Ratio fluorescence biosensor based on photonic crystal signal amplification and application thereof

Abstract

The invention provides a ratio fluorescence biosensor based on photonic crystal signal amplification and application thereof. The biosensor comprises a fluorescent magnetic bead modified by a capture antibody, a detection antibody modified by biotin, avidin modified by fluorescein and a substrate layer, wherein a photonic crystal layer is deposited on the substrate layer. The detection method provided by the invention has the advantages of small sample dosage, convenience in detection, rapidness, high efficiency, high sensitivity and low cost, and has the potential to be applied to various scenes requiring detection of low-abundance markers, such as drug research and development, early diagnosis of nerve diseases, tumor recurrence monitoring, discovery and confirmation of disease markers and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, and in particular to a ratiometric fluorescence biosensor based on photonic crystal signal amplification and application thereof. Background Art

[0002] Detection of protein biomarkers in body fluids such as serum, urine and saliva plays an important role in disease diagnosis, prognosis assessment, recurrence monitoring, and medication guidance. Among them, in the early stages of the disease, the concentration of protein markers that can be detected in body fluids is often very low, but the cure rate of many diseases, especially cancer, in early intervention treatment is much higher than that in the middle and late stages. At present, there are reports based on fluorescence detection, surface enhanced Raman, enzyme-linked immunosorbent, electrochemical sensing, immunoassay, chemiluminescence and other means for the detection of protein markers. Considering the importance of high-sensitivity detection of low-abundance proteins in early diagnosis, it is still necessary to develop low-cost, fast, efficient, highly sensitive and selective high-performance biosensors to further improve the sensitivity of detection and reduce the cost of detection to meet people's needs for medical testing in the context of "big health".

[0003] Direct detection of single molecules represents the limit of analytical detection sensitivity. Thanks to the development of single-photon imaging systems and microscopic imaging technology, single-molecule detection can be achieved by using total internal reflection microscopy, single-molecule fluorescence correlation spectroscopy and other technologies. However, in the detection of actual samples, the reading of single-molecule signals is often interfered by background signals, reading noise and matrix effects. Therefore, it is often necessary to improve the signal-to-noise ratio through signal amplification methods such as enzyme amplification and nucleic acid-related signal amplification to ensure detection sensitivity. However, this will bring more cumbersome operations, higher detection costs and longer detection times. This undoubtedly hinders the application of these methods in actual clinical detection. Therefore, it is urgent to develop new signal amplification analysis methods to shorten detection time, reduce operations and meet the needs of high-sensitivity detection.

[0004] Photonic crystal refers to an artificial periodic dielectric structure with photonic bandgap characteristics. This structure has the ability to converge light paths and enhance fluorescent signals, and has important applications in optical devices. This structure is often affected by the orientation of the modifier, nonspecific adsorption and matrix effects in biological detection and analysis, which restricts its practical application in clinical diagnosis. CN108508204A discloses a fluorescent immunoassay device and method combining photonic crystals with magnetic beads, including a photonic crystal; at least one magnetic bead, with multiple fluorescent molecules bonded to the surface, each of which is bound to a biological molecule to be detected, and the magnetic bead is located on the surface of the photonic crystal; a light source, providing an incident excitation light to resonate with the photonic crystal to enhance the electric field on the surface of the photonic crystal, and further exciting the fluorescent molecules to generate a fluorescent signal enhanced by the electric field; and a fluorescent sensor, receiving the fluorescent signal radiated by the fluorescent molecules to the fluorescent sensor and the fluorescent signal reflected by the surface of the photonic crystal, and processing it into a sensing image. However, the detection of a single magnetic bead in this patent requires a high concentration of the biomolecule to be tested (for example, 1000pg / mL, the area of ​​each magnetic bead is about 20*20pixels), resulting in low detection sensitivity. Another method in this patent is to detect the total intensity of the fluorescence signal of multiple magnetic beads / the number of magnetic bead particles, and enhance the fluorescence signal by aggregating the magnetic beads to obtain the fluorescence signal of a single magnetic bead. Although this method can reduce the concentration of the biomolecule to be tested (10 -4 pg / mL), but the detection accuracy also decreases, and absolute quantitative detection cannot be achieved. This is because the fluorescence signal of low-abundance proteins is easily interfered by background signals, which will cause large errors, and the total fluorescence intensity value is not an absolute value, but is related to the environment and conditions of each detection. The operation is cumbersome and the analysis error is large. Therefore, this method cannot achieve high sensitivity and high accuracy at the same time.

