CN113514397A - Device for enhancing fluorescence signal collection efficiency in immunoassay and preparation method - Google Patents
Device for enhancing fluorescence signal collection efficiency in immunoassay and preparation method Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The invention discloses a device for enhancing the fluorescent signal collection efficiency in immunodetection and a preparation method thereof, belonging to the field of immunofluorescence detection. The device for enhancing the fluorescent signal collection efficiency in the immunoassay reflects the angle information of the antigen or the antibody bound on the surface of the solid phase carrier to a certain degree; the preparation method of the invention changes a large part of fluorescence emission into conical directional emission to enter the glass substrate, and can observe strong fluorescence signals at the precise angle corresponding to the side surface of the prism, and the special layered structure can enhance the collection efficiency of the fluorescence signals in immunoassay and improve the detection sensitivity, and SiO2Layer protectionThe metal protection film is oxidized and is used as an isolation layer to prevent fluorescent molecules from directly contacting with metal to generate fluorescent quenching.
Description
Technical Field
The invention belongs to the technical field of optical films, and particularly relates to a device for enhancing fluorescence signal collection efficiency in immunoassay and a preparation method thereof.
Background
Metal particles and metal films exhibit rich, complex optoelectronic properties. When light irradiates the metal thin film at a very precise angle, i.e., the lateral component of the wave vector of the incident light matches the wave vector of the surface plasmon, the electromagnetic field is efficiently coupled to the surface plasmon, resulting in a great attenuation of the reflected light. The excited surface plasmon in the metal film generates an evanescent field with high intensity, and the evanescent field can penetrate through the dielectric medium on the metal surface and enter a hundreds of nanometers area of the sample to be coupled with the sample.
On the contrary, the fluorescence molecules bound on the surface of the thin metal surface are excited by light, and the excited fluorescence molecule groups generate an electromagnetic field which is strongly interacted with free electrons in the metal film, so that surface plasmons are generated. The frequency of these surface plasmons coincides with the emission frequency of the fluorescent molecule, and we observe a strong directional emission, called coupled emission of surface plasmons. This directional emission results from the coupling of the oscillating dipole of the excited fluorescent molecule with surface plasmons on the metal surface, followed by radiation onto the glass substrate. The coupled emission of surface plasmons exhibits the same spectral linetype as fluorescent molecules, but is highly correlated with the polarization state of the angle of emission back to the glass substrate. Simple optical systems based on surface plasmon coupled emission may provide 50% light collection efficiency and very high intrinsic wavelength resolution.
Most of the existing immunodetection technologies are designed on a transparent substrate with a layer of protein antibody bound on the surface, and the antibody protein molecules are detected by using surface fluorescent signals. In a free space environment, the fluorescent signal emitted by the fluorescent molecule is isotropic, and thus, the detection sensitivity is limited in part by the efficiency of fluorescent signal collection.
Meanwhile, in immunoassay, the strong fluorescence background of the biological sample due to the high-energy excitation light also limits the sensitivity of immunofluorescence assay. The emission of surface plasmon coupling is only related to fluorescent molecules that are tightly bound near the metal surface, and intense fluorescence can only be observed at precise angles.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a device for enhancing the fluorescent signal collection efficiency in immunoassay, which has simple structure; it is another object of the present invention to provide a method for preparing the same.
The utility model provides a device of fluorescence signal collection efficiency among reinforcing immunodetection, includes glass prism, glass slide, metallic film, silica protective layer, macromolecular layer, the sample groove that superpose the setting in proper order.
Furthermore, the glass prism is a hemispherical glass prism.
Further, the metal film is gold or silver, the thickness of the metal film ranges from 50nm to 100nm, and the frequency of the surface plasmon in the metal film is required to be consistent with the emission frequency of the fluorescent molecules.
Furthermore, the thickness of the silicon dioxide protective layer is 5-10 nm, and the silicon dioxide protective layer is also used as an isolation layer to prevent fluorescent molecules from directly contacting with metal to generate fluorescent quenching.
Furthermore, the thickness of the polymer layer is 10-50 nm, and the polymer layer is used for coupling biological molecules and is selected from nucleic acid, protein and polypeptide.
Furthermore, the sample groove is used for conveniently dripping the antigen or the antibody marked by the fluorescent molecule, and exciting light can be incident from the right upper part of the sample groove or from the lower part of the glass prism.
Further, the device is equipped with a fiber optic probe that rotates freely around the glass prism.
