Disclosure of Invention
Based on the prior art, the invention provides a plasmon enhanced fluorescence immunoassay chip and application thereof, and the chip can be used for immunoassay, such as detection of biomolecules such as nucleic acid, protein and polypeptide, and can significantly improve the flux and sensitivity of immunoassay.
The technical scheme adopted for realizing the above purpose of the invention is as follows:
a plasmon enhanced fluorescence immunoassay chip comprises a substrate, wherein a periodic nanometer pit structure is prepared on the substrate, nanometer pits in the periodic nanometer pit structure are distributed in a matrix mode and are in an inverted pyramid shape, a metal adhesion layer is deposited on the periodic nanometer pit structure, a metal enhancement layer is deposited on the metal adhesion layer, and a macromolecule layer is modified on the metal enhancement layer.
The top surface of the nano pit is square, the side surface of the nano pit is isosceles triangle, the side length of the top surface of the nano pit is 300-1400nm, the ratio of the side length of the top surface of the nano pit to the depth of the nano pit is 1:1.3-1.5, and the period of the nano pit structure is 500-2000 nm.
The metal used for the metal adhesion layer is chromium or titanium, and the thickness of the metal adhesion layer is 5-30 nm.
The metal selected for the metal enhancement layer is gold or silver, and the thickness of the metal is 200-300 nm.
The thickness of the polymer layer is 10-100nm, and the polymer structure units selected in the polymer layer are as follows:
the substrate is made of silicon.
When the substrate is made of silicon, the preparation method of the plasmon enhanced fluorescence immunoassay chip comprises the following steps:
1. taking a substrate A with a silicon oxide layer, coating an electron beam photoresist layer on the silicon oxide layer, processing a matrix formed by periodically arranging cylindrical grooves on the electron beam photoresist layer by using an electron beam exposure method, wherein the period of the cylindrical groove matrix is 500-2000nm, the diameter of the cylindrical groove is 300-1400nm, and the depth of the cylindrical groove is 200nm, and exposing the silicon oxide layer at the bottom of each cylindrical groove by developing;
2. placing the substrate A processed in the step 1 into reactive ion etching equipment, introducing trifluoromethane gas into the reactive ion etching equipment, etching the substrate A processed in the step 1, then replacing trifluoromethane into oxygen, introducing oxygen into the reactive ion etching equipment for etching again, taking out the substrate A processed by etching after etching is finished, dropwise adding trimethylchlorosilane into a vessel, placing the substrate A processed by etching into the vessel without contacting with the trimethylchlorosilane, sealing the vessel and standing, taking out the substrate A subjected to standing processing, cleaning and drying to obtain a nano-imprint template;
3. carrying out nano imprinting on the nano imprinting template by using a polystyrene film, transferring the periodic cylindrical groove matrix onto the polystyrene film, and obtaining a periodic cylindrical protrusion matrix on the polystyrene film;
4. taking a substrate B with a silicon oxide layer, coating an ultraviolet nano-imprinting adhesive layer on the silicon oxide layer, attaching one surface of a polystyrene film, which is printed with a periodic cylindrical convex matrix, to the ultraviolet nano-imprinting adhesive layer, carrying out nano-imprinting again, transferring the periodic cylindrical convex matrix to the ultraviolet nano-imprinting adhesive layer, and obtaining a periodic cylindrical groove matrix on the ultraviolet nano-imprinting adhesive layer;
5. putting the substrate B processed in the step 4 into a reactive ion etching device, introducing oxygen into the reactive ion etching device, etching off the residual ultraviolet nanoimprint lithography glue at the bottom of each cylindrical groove, exposing the silicon oxide layer at the bottom of each cylindrical groove, then replacing the oxygen with trifluoromethane, introducing the trifluoromethane into the reactive ion etching device for etching again, completely etching the exposed silicon oxide layer at the bottom of each cylindrical groove, exposing the substrate B at the bottom of each cylindrical groove, then replacing the trifluoromethane with the oxygen, introducing the oxygen into the reactive ion etching device, and etching off the residual ultraviolet nanoimprint lithography glue to obtain a substrate to be processed;
6. corroding the substrate to be treated with a potassium hydroxide solution, preparing a periodic nano pit structure on the substrate B to obtain a periodic nano pit structure, and removing the residual silicon oxide layer on the substrate B by using hydrofluoric acid to obtain a periodic nano pit structure substrate;
7. depositing a metal adhesion layer with the thickness of 5-30nm on the periodic nanometer pit structure substrate by adopting a physical vapor deposition method, and then depositing a metal enhancement layer with the thickness of 200-300nm on the metal adhesion layer to obtain the periodic metal nanometer pit structure substrate;
8. and coupling and modifying a high polymer layer with the thickness of 10-100nm on the substrate with the periodic metal nano pit structure.