[0005] Therefore, there is an urgent need to develop a high-performance biosensor that can detect low-abundance proteins with high sensitivity and accuracy. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a ratiometric fluorescence biosensor based on photonic crystal signal amplification and its application, which solves the problem that the prior art cannot simultaneously achieve high sensitivity and high accuracy in detection.

[0007] In one aspect, the present invention provides a ratiometric fluorescent biosensor based on photonic crystal signal amplification, comprising fluorescent magnetic beads modified with capture antibodies, detection antibodies modified with biotin, avidin modified with fluorescein, and a substrate layer on which a photonic crystal layer is deposited.

[0008] Furthermore, the preparation method of the fluorescent magnetic beads modified with the capture antibody includes: activating the fluorescent magnetic beads and then connecting them with the capture antibody;

[0009] Preferably, the fluorescent magnetic beads are activated by the EDC / NHS method, and the fluorescent magnetic beads are carboxyl fluorescent magnetic beads;

[0010] Preferably, the mass ratio of capture antibody to fluorescent magnetic beads is 1:100.

[0011] Furthermore, the method for preparing the biotin-modified detection antibody comprises: activating the biotin and reacting it with the detection antibody;

[0012] Preferably, biotin is activated by NHS to obtain NHS-biotin;

[0013] Preferably, the molar ratio of the detection antibody to NHS-biotin is 1:5-100.

[0014] Furthermore, the method for preparing the substrate layer includes: soaking the substrate in a washing liquid, washing and drying;

[0015] Preferably, the substrate is glass;

[0016] Preferably, the washing liquid is a piranha washing liquid; more preferably, the piranha washing liquid comprises concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass concentration of 30%, and the volume ratio of concentrated sulfuric acid to hydrogen peroxide is 7:3;

[0017] Preferably, the soaking temperature is 60-90°C and the soaking time is 30-60min;

[0018] Preferably, washing is performed using water, anhydrous ethanol, and methanol, respectively, and the number of washing times is 3-6 times;

[0019] Preferably, the drying temperature is 100-120° C. and the drying time is 10-20 h.

[0020] Furthermore, the preparation method of the photonic crystal layer includes: depositing the photonic crystal on the surface of the substrate and then curing it;

[0021] Preferably, the deposition time is 0.5-1.5h; the deposition humidity is 65-95%;

[0022] Preferably, the curing temperature is 65-85° C. and the curing time is 5-20 min.

[0023] Furthermore, the photonic band gap of the photonic crystal matches the emission wavelength of fluorescein.

[0024] Preferably, when the size of the photonic crystal monomer is between 200-220 nm, the forbidden band range is between 480-530 nm, and the fluorescent pigment includes atto 488, FAM, and Alexa 488.

[0025] Furthermore, the photonic crystal is prepared by emulsion polymerization;

[0026] Preferably, methyl methacrylate, acrylic acid and styrene are mixed and dissolved, and then subjected to polymerization reaction to obtain the product.

[0027] Furthermore, the fluorescence emission wavelength of the fluorescent magnetic beads does not match the photonic band gap of the photonic crystal.

[0028] Another aspect of the present invention provides the use of the ratiometric fluorescence biosensor based on photonic crystal signal amplification in detecting markers, wherein the detection is for non-diagnostic purposes.

[0029] In a specific embodiment of the present invention, the markers include proteins and nucleic acids.

