CN111744566A - Biochip, preparation method, application and kit thereof - Google Patents
Biochip, preparation method, application and kit thereof Download PDFInfo
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Abstract
The invention is suitable for the technical field of biological detection, and provides a biochip, a preparation method thereof, application thereof and a kit, wherein the biochip comprises: a rigid or flexible substrate; a double band gap photonic crystal film; the double-band-gap photonic crystal film is arranged on the substrate; the double-band-gap photonic crystal film is made of polymethyl methacrylate; a fluorescent film; the fluorescent film is arranged on the double-band-gap photonic crystal film; the fluorescent film is made of up-conversion fluorescent nano-particles; a polydopamine film; the polydopamine film is arranged on the fluorescent film. The invention utilizes the upconversion fluorescent film enhanced by the double-band-gap PMMA opal photonic crystal structure, and can be matched with a fluorescence resonance energy transfer detection method to detect tumor markers such as PSA and the like and detect nucleic acid, antibody and the like of viruses such as 2019-nCoV and the like. The detection method has good stability, convenience and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a biochip, a preparation method thereof, application thereof and a kit.
Background
Malignant tumor is one of the main diseases threatening the life safety of human beings, and the threat lies in the insufficiency of the reliability and the popularity of the early detection thereof, and in many cases, the tumor has already progressed to the malignant stage when obvious symptoms are found, and the problem greatly improves the harmfulness of the tumor. Therefore, the research on the early tumor detection technology has great significance in improving the reliability, convenience and operability of the early tumor detection technology.
Similarly, epidemic viruses such as 2019-nCoV threaten the life health of human beings, so the method is also of great significance for the research of detection technologies of antibodies, nucleic acids and the like of the epidemic viruses such as 2019-nCoV.
The concentration of tumor markers in blood is an important index in early detection of tumors, changes occur in early stages of tumors, one tumor usually corresponds to more than one marker, and the accuracy of detection results can be greatly improved by selecting multiple markers for detection. At present, the popular methods for early detection of tumor based on tumor markers mainly include a ligase immunoassay, an immunofluorescence assay and a chemiluminescence assay, and although the methods have already entered practical stage, the methods still have respective problems. The operation steps of the united enzyme immunoassay method are complicated, the detection process is long, the requirement on the environment is high, and the convenience of the united enzyme immunoassay method is limited; in the immunofluorescence method, a fluorescent dye is used as a fluorescent probe, although the fluorescent dye has a long application history, the luminescence property of the fluorescent dye is gradually reduced along with the luminescence time, and when a biological sample is detected, the excitation light of the fluorescent dye is also absorbed by some biological molecules to generate background fluorescence, so that the two defects limit the sample preservability and the detection accuracy of the detection method; the chemiluminescence method has the defects of insufficient selectivity and high risk of false positive. The high requirements of the methods on operation, equipment and environment all the more limit the convenience and the popularity of early tumor detection.
Disclosure of Invention
An object of the embodiments of the present invention is to provide a biochip, which is to solve the problems mentioned in the background art.
The embodiment of the invention is realized in such a way that the biochip comprises:
a rigid or flexible substrate;
a double band gap photonic crystal film; the double-band-gap photonic crystal film is arranged on the substrate; the double-band-gap photonic crystal film is made of polymethyl methacrylate (PMMA);
a fluorescent film; the fluorescent film is arranged on the double-band-gap photonic crystal film; the fluorescent film is made of up-conversion fluorescent nano-particles;
a polydopamine film; the polydopamine film is arranged on the fluorescent film.
Another objective of the embodiments of the present invention is to provide a method for preparing the above biochip, which comprises the following steps:
taking a substrate, placing the substrate in a PMMA nanosphere solution with the particle size of 240-280 nm, treating at the temperature of 20-60 ℃, taking out the substrate, and drying and reinforcing at the temperature of 100-180 ℃ to obtain the substrate attached with the first photonic crystal film;
taking the other substrate, placing the substrate in a PMMA nanosphere solution with the particle size of 380-480 nm, treating at the temperature of 20-60 ℃, taking out the substrate, and drying and reinforcing at the temperature of 100-180 ℃ to obtain the substrate attached with the second photonic crystal film; then, using deionized water to strip the substrate attached with the second photonic crystal film to obtain a second photonic crystal film;
covering the substrate attached with the first photonic crystal film with a second photonic crystal film to form a double-bandgap photonic crystal film, and obtaining the substrate attached with the double-bandgap photonic crystal film;
placing the substrate attached with the double-bandgap photonic crystal film in the upconversion fluorescent nanoparticle dispersion liquid, and treating at the temperature of 30-50 ℃ to form a fluorescent film on the double-bandgap photonic crystal film to obtain the substrate attached with the fluorescent film;
and placing the substrate attached with the fluorescent film in a buffer solution containing dopamine and having a pH value of 7.5-9.0 for reaction to form a polydopamine film on the fluorescent film, thereby obtaining the biochip.
As a preferable scheme of the embodiment of the present invention, the band gap position of the first photonic crystal thin film is 500nm to 580 nm; the band gap position of the second photonic crystal film is 800 nm-1000 nm.
As another preferable scheme of the embodiment of the invention, the preparation method of the PMMA nanosphere solution with the particle size of 240 nm-280 nm comprises the following steps:
cleaning methyl methacrylate with 3-15 mg/mL sodium hydroxide aqueous solution to obtain cleaned methyl methacrylate;
placing the cleaned methyl methacrylate, deionized water and potassium persulfate at the temperature of 70-100 ℃ for reaction to obtain PMMA nanosphere solution with the particle size of 240-280 nm; wherein the volume mass ratio of the cleaned methyl methacrylate to the potassium persulfate is (5-10): (30-40) in terms of mL/mg.