An application of plasmon enhanced fluorescence immunoassay chip in immunoassay.
Compared with the prior art, the invention has the beneficial effects and advantages that:
1. the chip of the invention is prepared with a special periodic nanometer pit structure, namely the nanometer pit is in an inverted pyramid shape, and the fluorescence signal can be enhanced by plasmon resonance in the special periodic nanometer pit structure and the metal layer on the nanometer pit structure, compared with other nanometer pits in common shapes, such as a cylinder shape, the fluorescence signal intensity is enhanced by about 35 times, thus the sensitivity of immunoassay can be greatly improved by adopting the special periodic nanometer pit structure.
2. The chip is used for detecting EMMPRIN protein (extracellular matrix metalloproteinase inducing factor), and compared with an ELISA technology, the detection sensitivity is improved by 26 times.
3. The preparation method of the chip is simple, the chip is manufactured by adopting the technologies of electron beam lithography, reactive ion etching, nano imprinting, physical vapor deposition and the like, the manufacturing process is simple and easy to operate, and the preparation cost is low.
Example 2
1. Taking a silicon substrate A (purchased commercially) with a silicon oxide layer with the thickness of 300nm, spinning and coating an electron beam photoresist layer (adopting PMMA photoresist) with the thickness of 200nm on the silicon oxide layer, processing a matrix formed by periodically arranging cylindrical grooves on the electron beam photoresist layer by using an electron beam exposure method, wherein the period of the periodic cylindrical groove matrix is 2000nm, the diameter of each cylindrical groove is 1400nm, the depth of each cylindrical groove is 200nm, and developing to expose the silicon oxide layer at the bottom of each cylindrical groove;
2. putting the silicon substrate A processed in the step 1 into reactive ion etching equipment, introducing trifluoromethane gas into the reactive ion etching equipment, and etching the substrate A processed in the step 1, wherein the etching conditions are as follows: the flow rate of trifluoromethane is 300sccm, the gas pressure is 1.6Pa, the radio frequency power is 150W, and the etching time is 250 s; then, replacing trifluoromethane as oxygen, introducing oxygen into the reactive ion etching equipment for etching again, wherein the etching conditions are as follows: the oxygen flow is 100sccm, the gas pressure is 1Pa, the radio frequency power is 100W, and the etching time is 60 s; after the etching is finished, taking out the etched substrate A, dropwise adding 0.5mL of trimethylchlorosilane into a vessel, placing the etched substrate A in the vessel without contacting with the trimethylchlorosilane, sealing the vessel and standing for 30min, taking out the substrate A subjected to standing treatment, cleaning with ethanol and drying by blowing to obtain the nano-imprint template, wherein the nano-imprint template is shown in a figure 2 (a);
3. performing nano imprinting on the nano imprinting template by using a polystyrene film, wherein the pressure of the nano imprinting is 40bar, the temperature is 150 ℃, as shown in fig. 2(b), transferring the periodic cylindrical groove matrix onto the polystyrene film, and obtaining a periodic cylindrical protrusion matrix on the polystyrene film, as shown in fig. 2 (c);
4. taking a silicon substrate B with a silicon oxide layer with the thickness of 100nm, spin-coating an ultraviolet nano imprinting adhesive layer with the thickness of 200nm on the silicon oxide layer, attaching one surface of a polystyrene film printed with a periodic cylindrical protrusion matrix to the ultraviolet nano imprinting adhesive layer as shown in figure 2(d), and then carrying out nano imprinting again, wherein the pressure of the nano imprinting is 30bar, the temperature is 65 ℃, as shown in figure 2(e), transferring the periodic cylindrical protrusion matrix to the ultraviolet nano imprinting adhesive layer, and obtaining a periodic cylindrical groove matrix on the ultraviolet nano imprinting adhesive layer as shown in figure 2 (f);