[0030] In another aspect, the present invention provides a method for detecting a marker using a ratiometric fluorescence biosensor based on photonic crystal signal amplification, comprising the following steps:

[0031] S1 Capture marker: Mix the capture antibody-modified fluorescent magnetic beads, the biotin-modified detection antibody and the marker and incubate them once to separate the capture marker fluorescent magnetic beads;

[0032] S2 performs fluorescein labeling: adding fluorescein-modified avidin to the fluorescent magnetic beads that capture the marker, performing secondary incubation, and separating the fluorescein-labeled fluorescent magnetic beads;

[0033] S3 is added to the basal layer for imaging analysis: fluorescent magnetic beads labeled with fluorescein are added to the basal layer, and the fluorescent signals of the fluorescent magnetic beads and the fluorescent signals of fluorescein are detected respectively, and the fluorescence ratio is calculated, and the concentration of the tumor marker to be tested is obtained through the standard curve;

[0034] The concentration of the capture antibody-modified fluorescent magnetic beads is 0.1-10 mg / mL, the concentration of the detection antibody is 0.1 μg / mL-100 μg / mL, and the concentration of the fluorescein-modified streptavidin is 0.1 μg / mL-10 μg / mL.

[0035] The technical principle of the present invention is:

[0036] In the prior art, the fluorescent marker-immunomagnetic bead coupling method detects antigens, and the fluorescence intensity of the magnetic conjugate is detected by a fluorescence detector to detect the antigen content in the sample to be detected. However, it is impossible to achieve high sensitivity and high accuracy of a single magnetic bead at the same time. After repeated experiments, the inventors found that combining fluorescent magnetic beads, fluorescent labeled antibodies and photonic crystals can achieve high sensitivity and high accuracy detection of a single magnetic bead. The mechanism mainly includes:

[0037] 1) Magnetic beads are used to separate the target object to be detected from the complex matrix, eliminating the matrix effect and reducing the background signal; at the same time, the protein is aggregated to the surface of the magnetic beads, and a single fluorescent magnetic bead is used as a separate detection unit. The single-particle near-field analysis technology is used to reduce the detection volume and further eliminate background interference; the single-molecule algorithm is realized through fluorescence ratio signal analysis.

[0038] 2) The protein to be detected is labeled with streptavidin with a fluorescent molecule tag using streptavidin-avidin interaction. Thanks to the super strong affinity between streptavidin-avidin and the one-to-many labeling ratio of the target protein and streptavidin, the fluorescence signal intensity of the single target label is guaranteed.

[0039] 3) The target protein is captured and enriched using antibody-modified large Stokes shift fluorescent magnetic beads, and the antibody sandwich method and streptavidin-avidin are used to amplify and label the fluorescent dye with high photostability and high light intensity.

[0040] 4) The emission range of the fluorescent dye matches the band gap of the photonic crystal, effectively amplifying its fluorescent signal. The inventors found that the fluorescent signal is related to the excitation and emission band of the fluorescent dye. The dyes excited at about 488nm and emitted at about 520nm have good amplification effects, such as Atto488, FAM, etc. Since photonic crystals also have good amplification effects in other bands, the inventors further prepared photonic crystals with amplification effects at about 560nm and 660nm, and emitted at about 590nm and 690nm. However, the inventors found that the photonic crystals with amplification effects on the dyes excited at about 560nm and emitted at about 590nm have a stronger amplification effect on the background matrix; the photonic crystals with amplification effects on the dyes excited at about 660nm and emitted at about 690nm will cross-color with the magnetic bead channel, which will affect the detection effect, resulting in inaccurate detection results and reduced sensitivity.

