) are:
and adding the third dispersion liquid into the first dispersion liquid, fully stirring and mixing, stirring in a dark place under the heating condition of 40-60 ℃, completely evaporating trichloromethane, adding deionized water, centrifugally washing for three times, and finally dispersing in the deionized water to obtain the hybrid nano biosensor.
7. The method according to claim 2, wherein in the first dispersion of step 1), the surface ligand is oil-soluble Mn-doped oleic acid2+The rare earth up-conversion luminescent nanoparticles comprise: NaYbF4:Mn/Er、NaYbF4:Mn/Er/Tm、NaYF4:Yb/Mn/Er、NaYF4:Yb/Mn/Er/Tm。
8. The use of the nanobiosensor for fluorescence detection of lactate dehydrogenase activity according to claim 1, wherein the hybridized nanobiosensor is used as a detection material for detecting the concentration of the lactate dehydrogenase activity marker, the amide adenine dinucleotide.
9. The use of the nanobiosensor for fluorescence detection of lactate dehydrogenase activity according to claim 1, wherein the hybrid nanobiosensor is used for anticancer drug screening for the detection of the therapeutic effect of anticancer drugs inhibiting lactate dehydrogenase activity.
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