As another preferable scheme of the embodiment of the invention, the preparation method of the PMMA nanosphere solution with the particle size of 380 nm-480 nm comprises the following steps:
placing the cleaned methyl methacrylate, the cleaned azodiisobutyronitrile, the deionized water and the PMMA nanosphere solution with the particle size of 240 nm-280 nm at the temperature of 70-100 ℃ for reaction to obtain the PMMA nanosphere solution with the particle size of 380 nm-480 nm; wherein the volume ratio of the cleaned methyl methacrylate to the PMMA nanosphere solution with the particle size of 240 nm-280 nm is (5-7) to (30-50); the volume-to-mass ratio of the cleaned methyl methacrylate to the azobisisobutyronitrile is (5-7): 30-40 (mL/mg).
As another preferable scheme of the embodiment of the present invention, the preparation method of the upconversion fluorescent nanoparticle dispersion liquid includes the following steps:
placing yttrium chloride, ytterbium trichloride, erbium chloride, octadecene and oleic acid at the temperature of 120-180 ℃, stirring, then placing ammonium fluoride and sodium hydroxide at the temperature of 280-320 ℃ for reaction, and then performing centrifugal separation to obtain a first solid; wherein the molar ratio of yttrium chloride, ytterbium trichloride and erbium chloride is (0.7-0.9): (0.15-0.25): 0.01-0.03);
placing yttrium chloride, ytterbium trichloride, neodymium trichloride, octadecene and oleic acid at the temperature of 120-180 ℃, stirring, then placing the mixture, ammonium fluoride, sodium hydroxide and the first solid at the temperature of 270-340 ℃ for reaction, and then carrying out centrifugal separation to obtain a second solid; wherein the molar ratio of yttrium chloride, ytterbium trichloride and neodymium trichloride is (0.6-0.8): (0.1-0.2): 0.1-0.3);
and dispersing the second solid in cyclohexane to obtain the upconversion fluorescent nanoparticle dispersion liquid.
Another objective of the embodiments of the present invention is to provide a biochip prepared by the above preparation method.
Another object of the embodiments of the present invention is to provide an application of the above biochip in the preparation of a kit for detecting tumor markers and/or viruses.
Among them, the tumor marker includes Prostate Specific Antigen (PSA), etc., but is not limited thereto; the virus includes nucleic acid or antibody of epidemic virus such as 2019-nCoV, etc., but is not limited thereto; in addition, the biochip can also be used for detecting other antigens, antibodies or proteins, and is not limited to the detection of tumor markers and viruses.
It is another object of embodiments of the present invention to provide a kit for detecting tumor markers and/or viruses, which includes the above-mentioned biochip.
As another preferable scheme of the embodiment of the invention, the kit further comprises gold nanoparticles.
The biochip provided by the embodiment of the invention utilizes the upconversion fluorescent film enhanced by the double-band-gap PMMA opal photonic crystal structure, and can be matched with a fluorescence resonance energy transfer detection method to detect tumor markers such as PSA (prostate specific antigen). The embodiment of the invention selects the core-shell structure to convert the fluorescent nano-particle NaYF4:Yb3+,Er3+@NaYF4:Yb3+,Nd3+As a fluorescent probe, the fluorescent probe can effectively avoid the problems of photobleaching, background fluorescence and high-energy ultraviolet light damaging biomolecules so as to improve the up-conversion luminescence quantum efficiency. In addition, the embodiment of the invention designs a novel nano structure, simultaneously uses two PMMA opal photonic crystals, and the band gap positions of the two PMMA opal photonic crystals are respectively 800-1000 nm and 500-580 nm which are respectively connected with NaYF4:Yb3 +,Er3+@NaYF4:Yb3+,Nd3+The excitation light wavelength corresponds to the emission light wavelength, the two photonic crystals are made into a double-layer structure, and NaYF is used4:Yb3+,Er3+@NaYF4:Yb3+,Nd3+The particles are settled on the surface of the photonic crystal through self-assembly to form a fluorescent film, and the double-layer structure can simultaneously carry out the treatment on NaYF4:Yb3+,Er3+@NaYF4:Yb3+,Nd3+The excitation light and the emission light of the up-conversion luminescence are enhanced, and the up-conversion luminescence intensity is further improved compared with that of the common photonic crystal, namely NaYF4:Yb3+,Er3+@NaYF4:Yb3+,Nd3+The oleic acid ligand on the surface of the nanoparticle can effectively enhance the water stability of photonic crystals, fluorescent particles are used as energy donors on the basis of enhancing up-conversion fluorescence, a polydopamine film is modified on the surface of the fluorescent film and used for connecting capture antibodies such as PSA (pressure swing adsorption) and the like, in addition, gold nanoparticles with absorption peaks of about 545nm are used as energy receptors and used for modifying detection antibodies such as PSA and the like, and the gold nanoparticles are connected by utilizing the antibodies, antigens and antibodiesThe distance between the particles and the fluorescent particles is shortened, so that the fluorescence resonance energy transfer effect occurs, and the concentration of the PSA waiting detection substance in the sample is determined by observing the reduction of the fluorescence intensity. The detection method has good stability and convenience. Besides, the method can be applied to various detection modes utilizing specific binding, such as antibody-antigen binding, antibody-antigen-antibody binding, specific binding of nucleic acid and the like, which means that the detection method has wide application prospect, and can also be used for detecting nucleic acid, antibody and the like of various viruses such as 2019-nCoV besides detecting tumor markers.
Drawings
FIG. 1 is a schematic structural diagram of a biochip according to example 1 of the present invention. In the figure, 1-substrate, 2-first photonic crystal film, 3-second photonic crystal film, 4-fluorescent film, 5-polydopamine film, and 6-capture antibody.
FIG. 2 is a schematic view of the biochip according to example 1 of the present invention in use. In the figure, 7-the analyte, 8-the detection antibody, and 9-the gold nanoparticles.
FIG. 3 is a scanning electron microscope image of a double-bandgap photonic crystal film obtained in example 1 of the present invention.