[0041] 5) The fluorescence emission spectrum of the beads does not match the band gap of the photonic crystal, but the fluorescence properties are not affected.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The present invention is a single-molecule, single-particle near-field analysis, in which each magnetic bead is a separate detection object, and is directly quantified by a digital quantitative method of the number of bright spots / number of magnetic beads or a ratio signal of the magnetic bead detection channel signal value / magnetic bead signal channel value. The detection method uses a small amount of sample, does not require calibration, and is absolutely quantitative. The detection is convenient, fast and efficient, has a large detection range, high sensitivity, high accuracy, and low cost. It has the potential to be applied to various scenarios that require the detection of low-abundance markers, such as drug development, early diagnosis of neurological diseases, tumor recurrence monitoring, and discovery and confirmation of disease markers.

[0044] (2) The present invention utilizes magnetic bead enrichment and separation technology and near-field magnetic bead single particle analysis technology to reduce background signals and improve sensitivity; utilizes streptavidin-avidin amplification and photonic crystal signal amplification to effectively, quickly and directly amplify the signal on a single magnetic bead, thereby making up for the defects of weak signal and complicated amplification operation in single molecule and single particle analysis, and high signal-to-noise ratio signal can be obtained in a short time without additional complex operation, thereby ensuring detection sensitivity; by analyzing the fluorescence ratio signal, that is, the ratio of the fluorescence marker signal intensity to the magnetic bead fluorescence signal intensity, the environmental influence of the fluorescence intensity signal is eliminated, single molecule calculation is realized, the accuracy and sensitivity are improved, absolute quantitative analysis is realized, and the influence of factors such as instruments, environment and operation on the absolute value of fluorescence is eliminated; the shortcomings of insufficient sensitivity and inability to achieve absolute quantification in the analysis of magnetic bead total fluorescence signals are made up for. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of the detection principle of the present invention;

[0046] Figure 2 This is a SEM image of the photonic crystal in Example 3 of the present invention;

[0047] Figure 3 This is a comparison diagram of imaging with and without photonic crystal amplification in Example 4 of the present invention;

[0048] Figure 4 This is a working curve diagram for detecting leukemia inhibitory factor (LIF) in Example 4 of the present invention;

[0049] Figure 5 It is a working curve diagram for detecting leukemia inhibitory factor (LIF) in the comparative example of the present invention; DETAILED DESCRIPTION

[0050] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0051] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Carboxyl functionalized fluorescent magnetic beads were purchased from Invitrogen, USA.

[0052] The principle of surface antibody modification of carboxyl magnetic beads by the EDC / NHS method: the carboxyl groups on the surface of the magnetic beads first react with the prepared EDC solution to generate unstable amino-active O-acylurea intermediates, which then react with NHS to generate semi-stable amino-reactive NHS esters. The products then react with the amino groups on the antibodies to generate immunomagnetic beads.

[0053] The detection principle of the present invention is as follows Figure 1 shown.

[0054] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; NHS: N-hydroxysuccinimide;

[0055] Example 1 Preparation of Antibody Modified Magnetic Beads

[0056] After dispersing the carboxyl functionalized fluorescent magnetic beads into 0.1M sodium hydroxide solution, ultrasonically clean them for 10s, and then wash them three times with water. After washing, disperse them into 90μL pH=4.5 buffer, then add 10μL EDC and NHS to get a mixed solution, the solvent is DMSO. At this time, the concentration of magnetic beads is 10mg / mL, the concentration of EDC is 2mM, and the concentration of NHS is 5mM. After 15min of reaction in the dark, quickly wash them with water three times, remove the solution, and quickly disperse the magnetic beads into 50μL PBS buffer containing 0.4mg / mL antibody (2mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , 136mM NaCl, 2.6mM KCl, pH=8). After 2h of reaction in the dark, the solution was removed and 100μL 50mM ethanolamine was added for blocking for 0.5h. After blocking, the solution was removed using a magnetic stand. After washing three times with a cleaning solution, the magnetic beads were dispersed in 100μL of a storage solution to obtain antibody-modified magnetic beads.

[0057] The cleaning solution formula is: 2mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , 136 mM NaCl, 2.6 mM KCl, pH = 7.4, 0.05% Tween 20. After washing five times, disperse into the preservation solution and store at 4°C.