5. putting the substrate B processed in the step 4 into reactive ion etching equipment, introducing oxygen into the reactive ion etching equipment, etching off the residual ultraviolet nano imprint glue at the bottom of each cylindrical groove, and exposing the silicon oxide layer at the bottom of each cylindrical groove, wherein the etching conditions are as follows: the oxygen flow is 100sccm, the gas pressure is 1Pa, the radio frequency power is 50W, and the etching time is 30 s; then, changing oxygen into trifluoromethane, introducing trifluoromethane into the reactive ion etching equipment for etching again, so that the silicon oxide layer exposed at the bottom of each cylindrical groove is completely etched, and the silicon at the bottom of each cylindrical groove is exposed, wherein as shown in fig. 2(g), the etching conditions are as follows: the flow rate of trifluoromethane is 300sccm, the gas pressure is 1.6Pa, the radio frequency power is 150W, and the etching time is 250 s; and finally, replacing trichloromethane as oxygen, introducing oxygen into the reactive ion etching equipment, etching the residual ultraviolet nanoimprint lithography glue to obtain a substrate to be processed, wherein the etching conditions are as follows as shown in figure 2 (h): the oxygen flow is 100sccm, the gas pressure is 1Pa, the radio frequency power is 100W, and the etching time is 60 s;
6. corroding the substrate to be treated with 28.6 mass percent potassium hydroxide solution for 15min, controlling the size of the inverted pyramid-shaped nano pits by controlling the corrosion time, preparing a periodic nano pit structure on the substrate B as shown in fig. 2(i), and removing residual silicon oxide by using hydrofluoric acid to obtain a periodic nano pit structure substrate as shown in fig. 2 (j);
7. depositing a metal adhesion layer (chromium is selected as metal) with the thickness of 30nm on the periodic nanometer pit structure substrate by adopting a physical vapor deposition method, and then depositing a metal enhancement layer (gold is selected as metal) with the thickness of 300nm on the metal adhesion layer to obtain the periodic metal nanometer pit structure substrate;
8. coupling and modifying a macromolecule layer on a periodic metal nano pit structure substrate:
8.1, uniformly mixing ethanol and water according to the volume ratio of the ethanol to the water of 4:1 to obtain a mixed solvent, adding mercaptoundecanol into the mixed solvent to prepare a mixed solution containing 10mmol/L mercaptoundecanol, soaking the periodic metal nano-pit structure substrate into the mixed solution for 12 hours to enable a coupling hydroxyl-based layer on the metal enhancement layer;
8.2, adding epichlorohydrin into 0.2mol/L sodium hydroxide solution to prepare 0.2mol/L epichlorohydrin alkaline solution, soaking the periodic metal nano pit structure substrate treated by 8.1 into the epichlorohydrin alkaline solution for 4 hours, activating a hydroxyl layer into an epoxy group layer, and soaking the periodic metal nano pit structure substrate containing the epoxy group layer into 0.3g/mL glucan solution or chitosan solution for 20 hours to obtain the periodic metal nano pit structure substrate with a glucan layer or chitosan layer with the thickness of 10 nm;
8.3, adding bromoacetic acid into 2mol/L NaOH solution to prepare 0.1mol/L bromoacetic acid alkaline solution, soaking the periodic metal nano-pit structure substrate treated by 8.3 into the bromoacetic acid alkaline solution for 12 hours, and modifying the glucan layer or the chitosan layer into a carboxylated glucan layer;
and 8.4, soaking the periodic metal nano pit structure substrate treated in the step 8.3 into an aqueous solution containing 0.1mol/L of N-hydroxysuccinimide and 0.1mol/L of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) for 1 hour to obtain the plasmon enhanced fluorescence immunoassay chip described in the example 1, and marking the chip as an immunoassay chip-S.