A method for preparing a device for enhancing the collection efficiency of a fluorescence signal in immunoassay comprises the following steps:
1) taking a glass slide, putting the glass slide into a glass cleaning solution, cleaning the glass slide in an ultrasonic environment, cleaning the glass slide in deionized water and absolute ethyl alcohol, and drying the glass slide; depositing a metal film with the thickness of 50nm by adopting a magnetron sputtering method, and depositing a silicon dioxide protective layer with the thickness of 5-10 nm on the metal film by adopting a chemical vapor deposition method to obtain a sandwich layer structure substrate;
2) preparing a mixed solvent of water and alcohol, adding 6-mercapto-1-hexanol into the mixed solvent to prepare a 6-mercapto-1-hexanol mixed solution, and soaking the substrate with the sandwich layer structure into the mixed solution to couple the hydroxyl base layer on the silicon dioxide protective layer;
3) soaking the sandwich layered structure substrate treated in the step 2) into an ethanol solution of epoxy chloropropane, activating a hydroxyl layer to form an epoxy group layer, soaking the sandwich layered structure substrate containing the epoxy group layer into a glucan solution or a chitosan solution to obtain a layered structure substrate with a glucan layer or a chitosan layer, then soaking the layered structure substrate into a bromoacetic acid solution, and modifying the glucan layer or the chitosan layer into a carboxylated glucan layer or a chitosan layer;
4) spin-coating a mixed aqueous solution of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride on the substrate with the sandwich structure processed in the step 3) to obtain a laminated structure substrate bound with a macromolecule layer;
5) the substrate bound with the polymer laminated structure is placed on a hemispherical glass prism, the glass prism is combined with a glass slide glass by using adhesive liquid, and a circular sample groove is prepared on the top.
Further, in the step 2), the volume ratio of water to alcohol is 1:4, the concentration of the 6-mercapto-1-hexanol mixed solution is 20-50 mmol/L, and the soaking time is 12-24 hours.
Further, the preparation method of the device for enhancing the fluorescence signal collection efficiency in the immunoassay comprises the following steps of 3), wherein in the step 3), the concentration of an ethanol solution of epoxy chloropropane is 0.2-0.5 mol/L, and the soaking time is 3-5 hours; the concentration of the glucan solution or the chitosan solution is 0.5-1 g/mL, and the soaking time is 20-30 hours; the concentration of the bromoacetic acid solution is 0.2-0.5 mol/L, and the soaking time is 10-12 hours;
further, in the step 4), the concentration of N-hydroxysuccinimide in the mixed aqueous solution is 0.1-0.5 mol/L, and the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.1-0.5 mol/L.
Further, in the step 5), the refractive index of the adhesive liquid is completely matched with that of the glass.
The invention principle is as follows: the directional emission of the surface plasmon coupling is generated due to the coupling of the oscillating dipole of the excited fluorescent molecule with the surface plasmon on the metal surface, followed by irradiation onto the glass substrate. The surface plasmon coupled emission exhibits the same spectral lineshape as the fluorescent molecular emission, but is highly correlated with the polarization state of the radiation to the glass substrate. The surface plasmons excited by the fluorescent molecules in the thin metal layer are radiated into the glass prism at a specific angle, and thus, strong fluorescent emission can be observed at a precise angle of one side of the prism, which is determined by the emission wavelength, the hierarchical structure, and the optical characteristics of the glass prism. It is possible to provide 50% light collection efficiency using a simple optical system based on the surface plasmon coupling launching technique. By using the directional emission of surface plasmon coupling to design the immunofluorescence detection platform, a great part of fluorescence emission can be converted into conical directional emission to enter the glass substrate, so that the signal collection efficiency is obviously improved, the background fluorescence of a biological sample is effectively reduced, and meanwhile, the angle information capable of reflecting the information of the biological molecular groups bound on the surface can be reserved.
Has the advantages that: compared with the prior art, the device for enhancing the fluorescent signal collection efficiency in the immunoassay reflects the angle information of the antigen or the antibody bound on the surface of the solid phase carrier to a certain degree; the preparation method of the invention changes a large part of fluorescence emission into conical directional emission to enter the glass substrate, and can observe strong fluorescence signals at the precise angle corresponding to the side surface of the prism, and the special layered structure can enhance the collection efficiency of the fluorescence signals in immunoassay and improve the detection sensitivity, and SiO2The layer protects the metal film from oxidation and simultaneously serves as an isolation layer to prevent fluorescent molecules from directly contacting with metalAnd (4) quenching fluorescence.