FIG. 4 is a graph of transmission spectra of two single-layer photonic crystals and a double-band-gap photonic crystal film.
FIG. 5 is a transmission electron micrograph of the upconversion fluorescent nanoparticles obtained in example 1 of the present invention.
FIG. 6 is a schematic structural diagram of a fluorescent film obtained in example 1 of the present invention.
FIGS. 7 to 8 are emission spectra of self-assembled fluorescent films prepared on the surfaces of a glass substrate, two single-layer photonic crystals and a double-bandgap photonic crystal film, respectively.
Fig. 9 is a schematic structural diagram of a polydopamine film obtained in example 1 of the present invention.
FIG. 10 is a transmission electron microscope image of gold nanoparticles obtained in example 1 of the present invention.
FIG. 11 is an absorption spectrum of gold nanoparticles after modification of detection antibody.
FIGS. 12 to 13 are graphs showing the comparison of fluorescence intensities in the detection of PSA at different concentrations.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
As shown in fig. 1, this embodiment provides a biochip comprising a rigid or flexible substrate 1, a double band gap photonic crystal film, a fluorescent film 4, and a polydopamine film 5; the double-band-gap photonic crystal film comprises a first photonic crystal film 2 and a second photonic crystal film 3, and the first photonic crystal film 2 and the second photonic crystal film 3 are sequentially arranged on the substrate 1; the first photonic crystal film 2 and the second photonic crystal film 3 are made of polymethyl methacrylate; in addition, the fluorescent film 4 is arranged on the second photonic crystal film 3, and is made of an up-conversion fluorescent nanoparticle material; the polydopamine film 5 is arranged on the fluorescent film 4. When the antigen needs to be detected, a capture antibody 6 can be arranged on the fluorescent film 4; for example, when it is desired to detect PSA, the capture antibody 6 may be a PSA capture antibody, but is not limited thereto.
Specifically, the preparation method of the biochip comprises the following steps:
s1, washing 25mL of methyl methacrylate with 150mL of 10mg/mL sodium hydroxide aqueous solution for 7 times to obtain washed methyl methacrylate; then, adding 6mL of cleaned methyl methacrylate, 80mL of deionized water and 36mg of potassium persulfate into a three-neck flask, then placing the three-neck flask into an oil bath kettle at 90 ℃ to carry out stirring reaction for 90min, and simultaneously carrying out condensation reflux by using a condenser pipe to obtain a PMMA nanosphere solution with the particle size of 240-280 nm for later use; in addition, 6mL of cleaned methyl methacrylate, 36mg of azodiisobutyronitrile, 40mL of deionized water and 40mL of the PMMA nanosphere solution with the particle size of 240nm to 280nm are added into a three-neck flask, then the three-neck flask is placed in an oil bath kettle at 90 ℃ for stirring reaction for 90min, and meanwhile, a condensing tube is used for condensation reflux, so that the PMMA nanosphere solution with the particle size of 380nm to 480nm can be obtained.
S2, taking a glass slide as a substrate, adding 60mL of deionized water and 3mL of PMMA nanosphere solution with the particle size of 240 nm-280 nm into a 100mL beaker, and uniformly stirring; and then vertically inserting the substrate into a beaker to enable the substrate to be in contact with PMMA nanosphere solution with the particle size of 240 nm-280 nm, placing the substrate at the temperature of 32 ℃ for holding treatment for 24 hours, taking out the substrate, placing the substrate at the temperature of 120 ℃ for drying and reinforcing for 1 hour to form a first photonic crystal film with the band gap position of 545nm, and obtaining the substrate attached with the first photonic crystal film.
S3, taking the other glass slide as a substrate, adding 60mL of deionized water and 3mL of PMMA nanosphere solution with the particle size of 380 nm-480 nm into a 100mL beaker, and uniformly stirring; then, putting the substrate in a beaker, enabling the substrate to be in contact with PMMA nanosphere solution with the particle size of 380 nm-480 nm, keeping the substrate at the temperature of 32 ℃ for processing for 24h, taking out the substrate, putting the substrate at the temperature of 120 ℃ for drying and reinforcing for 1h, forming a second photonic crystal film with the band gap position of 808nm, and obtaining the substrate attached with the second photonic crystal film.
S4, vertically fixing the substrate attached with the second photonic crystal film in a beaker, injecting deionized water into the beaker by using a trace liquid injection pump, adjusting the injection rate, enabling the liquid level of the deionized water in the beaker to rise at a rate of 1mm per hour, stripping the second photonic crystal film from the surface of the substrate, and enabling the second photonic crystal film to float on the surface of the deionized water, at the moment, fishing out the second photonic crystal film by using another substrate attached with the first photonic crystal film, and enabling the second photonic crystal film floating in the deionized water to cover the first photonic crystal film of the substrate to form a double-bandgap photonic crystal film, thereby obtaining the substrate attached with the double-bandgap photonic crystal film.
S5, mixing 0.78mmol of yttrium chloride hexahydrate, 0.2mmol of ytterbium trichloride hexahydrate, 0.02mmol of erbium chloride hexahydrate, 15mL of octadecene and 6mL of oleic acidPlacing the mixture into a 100mL three-necked flask, placing the mixture into a nitrogen protective atmosphere, heating the mixture to 140 ℃ by using a heating sleeve, stirring the mixture until the solid is completely dissolved, and cooling the mixture to 35 ℃; then, 0.148g of ammonium fluoride and 0.1g of sodium hydroxide were dissolved in 7mL of methanol to obtain a methanol solution; then, dropwise adding the methanol solution into the three-neck flask, stirring for 30min, heating to 120 ℃ to remove methanol, heating the heating jacket to 310 ℃ for 90min, cooling to 35 ℃, and adding a condensing tube for condensation and reflux in the process to obtain a reactant; then, 50mL of ethanol was added to the reaction mixture, centrifuged at 9500rpm for 15min using a centrifuge, the resulting solid was dispersed in 10mL of cyclohexane, 30mL of ethanol was added and centrifuged again at 9500rpm for 15min, and the resulting solid was centrifuged to obtain a first solid, designated NaYF4: Yb3+,Er3+And dispersed in 5mL of cyclohexane until used.