The plasmon-enhanced fluorescence immunoassay chip prepared in this example was scanned with a scanning electron microscope, and the obtained SEM image is shown in fig. 3, and as can be seen from fig. 3, the nano-pits on the plasmon-enhanced fluorescence immunoassay chip prepared in this example are in the shape of inverted pyramids, and the nano-pits are distributed in a matrix.
The first test is that the test method for detecting the Goat anti Mouse IgG antibody by the plasmon enhanced fluorescence immunoassay chip comprises the following steps:
1. preparing a goat anti-mouse antibody phosphate buffer solution with the concentration of 1mg/mL, immersing the plasmon-enhanced fluorescence immunoassay chip prepared in the embodiment 2 into the goat anti-mouse antibody phosphate buffer solution with the concentration of 60 minutes, taking out the chip, and soaking, cleaning and washing the chip for three times by using the phosphate buffer solution, wherein each time lasts for 5 minutes;
2. preparing a mouse antibody phosphate buffer solution with the concentration of 1mg/mL, soaking the plasmon-enhanced fluorescence immunoassay chip processed in the step 1 into the mouse antibody phosphate buffer solution for 30 minutes, taking out, and soaking and cleaning the chip with the phosphate buffer solution for three times, wherein each time lasts for 5 minutes;
3. preparing 1ug/mL goat anti-mouse antibody phosphate buffer solution marked by fluorescent Alexa 647, soaking the plasmon-enhanced fluorescence immunoassay chip processed in the step 2 into the goat anti-mouse antibody phosphate buffer solution marked by fluorescent Alexa 647 for 15 minutes, taking out, soaking and cleaning the chip with the phosphate buffer solution for three times, each time for 5 minutes, washing the chip with deionized water, and drying the chip with nitrogen;
4. detecting fluorescent signals of the plasmon enhanced fluorescence immunoassay chip processed in the step 3, which is provided with the periodic metal nano-pit structure area and the area without the periodic metal nano-pit structure (namely, the plane area) by using a fluorescence scanner (with an excitation wavelength of 632 nm);
and (3) test results:
after the plasmon enhanced fluorescence immunoassay chip prepared in example 2 is modified with the coat anti Mouse IgG antibody, the fluorescence detection result is shown in fig. 4, and as can be seen from fig. 4, the fluorescence of the chip on the region with the periodic metal nano-pit structure is enhanced by about 120 times compared with the region without the periodic metal nano-pit structure, and the detection sensitivity is higher as the fluorescence signal intensity is enhanced more.