Drawings
FIG. 1 is a schematic diagram of the apparatus configuration;
FIG. 2 is a flow chart of the device design;
FIG. 3 is a schematic illustration of angle measurement;
reference numerals: 1-glass prism, 2-glass slide glass, 3-metal film, 4-silicon dioxide protective layer, 5-macromolecule layer, 6-sample groove and 7-optical fiber detector.
Detailed Description
In order to further illustrate the present invention, the following embodiments are provided to describe the device and the method for enhancing the efficiency of collecting fluorescence signals in immunoassay.
The simple optical device is designed based on the principle of surface plasmon coupling emission, and comprises a glass prism 1, a glass slide 2, a metal film 3, a silica protective layer 4, a macromolecular layer 5, a sample groove 6 and an optical fiber detector 7. The glass prism 1 is arranged at the lowest part in a simple optical device structure diagram designed based on the principle of surface plasmon coupling emission, the glass slide glass 2 for depositing the metal film 3 is arranged on the glass prism 1, the glass prism 1 and the glass slide glass 2 are combined by adhesive liquid, the silicon dioxide protective layer 4 grows on the metal film 3, the high polymer layer 5 is modified on the silicon dioxide protective layer 4, and the O-shaped or rectangular sample groove 6 is arranged at the uppermost part.
The glass prism 1 is a hemispherical glass prism.
The metal selected for the metal film 3 is gold or silver, the thickness range of the metal is set to be 50-100 nm, and the frequency of the surface plasmon in the metal film 3 is required to be consistent with the emission frequency of the fluorescent molecules.
The adhesive liquid is a matching liquid with the same refractive index as the glass.
The thickness of the silicon dioxide protective layer 4 is 5-10 nm, and the silicon dioxide protective layer 4 is also used as an isolation layer to prevent fluorescent molecules from directly contacting with metal to generate fluorescent quenching.
The thickness of the polymer layer 5 is 10-50 nm, and the polymer layer 5 is used for coupling biological molecules such as nucleic acid, protein, polypeptide and the like.
The sample groove 6 is used for conveniently dripping the antigen or the antibody marked by the fluorescent molecule, and exciting light can be incident from the right upper part of the sample groove 6 or can be incident from the lower part of the glass prism 1.
The fiber detector 7 can freely rotate around the hemispherical prism.
Example 1
A schematic diagram of a device for enhancing the efficiency of fluorescent signal collection in immunoassay is shown in FIG. 1. A method for preparing a device for enhancing the efficiency of collecting fluorescence signals in immunoassay, as shown in fig. 2, comprising the steps of:
1) taking a glass slide 2, putting the glass slide into a glass cleaning solution, cleaning the glass slide for 30min under an ultrasonic environment, cleaning the glass slide in deionized water and absolute ethyl alcohol, and drying the glass slide; depositing a metal film 3 with the thickness of 50nm by adopting a magnetron sputtering method, and then depositing a silicon dioxide protective layer 4 with the thickness of 5nm on the metal film 3 by adopting a chemical vapor deposition method to obtain a sandwich layer structure substrate;
2) preparing a mixed solvent of water and alcohol, wherein the volume ratio of the water to the alcohol is 1:4, adding 6-mercapto-1-hexanol into the mixed solvent to prepare a mixed solution of 20 mmol/L6-mercapto-1-hexanol, soaking the substrate with the sandwich layer structure into the mixed solution for 12 hours to couple a hydroxyl base layer on the silicon dioxide protective layer 4;
3) adding epoxy chloropropane into the ethanol solution to prepare an epoxy chloropropane ethanol solution containing 0.2mol/L, soaking the sandwich substrate coupled with the hydroxyl base layer in epoxy chloropropane for 3 hours, and activating the hydroxyl layer into an epoxy group layer;
4) soaking the sandwich substrate containing the epoxy group layer in 0.5g/mL of glucan solution or chitosan solution for 20 hours to obtain a substrate bound with the glucan layer or the chitosan layer; then soaking the chitosan layer in 0.2mol/L bromoacetic acid solution for 10 hours to modify the glucan layer or the chitosan layer into a carboxylated glucan layer;
5) preparing a mixed aqueous solution of 0.1mol/L N-hydroxysuccinimide and 0.1mol/L (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), and spin-coating the mixed aqueous solution on the substrate with the sandwich structure processed by the steps to obtain a bound high molecular layer;
6) the substrate of the layered structure is placed on a hemispherical glass prism 1, the glass prism 1 and a glass slide 2 are combined by an adhesive solution, and a circular sample groove 6 is prepared on the uppermost design of the structure.