S6, placing 0.7mmol of yttrium chloride hexahydrate, 0.15mmol of ytterbium trichloride hexahydrate, 0.15mmol of neodymium trichloride hexahydrate, 15mL of octadecene and 6mL of oleic acid into a 100mL three-neck flask, placing the flask into a nitrogen protective atmosphere, heating the flask to 140 ℃ by using a heating sleeve, stirring until the solid is completely dissolved, cooling the flask to 35 ℃, and adding the obtained first solid into the three-neck flask; then, 0.148g of ammonium fluoride and 0.1g of sodium hydroxide were dissolved in 7mL of methanol to obtain a methanol solution; then, dropwise adding the methanol solution into the three-neck flask, stirring for 30min, heating to 120 ℃ to remove methanol, heating the heating jacket to 310 ℃ for 90min, cooling to 35 ℃, and adding a condensing tube for condensation and reflux in the process to obtain a reactant; then, adding 50mL of ethanol into the reactant, centrifuging the reactant for 15min at 9500rpm by using a centrifuge, dispersing the solid obtained after centrifugation in 10mL of cyclohexane, adding 30mL of ethanol, centrifuging the reactant for 15min at 9500rpm again, and centrifuging the mixture to obtain a second solid, namely the upconversion fluorescent nanoparticle which can be marked as NaYF4: Yb3+,Er3+@NaYF4:Yb3+,Nd3+And dispersing the second solid in 5mL of cyclohexane to obtain the upconversion fluorescent nanoparticle dispersion liquid for later use.
S7, putting 1mL of the obtained upconversion fluorescent nanoparticle dispersion liquid and 60mL of cyclohexane into a 100mL beaker, and uniformly mixing; and vertically inserting the substrate attached with the double-band-gap photonic crystal film into the beaker, enabling the substrate to be in contact with the dispersion liquid of the up-conversion fluorescent nano particles, and keeping the substrate at the temperature of 30 ℃ for 12 hours to perform self-assembly on the double-band-gap photonic crystal film to form a fluorescent film, thereby obtaining the substrate attached with the fluorescent film.
S8, vertically inserting the substrate with the fluorescent film into a buffer solution which contains 0.2mg/mL of dopamine and has the pH value of 8 to react for 2 hours, and actively polymerizing the dopamine on the surface of the fluorescent film to form a polydopamine film on the fluorescent film, so that the biochip can be obtained. The buffer solution may be a commercially available phosphate buffer solution.
In addition, the embodiment also provides a kit for detecting tumor markers and/or viruses, which comprises the biochip and gold nanoparticles. The preparation method of the gold nanoparticles comprises the following steps:
adding 50mL of 1mM chloroauric acid solution into a three-neck flask, stirring and heating to boil under an oil bath environment, quickly adding 5mL of 39mM sodium citrate solution into the solution after boiling, adding a condenser tube for condensation reflux in the process, gradually changing the solution into wine red, continuously heating for 15 minutes, removing a heat source, continuously stirring until the temperature is room temperature, and obtaining the aqueous solution containing gold nanoparticles, and keeping the aqueous solution at 4 ℃ in a dark place.
Example 2
This embodiment provides a biochip, and a method of preparing the biochip includes the steps of:
s1, washing 20mL of methyl methacrylate with 100mL of 3mg/mL sodium hydroxide aqueous solution for 6 times to obtain washed methyl methacrylate; then, adding 5mL of cleaned methyl methacrylate, 80mL of deionized water and 30mg of potassium persulfate into a three-neck flask, then placing the three-neck flask into an oil bath kettle at 70 ℃ for stirring reaction, and simultaneously using a condenser tube for condensation reflux to obtain a PMMA nanosphere solution with the particle size of 240 nm-280 nm for later use; in addition, 5mL of cleaned methyl methacrylate, 30mg of azodiisobutyronitrile, 30mL of deionized water and 30mL of the PMMA nanosphere solution with the particle size of 240nm to 280nm are added into a three-neck flask, then the three-neck flask is placed into an oil bath kettle at 70 ℃ for stirring reaction, and a condensing tube is used for condensation reflux at the same time, so that the PMMA nanosphere solution with the particle size of 380nm to 480nm can be obtained.
S2, taking a glass slide as a substrate, adding 60mL of deionized water and 3mL of PMMA nanosphere solution with the particle size of 240 nm-280 nm into a 100mL beaker, and uniformly stirring; and then vertically inserting the substrate into a beaker to enable the substrate to be in contact with PMMA nanosphere solution with the particle size of 240 nm-280 nm, placing the substrate at the temperature of 20 ℃ for treatment for 32 hours, taking out the substrate, placing the substrate at the temperature of 100 ℃ for drying and reinforcing to form a first photonic crystal film with the band gap position of 500nm, and obtaining the substrate attached with the first photonic crystal film.
S3, taking the other glass slide as a substrate, adding 60mL of deionized water and 3mL of PMMA nanosphere solution with the particle size of 380 nm-480 nm into a 100mL beaker, and uniformly stirring; then, placing the substrate in a beaker, enabling the substrate to be in contact with PMMA nanosphere solution with the particle size of 380 nm-480 nm, placing the substrate at the temperature of 20 ℃ for treatment for 32 hours, taking out the substrate, placing the substrate at the temperature of 100 ℃ for drying and reinforcing to form a second photonic crystal film with the band gap position of 800nm, and obtaining the substrate attached with the second photonic crystal film.