Second, detection limit test of plasmon enhanced fluorescence immunoassay chip of the present invention
In this experiment, the plasmon enhanced fluorescence immunoassay chip prepared in example 2 and a commercial kit were used (R&D male Scuman Emmpin Duo Set kit) The two antibodies are compared with the detection limit of the EMMPRIN antibody, and the specific test method is as follows:
1. the detection method of the plasmon-enhanced fluorescence immunoassay chip prepared in example 2 is as follows:
1.1, preparing 5 parts of goat anti-human EMMPRIN antibody phosphate buffer solution with the concentration of 250ug/mL, respectively soaking 5 pieces of plasmon enhanced fluorescence immunoassay chips prepared according to the method in the embodiment 2 into 5 parts of goat anti-human EMMPRIN antibody phosphate buffer solution for 60min, taking out 5 pieces of plasmon enhanced fluorescence immunoassay chips, and then soaking and cleaning the chips for three times by using the respective phosphate buffer solutions, wherein each time lasts for 5 min;
1.2, preparing EMMPRIN phosphate buffer solutions with the concentrations of 150pg/mL, 15pg/mL, 1.5pg/mL, 0.15pg/mL and 0pg/mL respectively, soaking the 5 pieces of plasmon enhanced fluorescence immunoassay chips processed in the step 1 into the EMMPRIN phosphate buffer solutions with the concentrations of 150pg/mL, 15pg/mL, 1.5pg/mL, 0.15pg/mL and 0pg/mL respectively for 120min, taking out the 5 pieces of plasmon enhanced fluorescence immunoassay chips, and soaking and washing the chips with the phosphate buffer solutions for three times, 5 minutes each time;
1.3, preparing 5 parts of mouse anti-human EMMPRIN antibody phosphate buffer solution marked by fluorescent Alexa 647, respectively soaking 5 pieces of plasmon enhanced fluorescence immunoassay chips processed in the step 2 into 5 parts of mouse anti-human EMMPRIN antibody phosphate buffer solution marked by fluorescent Alexa 647, wherein the soaking time is 30 minutes, taking out 5 pieces of plasmon enhanced fluorescence immunoassay chips, soaking and cleaning the chips with the respective phosphate buffer solutions for three times, 5 minutes each time, washing with deionized water, and drying with nitrogen;
1.4, detecting the fluorescence signals of the 5 plasmon enhanced fluorescence immunoassay chips processed in the step 3 by using a fluorescence scanner (the excitation wavelength is 632nm, and the emission wavelength is 677 nm);
and (3) test results:
1. plotting the fluorescence signal intensity as a vertical coordinate and the concentration of EMMPRIN phosphate buffer solution as a horizontal coordinate according to data detected by 5 pieces of plasmon enhanced fluorescence immunoassay chips, wherein the obtained point line graph is shown in figure 5, a standard curve is obtained after fitting, and the detection limit of the plasmon enhanced fluorescence immunoassay chip prepared in the embodiment 2 is 1.5pg/mL according to the standard curve;
2. the fluorescence signal intensity is used as a vertical coordinate, the concentration of EMMPRIN phosphate buffer solution is used as a horizontal coordinate, the detected data of the commercial kit are plotted, the obtained point line graph is shown in figure 6, a standard curve is obtained after fitting, and 39pg/mL of the commercial kit is obtained according to the standard curve; therefore, compared with a kit adopting an ELISA technology, the detection limit of the plasmon enhanced fluorescence immunoassay chip is greatly reduced, the detection sensitivity is improved by 26 times, and the detection sensitivity is greatly improved.
Comparative example 1
The structure of the plasmon-enhanced fluorescence immunoassay chip prepared in this comparative example is shown in fig. 7, and includes a substrate, in this example, a silicon oxide substrate is used as the substrate.
As shown in fig. 7, a periodic nano pit structure is prepared on a substrate 7, nano pits in the periodic nano pit structure are distributed in a honeycomb shape, the period of the nano pit structure is 500nm, nano pits 2 are cylindrical, the diameter of the nano pits 2 is 400nm, and the depth of the nano pits 2 is 300 nm.
And a metal adhesion layer 3 with the thickness of 10nm is deposited on the periodic nanometer pit structure, and the metal selected for the metal adhesion layer 3 is chromium.
A metal enhancement layer 4 with the thickness of 100nm is deposited on the metal adhesion layer 3, and the metal selected for the metal enhancement layer 4 is gold.