[0058] Storage solution formula: 2mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , 136mM NaCl, 2.6mM KCl, pH=7.4, 0.05% Tween20, 1% BSA, 0.05% Proclean.

[0059] Example 2 Preparation of detection antibodies

[0060] Mix the detection antibody and NHS-biotin in a PBS solution at pH = 8, with a molar ratio of antibody: NHS-biotin = 1:5 to 1:100. After stirring at room temperature for 2 hours, use a 30K ultrafiltration tube to centrifuge and change the solution five times to remove excess NHS-biotin. The centrifugal speed is 10000g for a fixed angle centrifuge and 4500g for a hanging basket centrifuge. After the detection antibody is divided, store it in a -20 refrigerator for later use.

[0061] Example 3 Preparation of Photonic Crystal Signal Rapid Amplification Base Layer

[0062] 1. Preparation of photonic crystals

[0063] Polystyrene methyl methacrylate-acrylic acid microspheres (poly(St-MMA-AA)) were prepared by emulsion polymerization. Methyl methacrylate (10.00mmol), acrylic acid (13.89mmol) and styrene (182.60mmol) were dissolved in 100 ml of water (containing trace emulsifiers sodium dodecylbenzene sulfonate and ammonium bicarbonate). The reaction mixture was kept at 70°C for 0.5 hours. Then, an aqueous solution of ammonium persulfate (APS, 2.12mmol) was added and stirred at 80°C for 10 hours to obtain polymer microspheres of a specific size, highly uniform and highly dispersed. The size is about 200-220nm, and the SEM image of the photonic crystal is shown in the figure. Figure 2 shown.

[0064] 2. Preparation of Photonic Crystal Signal Rapid Amplification Substrate

[0065] Soak the glass slide or cover glass in 80mL of "piranha" washing solution. The preferred washing solution formula is 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3. Carefully mix in an open container and stir quickly to dissipate heat. Soak in a 75℃ water bath for 30min, then use a large amount of ultrapure water, anhydrous ethanol, and methanol to wash thoroughly 3 times, blow dry with nitrogen, and blow dry at 110℃ for 12h. After cooling, add 1uL of the uniformly dispersed polystyrene methyl methacrylate-acrylic acid microsphere colloidal solution with a particle size of 220nm prepared in step 1, stand in a 95% constant humidity environment for 30min, and heat and cure at 80℃ for 5min to obtain the signal amplification substrate. The substrate without the deposition of the photonic crystal film is set as the control group.

[0066] Similarly, a similar signal amplification substrate can be obtained by dropping a polystyrene microsphere dispersion, a silica microsphere solution or other nanoparticle dispersion of a suitable concentration onto a glass substrate and allowing the dispersion to naturally deposit and solidify in a constant humidity environment.

[0067] Example 4 Method for detecting leukemia inhibitory factor

[0068] 1. Target capture

[0069] 5 μL of 0.1 mg / mL capture antibody-modified fluorescent magnetic beads were incubated with 45 μL of target protein solution and 5 μL of 10 μg / mL biotinylated detection antibody solution at room temperature for 1 hour, and then the magnetic beads were separated from the solution using a commercially available magnetic separation stand, the solution was discarded, and the magnetic beads were washed 3 times with a cleaning solution to remove free detection antibodies. At this point, the capture antibody-modified magnetic beads, target protein, and biotinylated detection antibody formed a sandwich structure.

[0070] 2. Fluorescent labeling

[0071] The magnetic beads were then dispersed into 30 μL of phosphate buffer containing 1 μg / mL streptavidin (SA) labeled with atto 488, and incubated at room temperature for 15 min with rotation and shaking to label the fluorescent molecules; the magnetic beads were separated with a magnetic stand, and the solution was carefully removed with a pipette, and washed three times with a cleaning solution, and the washed magnetic beads were dispersed into 5 μL of solution, and then dropped onto the imaging substrate, and the magnetic beads were quickly adsorbed onto the imaging substrate using a magnet to prevent the solution from evaporating and produce a coffee ring effect. A cover glass was placed on top of the substrate for imaging.