S4, vertically fixing the substrate attached with the second photonic crystal film in a beaker, injecting deionized water into the beaker by using a trace liquid injection pump, adjusting the injection rate, enabling the liquid level of the deionized water in the beaker to rise at a rate of 1mm per hour, stripping the second photonic crystal film from the surface of the substrate, and enabling the second photonic crystal film to float on the surface of the deionized water, at the moment, fishing out the second photonic crystal film by using another substrate attached with the first photonic crystal film, and enabling the second photonic crystal film floating in the deionized water to cover the first photonic crystal film of the substrate to form a double-bandgap photonic crystal film, thereby obtaining the substrate attached with the double-bandgap photonic crystal film.
S5, 0.Placing 7mmol of yttrium chloride hexahydrate, 0.15mmol of ytterbium trichloride hexahydrate, 0.01mmol of erbium chloride hexahydrate, 15mL of octadecene and 5mL of oleic acid in a 100mL three-neck flask, placing the flask in a nitrogen protective atmosphere, heating to 120 ℃ by using a heating sleeve, stirring until the solid is completely dissolved, and cooling to 35 ℃; then, 0.148g of ammonium fluoride and 0.1g of sodium hydroxide were dissolved in 7mL of methanol to obtain a methanol solution; then, dropwise adding the methanol solution into the three-neck flask, stirring for 30min, heating to 120 ℃ to remove methanol, heating the heating jacket to 270 ℃ for 90min, cooling to 35 ℃, and adding a condensing tube for condensation and reflux in the process to obtain a reactant; then, 50mL of ethanol was added to the reaction mixture, centrifuged at 9500rpm for 15min using a centrifuge, the resulting solid was dispersed in 10mL of cyclohexane, 30mL of ethanol was added and centrifuged again at 9500rpm for 15min, and the resulting solid was centrifuged to obtain a first solid, designated NaYF4: Yb3+,Er3+And dispersed in 5mL of cyclohexane until used.
S6, placing 0.6mmol of yttrium chloride hexahydrate, 0.1mmol of ytterbium trichloride hexahydrate, 0.1mmol of neodymium trichloride hexahydrate, 15mL of octadecene and 5mL of oleic acid in a 100mL three-neck flask, placing in a nitrogen protective atmosphere, heating to 120 ℃ by using a heating sleeve, stirring until the solid is completely dissolved, cooling to 35 ℃, and adding the obtained first solid into the three-neck flask; then, 0.148g of ammonium fluoride and 0.1g of sodium hydroxide were dissolved in 7mL of methanol to obtain a methanol solution; then, dropwise adding the methanol solution into the three-neck flask, stirring for 30min, heating to 120 ℃ to remove methanol, heating the heating jacket to 270 ℃ for 90min, cooling to 35 ℃, and adding a condensing tube for condensation and reflux in the process to obtain a reactant; then, 50mL of ethanol was added to the reaction mixture, centrifuged at 9500rpm for 15min using a centrifuge, the resulting solid was dispersed in 10mL of cyclohexane, 30mL of ethanol was added and centrifuged again at 9500rpm for 15min, and a second solid was obtained by centrifugation, which was designated NaYF4: Yb3+,Er3+@NaYF4:Yb3+,Nd3+And dispersing the second solid in 5mL of cyclohexane to obtain the upconversion fluorescent nanoparticle dispersion liquid for later use.
S7, putting 1mL of the obtained upconversion fluorescent nanoparticle dispersion liquid and 60mL of cyclohexane into a 100mL beaker, and uniformly mixing; and vertically inserting the substrate attached with the double-band-gap photonic crystal film into the beaker, enabling the substrate to be in contact with the dispersion liquid of the up-conversion fluorescent nano particles, and keeping the substrate at the temperature of 30 ℃ for 20 hours to perform self-assembly on the double-band-gap photonic crystal film to form a fluorescent film, thereby obtaining the substrate attached with the fluorescent film.
S8, vertically inserting the substrate with the fluorescent film into a buffer solution which contains 0.1mg/mL of dopamine and has a pH value of 7.5 to react for 2 hours, and actively polymerizing the dopamine on the surface of the fluorescent film to form a poly-dopamine film on the fluorescent film, so that the biochip can be obtained. The buffer solution may be a commercially available phosphate buffer solution.
In addition, the embodiment also provides a kit for detecting tumor markers and/or viruses, which comprises the biochip and gold nanoparticles. The preparation method of the gold nanoparticles comprises the following steps:
adding 50mL of 1mM chloroauric acid solution into a three-neck flask, stirring and heating to boil under an oil bath environment, quickly adding 5mL of 39mM sodium citrate solution into the solution after boiling, adding a condenser tube for condensation reflux in the process, gradually changing the solution into wine red, continuously heating for 15 minutes, removing a heat source, continuously stirring until the temperature is room temperature, and obtaining the aqueous solution containing gold nanoparticles, and keeping the aqueous solution at 4 ℃ in a dark place.
Example 3
This embodiment provides a biochip, and a method of preparing the biochip includes the steps of:
s1, washing 30mL of methyl methacrylate with 200mL of sodium hydroxide aqueous solution with the concentration of 15mg/mL for 8 times to obtain washed methyl methacrylate; then, adding 10mL of cleaned methyl methacrylate, 100mL of deionized water and 40mg of potassium persulfate into a three-neck flask, then placing the three-neck flask into an oil bath kettle at 100 ℃ for stirring reaction, and simultaneously using a condenser tube for condensation reflux to obtain a PMMA nanosphere solution with the particle size of 240 nm-280 nm for later use; in addition, after 7mL of cleaned methyl methacrylate, 40mg of azodiisobutyronitrile, 50mL of deionized water and 50mL of the PMMA nanosphere solution with the particle size of 240nm to 280nm are added into a three-neck flask, the three-neck flask is placed in an oil bath kettle at 100 ℃ for stirring reaction, and a condensing tube is used for condensation reflux at the same time, so that the PMMA nanosphere solution with the particle size of 380nm to 480nm can be obtained.