The metal enhancement layer 4 is modified with a polymer layer 5 with the thickness of 10nm, and the structural units of the polymer used in the polymer layer 5 are as follows:
comparative example 2
1. Taking a silicon substrate A with a silicon oxide layer with the thickness of 300nm, spin-coating an electron beam photoresist layer (adopting PMMA photoresist) with the thickness of 200nm on the silicon oxide layer, processing a honeycomb array formed by periodically arranging cylindrical grooves on the electron beam photoresist layer by using an electron beam exposure method, wherein the period of the honeycomb array of the periodic cylindrical grooves is 500nm, the diameter of the cylindrical grooves is 400nm, the depth of the cylindrical grooves is 200nm, and then exposing the silicon oxide layer at the bottom of each cylindrical groove through development;
2. putting the silicon substrate A processed in the step 1 into reactive ion etching equipment, introducing trifluoromethane gas into the reactive ion etching equipment, and etching the substrate A processed in the step 1, wherein the etching conditions are as follows: the flow rate of trifluoromethane is 300sccm, the gas pressure is 1.6Pa, the radio frequency power is 150W, and the etching time is 250 s; then, replacing trifluoromethane as oxygen, introducing oxygen into the reactive ion etching equipment for etching again, wherein the etching conditions are as follows: the oxygen flow is 100sccm, the gas pressure is 1Pa, the radio frequency power is 100W, and the etching time is 60 s; after the etching is finished, taking out the etched substrate A, dropwise adding 0.5mL of trimethylchlorosilane into a vessel, placing the etched substrate A in the vessel without contacting with the trimethylchlorosilane, sealing the vessel and standing for 30min, taking out the substrate A subjected to standing treatment, cleaning with ethanol and drying by blowing to obtain a nano-imprint template, wherein the nano-imprint template is shown in fig. 8 (a);
3. performing nano imprinting on the nano imprinting template by using a polystyrene film, wherein the pressure of the nano imprinting is 40.5bar, the temperature is 150 ℃, as shown in fig. 8(b), transferring the periodic cylindrical groove matrix onto the polystyrene film, and obtaining a periodic cylindrical protrusion matrix on the polystyrene film, as shown in fig. 8 (c);
4. taking a silicon substrate B with a silicon oxide layer with the thickness of 100nm, spin-coating an ultraviolet nano imprinting adhesive layer with the thickness of 200nm on the silicon oxide layer, attaching one surface of a polystyrene film printed with a periodic cylindrical protrusion matrix to the ultraviolet nano imprinting adhesive layer as shown in fig. 8(d), and then carrying out nano imprinting again, wherein the pressure of the nano imprinting is 30bar, the temperature is 65 ℃, as shown in fig. 8(e), transferring the periodic cylindrical protrusion matrix to the ultraviolet nano imprinting adhesive layer, and obtaining a periodic cylindrical groove matrix on the ultraviolet nano imprinting adhesive layer as shown in fig. 8 (f);
5. putting the substrate B processed in the step 4 into reactive ion etching equipment, introducing oxygen into the reactive ion etching equipment, etching off the residual ultraviolet nano imprint glue at the bottom of each cylindrical groove, and exposing the silicon oxide layer at the bottom of each cylindrical groove, wherein the etching conditions are as follows: the oxygen flow is 100sccm, the gas pressure is 1Pa, the radio frequency power is 50W, and the etching time is 30 s; then, changing oxygen into trifluoromethane, introducing trifluoromethane into the reactive ion etching equipment for etching again, so that the silicon oxide layer exposed at the bottom of each cylindrical groove is completely etched, and the silicon at the bottom of each cylindrical groove is exposed, as shown in fig. 8(g), the etching conditions are as follows: the flow rate of trifluoromethane is 300sccm, the gas pressure is 1.6Pa, the radio frequency power is 150W, and the etching time is 250 s; and finally, replacing trichloromethane as oxygen, introducing oxygen into the reactive ion etching equipment, etching the residual ultraviolet nanoimprint lithography glue to obtain a substrate to be processed, wherein the etching conditions are as follows as shown in fig. 8 (h): the oxygen flow is 100sccm, the gas pressure is 1Pa, the radio frequency power is 100W, and the etching time is 60 s;