[0072] 3. Imaging and data processing: Place the substrate under a microscope and use a 20x magnification, NA = 0.4 air lens for imaging. The dye excitation light is a 488nm wavelength monochromatic laser with a power of 1mW. Use a FAM module filter to collect the signal. The magnetic beads use the same excitation light and a cy5 module filter to collect the signal. The fluorescence signal is shown in the figure below. Figure 3 shown.

[0073] Data processing: MATLAB software was used to calculate the fluorescence values ​​of the FAM channel and the Cy5 channel in a pixel of the size of a magnetic bead, and the ratio of the two was calculated. The working curve was obtained by taking the concentration of the standard as the horizontal axis and the ratio of the fluorescence intensity of the two as the vertical axis. Figure 4 As shown, the standard solutions with known concentrations were subsequently tested, and the yields were determined to be between 92.1% and 110.6%;

[0074] Among them, the working curve of LIF is y=44.02x-1.91, R2=0.985; the detection limit is 7fg / mL.

[0075] Comparative Example: Similar to Example 4, except that the fluorescent magnetic beads are replaced with magnetic beads not stained with fluorescent molecules. The specific steps are as follows:

[0076] 1. Target capture

[0077] 5 μL 0.1 mg / mL capture antibody-modified magnetic beads were incubated with 45 μL target protein solution and 5 μL 10 μg / mL biotinylated detection antibody solution at room temperature for 1 hour, and then the magnetic beads were separated from the solution using a commercially available magnetic separation stand, the solution was discarded, and the magnetic beads were washed 3 times with a cleaning solution to remove the free detection antibody. At this point, the capture antibody-modified magnetic beads, the target protein, and the biotinylated detection antibody formed a sandwich structure.

[0078] 2. Fluorescent labeling

[0079] The magnetic beads were then dispersed into 30 μL of phosphate buffer containing 1 μg / mL SA labeled with atto 488, and incubated at room temperature for 15 min with rotation and shaking to label the fluorescent molecules. The magnetic beads were separated with a magnetic stand, and the solution was carefully removed with a pipette. The beads were washed three times with a cleaning solution, and the washed beads were dispersed into 5 μL of solution, and then dropped onto the imaging substrate. The magnetic beads were quickly adsorbed onto the imaging substrate using a magnet to prevent the solution from evaporating and produce a coffee ring effect. A cover glass was placed on top of the substrate for imaging.

[0080] 3. Imaging and data processing: Place the substrate under a microscope and use a 20x magnification, NA = 0.4 air lens for imaging. The dye excitation light is a monochromatic laser with a wavelength of 488nm and a power of 1mW. Use a FAM module filter to collect the signal.

[0081] Data processing: MATLAB software was used to calculate the fluorescence value of the FAM channel, and a graph was drawn with the standard concentration as the horizontal axis and the fluorescence intensity as the vertical axis to obtain a working curve. The working curve is shown in Figure 5 As shown, a standard solution of known concentration was subsequently tested, and the yield was determined to be between 78% and 132%;

[0082] Among them, the working curve of LIF is y=17.9x+0.97, R 2 =0.983; detection limit is 2.5pg / mL.

[0083] It can be seen that the detection accuracy and detection sensitivity in Example 4 are significantly better than those in the comparative example.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A ratiometric fluorescence biosensor based on photonic crystal signal amplification, characterized in that: The method comprises fluorescent magnetic beads modified with capture antibodies, detection antibodies modified with biotin, avidin modified with fluorescein, and a base layer, on which a photonic crystal layer is deposited.

2. The ratiometric fluorescence biosensor based on photonic crystal signal amplification according to claim 1, characterized in that: The preparation method of the fluorescent magnetic beads modified with the capture antibody comprises: activating the fluorescent magnetic beads and then connecting them with the capture antibody; Preferably, the fluorescent magnetic beads are activated by the EDC / NHS method, and the fluorescent magnetic beads are carboxyl fluorescent magnetic beads; Preferably, the mass ratio of capture antibody to fluorescent magnetic beads is 1:

100.