S2, fixing a polyethylene terephthalate (PET) flexible film subjected to ultraviolet ozone treatment on a glass slide as a substrate, adding 60mL of deionized water and 3mL of PMMA nanosphere solution with the particle size of 240-280 nm into a 100mL beaker, and uniformly stirring; and then vertically inserting the substrate into a beaker to enable the substrate to be in contact with PMMA nanosphere solution with the particle size of 240 nm-280 nm, placing the substrate at the temperature of 60 ℃ for treatment for 18 hours, taking out the substrate, placing the substrate at the temperature of 180 ℃ for drying and reinforcing to form a first photonic crystal film with the band gap position of 580nm, and obtaining the substrate attached with the first photonic crystal film.
S3, taking the other glass slide as a substrate, adding 60mL of deionized water and 3mL of PMMA nanosphere solution with the particle size of 380 nm-480 nm into a 100mL beaker, and uniformly stirring; then, placing the substrate in a beaker, enabling the substrate to be in contact with PMMA nanosphere solution with the particle size of 380 nm-480 nm, placing the substrate at the temperature of 60 ℃ for maintaining for 18h, taking out the substrate, placing the substrate at the temperature of 180 ℃ for drying and reinforcing to form a second photonic crystal film with the band gap position of 1000nm, and obtaining the substrate attached with the second photonic crystal film.
S4, vertically fixing the substrate attached with the second photonic crystal film in a beaker, injecting deionized water into the beaker by using a trace liquid injection pump, adjusting the injection rate, enabling the liquid level of the deionized water in the beaker to rise at a rate of 1mm per hour, stripping the second photonic crystal film from the surface of the substrate, and enabling the second photonic crystal film to float on the surface of the deionized water, at the moment, fishing out the second photonic crystal film by using another substrate attached with the first photonic crystal film, and enabling the second photonic crystal film floating in the deionized water to cover the first photonic crystal film of the substrate to form a double-bandgap photonic crystal film, thereby obtaining the flexible substrate attached with the double-bandgap photonic crystal film.
S5, placing 0.9mmol of yttrium chloride hexahydrate, 0.25mmol of ytterbium trichloride hexahydrate, 0.03mmol of erbium chloride hexahydrate, 20mL of octadecene and 10mL of oleic acid into a 100mL three-neck flask, placing the flask into a nitrogen protective atmosphere, heating to 170 ℃ by using a heating sleeve, stirring until the solid is completely dissolved, and cooling to 35 ℃; then, 0.148g of ammonium fluoride and 0.1g of sodium hydroxide were dissolved in 7mL of methanol to obtain a methanol solution; then, dropwise adding the methanol solution into the three-neck flask, stirring for 30min, heating to 120 ℃ to remove methanol, heating the heating jacket to 340 ℃ for 90min, cooling to 35 ℃, and adding a condensing tube for condensation and reflux in the process to obtain a reactant; then, 50mL of ethanol was added to the reaction mixture, centrifuged at 9500rpm for 15min using a centrifuge, the resulting solid was dispersed in 10mL of cyclohexane, 30mL of ethanol was added and centrifuged again at 9500rpm for 15min, and the resulting solid was centrifuged to obtain a first solid, designated NaYF4: Yb3+,Er3+And dispersed in 5mL of cyclohexane until used.
S6, placing 0.8mmol of yttrium chloride hexahydrate, 0.2mmol of ytterbium trichloride hexahydrate, 0.2mmol of neodymium trichloride hexahydrate, 20mL of octadecene and 10mL of oleic acid in a 100mL three-neck flask, placing in a nitrogen protective atmosphere, heating to 170 ℃ by using a heating sleeve, stirring until the solid is completely dissolved, cooling to 35 ℃, and adding the obtained first solid into the three-neck flask; then, 0.148g of ammonium fluoride and 0.1g of sodium hydroxide were dissolved in 7mL of methanol to obtain a methanol solution; then, dropwise adding the methanol solution into the three-neck flask, stirring for 30min, heating to 120 ℃ to remove methanol, heating the heating jacket to 340 ℃ for 90min, cooling to 35 ℃, and adding a condensing tube for condensation and reflux in the process to obtain a reactant; then, 50mL of ethanol was added to the reaction mixture, centrifuged at 9500rpm for 15min using a centrifuge, the resulting solid was dispersed in 10mL of cyclohexane, 30mL of ethanol was added and centrifuged again at 9500rpm for 15min, and a second solid was obtained by centrifugation, which was designated NaYF4: Yb3+,Er3+@NaYF4:Yb3+,Nd3+And dispersing the second solid in 5mL of cyclohexane to obtain the upconversion fluorescent nanoparticle dispersion liquid for later use.
S7, putting 1mL of the obtained upconversion fluorescent nanoparticle dispersion liquid and 60mL of cyclohexane into a 100mL beaker, and uniformly mixing; and vertically inserting the substrate attached with the double-band-gap photonic crystal film into the beaker, enabling the substrate to be in contact with the dispersion liquid of the up-conversion fluorescent nano particles, and keeping the substrate at the temperature of 50 ℃ for 8 hours to perform self-assembly on the double-band-gap photonic crystal film to form a fluorescent film, thereby obtaining the flexible substrate attached with the fluorescent film.
S8, vertically inserting the substrate with the fluorescent film into a buffer solution which contains 0.3mg/mL of dopamine and has a pH value of 9.0 to react for 2 hours, and actively polymerizing the dopamine on the surface of the fluorescent film to form a poly-dopamine film on the fluorescent film, so that the biochip can be obtained. The buffer solution may be commercially available tris buffer solution.
In addition, the embodiment also provides a kit for detecting tumor markers and/or viruses, which comprises the biochip and gold nanoparticles. The preparation method of the gold nanoparticles comprises the following steps:
adding 50mL of 1mM chloroauric acid solution into a three-neck flask, stirring and heating to boil under an oil bath environment, quickly adding 5mL of 39mM sodium citrate solution into the solution after boiling, adding a condenser tube for condensation reflux in the process, gradually changing the solution into wine red, continuously heating for 15 minutes, removing a heat source, continuously stirring until the temperature is room temperature, and obtaining the aqueous solution containing gold nanoparticles, and keeping the aqueous solution at 4 ℃ in a dark place.