7. depositing a metal adhesion layer (chromium is selected as metal) with the thickness of 10nm on the periodic nanometer pit structure substrate by adopting a physical vapor deposition method, and then depositing a metal enhancement layer (gold is selected as metal) with the thickness of 100nm on the metal adhesion layer to obtain the periodic metal nanometer pit structure substrate;
8. coupling and modifying a macromolecule layer on a periodic metal nano pit structure substrate:
8.1, uniformly mixing ethanol and water according to the volume ratio of the ethanol to the water of 4:1 to obtain a mixed solvent, adding mercaptoundecanol into the mixed solvent to prepare a mixed solution containing 10mmol/L mercaptoundecanol, soaking the periodic metal nano-pit structure substrate into the mixed solution for 12 hours to enable a coupling hydroxyl-based layer on the metal enhancement layer;
8.2, adding epichlorohydrin into 0.2mol/L sodium hydroxide solution to prepare 0.2mol/L epichlorohydrin alkaline solution, soaking the periodic metal nano pit structure substrate treated by 8.1 into the epichlorohydrin alkaline solution for 4 hours, activating a hydroxyl layer into an epoxy group layer, and soaking the periodic metal nano pit structure substrate containing the epoxy group layer into 0.3g/mL glucan solution or chitosan solution for 20 hours to obtain the periodic metal nano pit structure substrate with a glucan layer or chitosan layer with the thickness of 10 nm;
8.3, adding bromoacetic acid into 2mol/L NaOH solution to prepare 0.1mol/L bromoacetic acid alkaline solution, soaking the periodic metal nano-pit structure substrate treated by 8.3 into the bromoacetic acid alkaline solution for 12 hours, and modifying the glucan layer or the chitosan layer into a carboxylated glucan layer;
and 8.4, soaking the periodic metal nano pit structure substrate treated in the step 8.3 into an aqueous solution containing 0.1mol/L of N-hydroxysuccinimide and 0.1mol/L of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) for 1 hour to obtain the plasmon enhanced fluorescence immunoassay chip described in the example 1, and marking the chip as an immunoassay chip-D.
The plasmon-enhanced fluorescence immunoassay chip prepared in the comparative example was scanned with a scanning electron microscope, and the obtained SEM image is shown in fig. 9, and as can be seen from fig. 9, the nano-pits on the plasmon-enhanced fluorescence immunoassay chip prepared in the comparative example were cylindrical and were distributed in a honeycomb shape.
Third, Effect test of different plasmon enhanced fluorescence immunoassay chip immunoassay
The test method comprises the following steps:
1. preparing two goat anti-mouse antibody phosphate buffer solutions with the concentration of 1mg/mL, respectively immersing the immunoassay chip-S prepared in the example 2 and the immunoassay chip-D prepared in the comparative example 2 into the two goat anti-mouse antibody phosphate buffer solutions, immersing for 60 minutes, taking out the two immunoassay chips, respectively immersing and cleaning the two immunoassay chips with the phosphate buffer solutions for three times, and 5 minutes each time;
2. preparing two mouse antibody phosphate buffer solutions with the concentration of 1ug/mL, respectively soaking the immunodetection chip-S and the immunodetection chip-D processed in the step 1 into the two mouse antibody phosphate buffer solutions for 30 minutes, taking out the two immunodetection chips, respectively soaking and cleaning the two immunodetection chips in the phosphate buffer solutions for three times, and 5 minutes each time;
3. preparing two parts of fluorescent Alexa 647-labeled 1ug/mL goat anti-mouse antibody phosphate buffer, respectively soaking the immunodetection chip-S and the immunodetection chip-D processed in the step 2 into the two parts of Alexa 647-labeled goat anti-mouse antibody phosphate buffer for 15 minutes, taking out the two immunodetection chips, respectively soaking and cleaning the chips with the phosphate buffer for three times, 5 minutes each time, washing with deionized water, and drying with nitrogen;
4. detecting the fluorescence signals of the immunodetection chip-S and the immunodetection chip-D processed in the step 3 by using a fluorescence scanner (the excitation wavelength is 632nm and the emission wavelength is 677 nm);
and (3) test results:
the fluorescence detection results of the plasmon-enhanced fluorescence immunoassay chips prepared in example 2 and comparative example 2 after the Goat anti mouse IgG antibody is modified are shown in FIG. 9, and it can be seen from FIG. 9 that the fluorescence signal of the immunoassay chip S prepared in example 2 is enhanced by 30 times, and the stronger the fluorescence signal, the higher the detection sensitivity, compared to the immunoassay chip D prepared in comparative example 2.