Example 2
1) Taking a glass slide 2, putting the glass slide into a glass cleaning solution, cleaning the glass slide for 30min under an ultrasonic environment, cleaning the glass slide in deionized water and absolute ethyl alcohol, and drying the glass slide; depositing a metal film 3 with the thickness of 50nm by adopting a magnetron sputtering method, and then depositing a silicon dioxide protective layer 4 with the thickness of 8nm on the metal film 3 by adopting a chemical vapor deposition method to obtain a sandwich layer structure substrate;
2) preparing a mixed solvent of water and alcohol, wherein the volume ratio of the water to the alcohol is 1:4, adding 6-mercapto-1-hexanol into the mixed solvent to prepare a 30 mmol/L6-mercapto-1-hexanol mixed solution, soaking the sandwich layer structure substrate into the mixed solution for 20 hours to couple a hydroxyl base layer on the silicon dioxide protective layer 4;
3) adding epoxy chloropropane into an ethanol solution to prepare an epoxy chloropropane ethanol solution containing 0.3mol/L, soaking the sandwich substrate coupled with the hydroxyl base layer in epoxy chloropropane for 4 hours, and activating the hydroxyl layer into an epoxy group layer;
4) soaking the sandwich substrate containing the epoxy group layer in 0.8g/mL of glucan solution or chitosan solution for 25 hours to obtain a substrate bound with the glucan layer or the chitosan layer; then soaking the chitosan layer in 0.3mol/L bromoacetic acid solution for 10 hours to modify the glucan layer or the chitosan layer into a carboxylated glucan layer;
5) preparing a mixed aqueous solution of 0.3mol/L N-hydroxysuccinimide and 0.3mol/L (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), and spin-coating the mixed aqueous solution on the substrate with the sandwich structure processed by the steps to obtain a bound high molecular layer;
6) the substrate of the layered structure is placed on a hemispherical glass prism 1, the glass prism 1 and a glass slide 2 are combined by an adhesive solution, and a circular sample groove 6 is prepared on the uppermost design of the structure.
Example 3
1) Taking a glass slide 2, putting the glass slide into a glass cleaning solution, cleaning the glass slide for 30min under an ultrasonic environment, cleaning the glass slide in deionized water and absolute ethyl alcohol, and drying the glass slide; depositing a metal film 3 with the thickness of 50nm by adopting a magnetron sputtering method, and then depositing a silicon dioxide protective layer 4 with the thickness of 10nm on the metal film 3 by adopting a chemical vapor deposition method to obtain a sandwich layer structure substrate;
2) preparing a mixed solvent of water and alcohol, wherein the volume ratio of water to alcohol is 1:4, adding 6-mercapto-1-hexanol into the mixed solvent to prepare a 50 mmol/L6-mercapto-1-hexanol mixed solution, soaking the sandwich layer structure substrate into the mixed solution for 24 hours to couple a hydroxyl base layer on the silicon dioxide protective layer 4;
3) adding epoxy chloropropane into the ethanol solution to prepare an epoxy chloropropane ethanol solution containing 0.5mol/L, soaking the sandwich substrate coupled with the hydroxyl base layer in epoxy chloropropane for 5 hours, and activating the hydroxyl layer into an epoxy group layer;
4) soaking the sandwich substrate containing the epoxy group layer in a 1g/mL glucan solution or a chitosan solution for 30 hours to obtain a substrate bound with the glucan layer or the chitosan layer; then soaking the chitosan layer in 0.5mol/L bromoacetic acid solution for 12 hours, and modifying the glucan layer or the chitosan layer into a carboxylated glucan layer;
5) preparing a mixed aqueous solution of 0.5mol/L N-hydroxysuccinimide and 0.5mol/L (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), and spin-coating the mixed aqueous solution on the substrate with the sandwich structure processed by the steps to obtain a bound high molecular layer;
6) the substrate of the layered structure is placed on a hemispherical glass prism 1, the glass prism 1 and a glass slide 2 are combined by an adhesive solution, and a circular sample groove 6 is prepared on the uppermost design of the structure.
As shown in FIG. 3, (a) is an experimental setup diagram for fluorescence angular emission measurement, and (b) is a distribution diagram of angular emission intensity. The apparatus used in this experiment was the one prepared in example 3, a metal thin filmGold with a thickness of 50nm and a high delamination thickness of about 30nm is selected for deposition of SiO2The layer thickness was 10nm and the sample was Alexa Fluor 488-labeled goat anti-mouse IgG (H + L) phosphate buffer. The excitation light in fig. 3(a) has a wavelength of 495nm, and the fluorescence having a wavelength around 520nm is detected at a specific angle by a movable fiber optic method. Fig. 3(b) is a distribution diagram of angular emission intensity. The graph shows the intensity of fluorescence detected by the fiber as a function of the azimuthal angle θ. The figure shows that the observed fluorescence intensity increases dramatically at an azimuth angle of around 47 °.