Experimental example:
first, the double-bandgap photonic crystal film obtained in example 1 is observed by a scanning electron microscope, and the obtained scanning electron microscope image is shown in fig. 3. As can be seen from the figure, the photonic crystals of the double-layer structure of the double-band-gap photonic crystal film prepared by the embodiment of the invention are not damaged and are still arranged orderly.
Secondly, the first photonic crystal film, the second photonic crystal film and the double-bandgap photonic crystal film obtained in the above embodiment 1 are respectively subjected to a transmission spectrum test, the obtained transmission spectrum is shown in fig. 4, and the curves in fig. 4 are the transmission spectra of the first photonic crystal film, the second photonic crystal film and the double-bandgap photonic crystal film from top to bottom in sequence. As can be seen from the figure, the double-band-gap photonic crystal film has two optical forbidden bands at about 808nm, about 540 nm.
Thirdly, the upconversion fluorescent nanoparticle obtained in the above example 1 (NaYF4: Yb)3+,Er3+@NaYF4:Yb3+,Nd3 +) The transmission electron microscope image obtained by observing the sample with a transmission electron microscope is shown in the attached figure 5. As can be seen from the figure, the size of the upconversion fluorescent nanoparticle is about 35 nm.
Fourthly, the fluorescent film obtained in the above example 1 was observed, and the schematic structural view thereof is shown in FIG. 6. As can be seen from the figure, the nanoparticles in the fluorescent film settle in the gaps of the double-bandgap photonic crystal film.
Fifthly, the upconversion fluorescent nanoparticles obtained in the embodiment 1 are respectively placed on a GLASS substrate (GLASS), a first photonic crystal film (OPC545) and a second photonic crystal film (OPC808) to be self-assembled to form fluorescent films, and emission spectrum tests are carried out on the fluorescent films correspondingly obtained and the fluorescent films (OPC808/545) formed on the double-bandgap photonic crystal film in the embodiment 1, wherein the test results are shown in attached figures 7-8. It can be seen from the figure that the integral area of the wavelength range of 520nm to 580nm is taken to calculate the enhancement factor, wherein the enhancement effect of the fluorescent thin film formed on the double-bandgap photonic crystal thin film in the above example 1 is the best, and the enhancement factor reaches 171 times.
Sixthly, the polydopamine film obtained in example 1 is observed, and the schematic structural diagram is shown in fig. 9. As can be seen from the figure, the polydopamine film formed on the fluorescent film in the embodiment of the invention is dense and uniform.
Seventhly, the gold nanoparticles obtained in example 1 were observed by a transmission electron microscope, and the transmission electron microscope image obtained is shown in FIG. 10. As can be seen from the figure, the size of the gold nanoparticles prepared in the example of the present invention is about 15 nm.
Eighthly, taking 1mL of the gold nanoparticles obtained in the example 1, centrifuging at 12000r/min, washing twice with deionized water, dispersing again with 1mL of deionized water, adding 60uL of 0.1mg/mL detection antibody, oscillating for 1h, placing in a refrigerator at 4 ℃ for 10h to ensure that the detection antibody is connected with the gold nanoparticles through electrostatic adsorption, centrifuging at 13000r/min to remove free antibody, and dispersing in 1mL of deionized water for standby to obtain the dispersion liquid of the gold nanoparticles after the detection antibody is modified. Wherein, the gold nanoparticles after the modification of the detection antibody are subjected to absorption spectrum test, and the test result is shown in figure 11. As can be seen from the figure, the gold nanoparticles after modification of the detection antibody were compared with the upconversion fluorescent nanoparticles provided in example 1 above (NaYF4: Yb)3+,Er3+@NaYF4:Yb3+,Nd3+) Corresponds to the emission peak position.
Ninthly, PSA detection experiment: as shown in fig. 1 to 2, six detection regions were selected on the surface of the fluorescent thin film 4 obtained in example 1, the concentration of the capture antibody 6 for PSA was adjusted to 1mg/mL with phosphate buffer (PBS buffer, pH 7.2 to 7.4), 50 μ L of the capture antibody was dropped on the detection region of the fluorescent thin film 4, the sample was washed with PBS buffer after 15min reaction, 50 μ L of PSA samples (i.e., the specimen 7) having concentrations of 0ng/mL, 0.01ng/mL, 0.1ng/mL, 2ng/mL, 5ng/mL, and 12.5ng/mL were dropped on the detection region, the sample was washed again with PBS buffer after 15min reaction, 50 μ L of the dispersion of the gold-modified detection antibody 8 prepared in the above example was dropped on the detection region, the sample was washed with PBS buffer after 15min reaction, the detection region was dried and then subjected to spectroscopic detection, the detection results are shown in FIGS. 12-13. As can be seen from FIGS. 12 to 13, the fluorescence intensity in the range of 0.01ng/mL to 12.5ng/mL of PSA concentration is reduced with the increase of the PSA concentration in the analyte, and the linear relationship is good, so that the detection of PSA by using the biochip and the kit provided by the embodiment of the invention has good stability and convenience. It should be noted that the embodiment of the present invention is only an example of the detection experiment of PSA, and is not limited thereto, that is, the biochip and the kit provided in the embodiment of the present invention can be applied to detection methods using specific binding, such as antibody-antigen binding, antibody-antigen-antibody binding, and specific binding of nucleic acid, and therefore, can also be used for detection of nucleic acid, antibody, and the like of viruses such as 2019-nCoV, or detection of other types of antigens and antibodies.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A biochip comprising a rigid or flexible substrate, further comprising:
a double band gap photonic crystal film; the double-band-gap photonic crystal film is arranged on the substrate; the double-band-gap photonic crystal film is made of polymethyl methacrylate;
a fluorescent film; the fluorescent film is arranged on the double-band-gap photonic crystal film; the fluorescent film is made of up-conversion fluorescent nano-particles;
a polydopamine film; the polydopamine film is arranged on the fluorescent film.