3. The ratiometric fluorescence biosensor based on photonic crystal signal amplification according to claim 1, characterized in that: The method for preparing the biotin-modified detection antibody comprises: activating the biotin and reacting it with the detection antibody; Preferably, biotin is activated by NHS to obtain NHS-biotin; Preferably, the molar ratio of the detection antibody to NHS-biotin is 1:5-100.

4. The ratiometric fluorescence biosensor based on photonic crystal signal amplification according to claim 1, characterized in that: The method for preparing the base layer comprises: soaking the base in a washing liquid, washing and drying; Preferably, the substrate is glass; Preferably, the washing liquid is a piranha washing liquid; more preferably, the piranha washing liquid comprises concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass concentration of 30%, and the volume ratio of concentrated sulfuric acid to hydrogen peroxide is 7:3; Preferably, the soaking temperature is 60-90°C and the soaking time is 30-60min; Preferably, washing is performed using water, anhydrous ethanol, and methanol, respectively, and the number of washing times is 3-6 times; Preferably, the drying temperature is 100-120° C. and the drying time is 10-20 h.

5. The ratiometric fluorescence biosensor based on photonic crystal signal amplification according to claim 1, characterized in that: The preparation method of the photonic crystal layer comprises: depositing the photonic crystal on the surface of the substrate and then curing it; Preferably, the deposition time is 0.5-1.5h; the deposition humidity is 65-95%; Preferably, the curing temperature is 65-85° C. and the curing time is 5-20 min.

6. The ratiometric fluorescence biosensor based on photonic crystal signal amplification according to claim 1, characterized in that: The photonic band gap of the photonic crystal matches the emission wavelength of fluorescein. Preferably, when the size of the photonic crystal monomer is between 200-220 nm, the forbidden band range is between 480-530 nm, and the fluorescent pigment includes atto 488, FAM, and Alexa 488.

7. The ratiometric fluorescence biosensor based on photonic crystal signal amplification according to claim 1, characterized in that: The preparation method of the photonic crystal is an emulsion polymerization method; Preferably, methyl methacrylate, acrylic acid and styrene are mixed and dissolved, and then subjected to polymerization reaction to obtain the product.

8. The ratiometric fluorescence biosensor based on photonic crystal signal amplification according to claim 1, characterized in that: The fluorescence emission wavelength of the fluorescent magnetic beads does not match the photonic band gap of the photonic crystal.

9. Use of the ratiometric fluorescence biosensor based on photonic crystal signal amplification according to any one of claims 1 to 8 in detecting markers, wherein the detection is for non-diagnostic purposes.

10. A method for detecting a marker using a ratiometric fluorescence biosensor based on photonic crystal signal amplification, comprising the following steps: S1 Capture marker: Mix the capture antibody-modified fluorescent magnetic beads, the biotin-modified detection antibody and the marker and incubate them once to separate the capture marker fluorescent magnetic beads; S2 performs fluorescein labeling: adding fluorescein-modified avidin to the fluorescent magnetic beads that capture the marker, performing secondary incubation, and separating and obtaining fluorescein-labeled fluorescent magnetic beads; S3 Adding the basal layer for imaging analysis: adding fluorescent magnetic beads labeled with fluorescein to the basal layer where the photonic crystal layer is deposited, detecting the fluorescent signals of the fluorescent magnetic beads and the fluorescent signals of fluorescein respectively, calculating the fluorescence ratio, and obtaining the concentration of the tumor marker to be tested through the standard curve; Preferably, the concentration of the capture antibody-modified fluorescent magnetic beads is 0.1-10 mg / mL, the concentration of the detection antibody is 0.1 μg / mL-100 μg / mL, and the concentration of the fluorescein-modified streptavidin is 0.1 μg / mL-10 μg / mL.

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