Claims (10)
1. The device for enhancing the collection efficiency of the fluorescence signal in the immunoassay is characterized by comprising a glass prism (1), a glass slide (2), a metal film (3), a silicon dioxide protective layer (4), a high polymer layer (5) and a sample groove (6) which are sequentially overlapped.
2. The device for enhancing the efficiency of collecting fluorescence signals in immunoassay according to claim 1, wherein the glass prism (1) is a hemispherical glass prism.
3. The device for enhancing the collection efficiency of fluorescence signals in immunoassay according to claim 1, wherein the metal thin film (3) is gold or silver, the thickness of the metal thin film is 50 to 100nm, and the frequency of surface plasmons in the metal thin film (3) is consistent with the emission frequency of fluorescent molecules.
4. The device for enhancing fluorescence signal collection efficiency in immunoassay according to claim 1, wherein the thickness of the silica protective layer (4) is 5 to 10 nm.
5. The device for enhancing fluorescence signal collection efficiency in immunoassay according to claim 1, wherein the thickness of the polymer layer (5) is 10 to 50nm, and the polymer layer (5) is selected from nucleic acid, protein, polypeptide.
6. The device for enhancing the efficiency of collecting fluorescence signals in immunoassay according to claim 1, wherein the excitation light is incident from directly above the sample well (6) or from below the glass prism (1).
7. The device for enhancing the efficiency of fluorescence signal collection in immunoassay according to claim 1, wherein the device is equipped with a fiber optic probe (7), said fiber optic probe 7 being freely rotatable around the glass prism (1).
8. The method of claim 1 for preparing a device for enhancing the efficiency of fluorescent signal collection in an immunoassay, comprising the steps of:
1) taking a glass slide (2), putting the glass slide into a glass cleaning solution, cleaning the glass slide in an ultrasonic environment, cleaning the glass slide in deionized water and absolute ethyl alcohol, and drying the glass slide; depositing a metal film (3) with the thickness of 50-100 nm by adopting a magnetron sputtering method, and depositing a silicon dioxide protective layer (4) with the thickness of 5-10 nm on the metal film (3) by adopting a chemical vapor deposition method to obtain a sandwich layer structure substrate;
2) preparing a mixed solvent of water and alcohol, adding 6-mercapto-1-hexanol into the mixed solvent to prepare a 6-mercapto-1-hexanol mixed solution, and soaking the substrate with the sandwich layer structure into the mixed solution to couple a hydroxyl base layer on the silicon dioxide protective layer (4);
3) soaking the sandwich layered structure substrate treated in the step 2) into an ethanol solution of epoxy chloropropane, activating a hydroxyl layer to form an epoxy group layer, soaking the sandwich layered structure substrate containing the epoxy group layer into a glucan solution or a chitosan solution to obtain a layered structure substrate with a glucan layer or a chitosan layer, then soaking the layered structure substrate into a bromoacetic acid solution, and modifying the glucan layer or the chitosan layer into a carboxylated glucan layer or a chitosan layer;
4) spin-coating a mixed aqueous solution of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride on the substrate with the sandwich structure processed in the step 3) to obtain a laminated structure substrate bound with a macromolecule layer;
5) the substrate bound with the polymer laminated structure is placed on a hemispherical glass prism (1), the glass prism (1) and a glass slide glass (2) are combined by using adhesive liquid, and a circular sample groove (6) is designed and prepared at the top.
9. The method for preparing a device for enhancing fluorescence signal collection efficiency in immunoassay according to claim 8, wherein in the step 2), the volume ratio of water to alcohol is 1:4, the concentration of the 6-mercapto-1-hexanol mixed solution is 20 to 50mmol/L, and the soaking time is 12 to 24 hours; in the step 3), the concentration of the ethanol solution of the epoxy chloropropane is 0.2-0.5 mol/L, and the soaking time is 3-5 hours; the concentration of the glucan solution or the chitosan solution is 0.5-1 g/mL, and the soaking time is 20-30 hours; the concentration of the bromoacetic acid solution is 0.2-0.5 mol/L, and the soaking time is 10-12 hours.
10. The method for preparing a device for enhancing fluorescence signal collection efficiency in immunoassay according to claim 8, wherein in the step 4), the concentration of N-hydroxysuccinimide and the concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the mixed aqueous solution are respectively 0.1-0.5 mol/L and 0.1-0.5 mol/L, respectively.
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