2. A method for preparing the biochip according to claim 1, comprising the steps of:
taking a substrate, placing the substrate in a PMMA nanosphere solution with the particle size of 240-280 nm, treating at the temperature of 20-60 ℃, taking out the substrate, and drying and reinforcing at the temperature of 100-180 ℃ to obtain the substrate attached with the first photonic crystal film;
taking the other substrate, placing the substrate in a PMMA nanosphere solution with the particle size of 380-480 nm, treating at the temperature of 20-60 ℃, taking out the substrate, and drying and reinforcing at the temperature of 100-180 ℃ to obtain the substrate attached with the second photonic crystal film; then, using deionized water to strip the substrate attached with the second photonic crystal film to obtain a second photonic crystal film;
covering the substrate attached with the first photonic crystal film with a second photonic crystal film to form a double-bandgap photonic crystal film, and obtaining the substrate attached with the double-bandgap photonic crystal film;
placing the substrate attached with the double-bandgap photonic crystal film in the upconversion fluorescent nanoparticle dispersion liquid, and treating at the temperature of 30-50 ℃ to form a fluorescent film on the double-bandgap photonic crystal film to obtain the substrate attached with the fluorescent film;
and placing the substrate attached with the fluorescent film in a buffer solution containing dopamine and having a pH value of 7.5-9.0 for reaction to form a polydopamine film on the fluorescent film, thereby obtaining the biochip.
3. The method for preparing a biochip according to claim 2, wherein the band gap position of the first photonic crystal thin film is 500nm to 580 nm; the band gap position of the second photonic crystal film is 800 nm-1000 nm.
4. The method for preparing a biochip according to claim 2, wherein the method for preparing the PMMA nanosphere solution with the particle size of 240nm to 280nm comprises the following steps:
cleaning methyl methacrylate with 3-15 mg/mL sodium hydroxide aqueous solution to obtain cleaned methyl methacrylate;
placing the cleaned methyl methacrylate, deionized water and potassium persulfate at the temperature of 70-100 ℃ for reaction to obtain PMMA nanosphere solution with the particle size of 240-280 nm; wherein the volume mass ratio of the cleaned methyl methacrylate to the potassium persulfate is (5-10): (30-40) in terms of mL/mg.
5. The method for preparing biochip according to claim 4, wherein the method for preparing PMMA nanosphere solution with particle size of 380 nm-480 nm comprises the following steps:
placing the cleaned methyl methacrylate, the cleaned azodiisobutyronitrile, the deionized water and the PMMA nanosphere solution with the particle size of 240 nm-280 nm at the temperature of 70-100 ℃ for reaction to obtain the PMMA nanosphere solution with the particle size of 380 nm-480 nm; wherein the volume ratio of the cleaned methyl methacrylate to the PMMA nanosphere solution with the particle size of 240 nm-280 nm is (5-7) to (30-50); the volume-to-mass ratio of the cleaned methyl methacrylate to the azobisisobutyronitrile is (5-7): 30-40 (mL/mg).
6. The method for preparing a biochip according to claim 2, wherein the method for preparing the dispersion of upconversion fluorescent nanoparticles comprises the following steps:
placing yttrium chloride, ytterbium trichloride, erbium chloride, octadecene and oleic acid at the temperature of 120-170 ℃ for stirring, then placing ammonium fluoride and sodium hydroxide at the temperature of 270-340 ℃ for reaction, and then performing centrifugal separation to obtain a first solid; wherein the molar ratio of yttrium chloride, ytterbium trichloride and erbium chloride is (0.7-0.9): (0.15-0.25): 0.01-0.03);
placing yttrium chloride, ytterbium trichloride, neodymium trichloride, octadecene and oleic acid at the temperature of 120-170 ℃ for stirring, then placing the yttrium chloride, ytterbium trichloride, neodymium trichloride, octadecene and oleic acid at the temperature of 270-340 ℃ for reacting with ammonium fluoride, sodium hydroxide and the first solid, and then carrying out centrifugal separation to obtain a second solid; wherein the molar ratio of yttrium chloride, ytterbium trichloride and neodymium trichloride is (0.6-0.8): (0.1-0.2): 0.1-0.3);
and dispersing the second solid in cyclohexane to obtain the upconversion fluorescent nanoparticle dispersion liquid.
7. A biochip obtained by the method according to any one of claims 1 to 6.
8. Use of the biochip according to claim 1 or 7 for the preparation of a kit for the detection of tumor markers and/or viruses.
9. A kit for detecting a tumor marker and/or a virus, comprising the biochip of claim 1 or 7.
10. A kit as claimed in claim 9, further comprising gold nanoparticles.
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| HENG LI ETAL: "Fluorescence enhancement by heterostructure colloidal photonic crystals with dual stopbands", 《JOURNAL OF COLLOID AND INTERFACE SCIENCE》 * |
| XIAOXIA HU ETAL: "Naked eye detection of multiple tumor-related mRNAs from patients with photonic-crystal micropattern supported dual-modal upconversion bioprobes", 《CHEMICAL SCIENCE》 * |
| 周平伟: "三维光子晶体的光致发光增强性质以及对稀土掺杂无机物的发光调控", 《中国博士学位论文全文数据库》 * |
| 许红威: "基于磁性/荧光复合纳米材料和微流控芯片的循环肿瘤细胞检测应用", 《中国优秀硕士学位论文全文数据库》 * |
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| CN112342115A (en) * | 2020-10-21 | 2021-02-09 | 吉林大学 | Microfluidic chip for capturing and/or counting cells and preparation method and application thereof |
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