Method for enhancing animal virus detection sensitivity by surface modification of optical fiber SPR (surface plasmon resonance) probe

Abstract

The invention relates to the technical field of virus detection, in particular to a method for enhancing animal virus detection sensitivity through optical fiber SPR probe surface modification, which comprises the following steps: S1, optical fiber SPR probe surface pretreatment: obtaining a hydroxylated surface; s2, gradient nanostructure modification: forming a gradient aperture surface topological structure; s3, carboxyl functionalization: immersing the substrate into a 3-aminopropyltriethoxysilane / methacrylic acid mixed solution, and constructing a carboxyl activation layer on the surface; s4, fixation of the aptamer: realizing directional fixation of the aptamer through EDC / NHS mediated carboxyl-amino coupling reaction; s5, non-specific site closing: carrying out cross-linking closing on the non-binding sites by adopting a thioacetamide / polydopamine composite solution; s6, virus detection: acquiring the surface refractive index variation through a phase modulation type SPR detection system; according to the invention, accurate fitting and quantitative correction of virus concentration are realized, and the stability and accuracy of detection are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and particularly to a method for enhancing the detection sensitivity of animal viruses by surface modification of an optical fiber SPR probe. Background Art

[0002] With the frequent outbreaks of newly emerging and sudden viral diseases, the rapid detection of animal viruses is of great significance in the prevention and control of animal diseases, biosafety monitoring, and public health fields. At present, surface plasmon resonance (SPR) technology has become an important tool in the field of biosensing detection due to its high sensitivity and real-time label-free detection ability. Among them, optical fiber SPR probes have received extensive attention in the application of virus detection due to their miniaturization, portability, and high refractive index sensitivity. By functionalizing the surface of the optical fiber SPR probe, its specific recognition ability for target viruses can be improved, thereby enhancing the sensitivity and accuracy of detection. However, in existing detection methods, there are still many challenges in the structural design of the probe surface, the recognition molecule immobilization strategy, and the signal processing algorithm, which affect the stability and reliability of detection.

[0003] In the prior art, conventional methods for surface modification of SPR probes usually adopt single-layer nanomaterial modification or random aptamer immobilization strategies, resulting in a limited specific surface area and insufficient virus capture ability. At the same time, it is difficult to ensure the uniformity and stability of the nanomodification layer, which affects the reliability of SPR signals. In addition, most aptamer immobilization methods rely on physical adsorption or unoptimized chemical coupling, resulting in low immobilization efficiency and poor directionality, thereby affecting the specific recognition of viruses. On the other hand, the signal processing methods of traditional SPR detection mainly rely on simple phase change measurement, without fully considering the influence of background noise interference and non-specific adsorption, resulting in limited specificity and accuracy of detection. In addition, existing methods have insufficient sensitivity in detecting low-concentration viruses and are difficult to meet the requirements of high-precision virus detection.

[0004] The purpose of the present invention is to overcome the above technical defects and provide a method for enhancing the detection sensitivity of animal viruses by surface modification of an optical fiber SPR probe, effectively improving the sensitivity and accuracy of detection, and providing an efficient and reliable technical solution for the rapid and accurate detection of animal viruses. Summary of the Invention

[0005] The present invention provides a method for enhancing the detection sensitivity of animal viruses by surface modification of an optical fiber SPR probe.

[0006] The method for enhancing the detection sensitivity of animal viruses by surface modification of an optical fiber SPR probe includes the following steps:

[0007] S1, surface pretreatment of the optical fiber SPR probe: Immerse the etched section of the optical fiber in piranha solution and perform ultrasonic treatment to obtain a hydroxylated surface;

[0008] S2, Gradient nanostructure modification: Layer-by-layer modification of three-dimensional porous silica nanospheres on the hydroxylated surface to form a surface topology with a gradient pore size;

[0009] S3, Carboxyl functionalization: Based on the probe modified with the surface topology of the gradient pore size, immerse it in a mixed solution of 3-aminopropyltriethoxysilane / methacrylic acid to construct a carboxyl activation layer on the surface;

[0010] S4, Aptamer immobilization: Pass the virus-specific aptamer solution into the surface carboxyl activation layer, and achieve the directional immobilization of the aptamer through the carboxyl-amino coupling reaction mediated by EDC / NHS;

[0011] S5, Blocking of non-specific sites: Use a composite solution of thioacetamide / polydopamine to crosslink and block the unbound sites;

[0012] S6, Virus detection: Inject the sample to be tested into the detection chamber, and obtain the change in surface refractive index through a phase modulation type SPR detection system.

[0013] Optionally, the surface pretreatment of the optical fiber SPR probe in S1 includes:

[0014] S11, Solution preparation: Prepare a mixed solution of 98% sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1 to form a piranha solution, and let it stand at room temperature to release oxidizing gases;

[0015] S12, Optical fiber cleaning: Place the etched section of the optical fiber in deionized water and ultrasonically clean it for 10 minutes. After removing the surface contaminants, rinse it successively with absolute ethanol and acetone, and dry it under a nitrogen stream;

[0016] S13, Hydroxylation treatment: Immerse the cleaned etched section of the optical fiber completely in the pre-prepared piranha solution, and perform ultrasonic treatment for 3-5 minutes at room temperature or 50 °C to form a uniform hydroxylation layer on the surface of the optical fiber;

[0017] S14, Post-treatment: Take out the ultrasonically treated optical fiber, rinse it successively with deionized water and absolute ethanol to remove the residual piranha solution, and finally dry it under a nitrogen stream for standby.

[0018] Optionally, the gradient nanostructure modification in S2 includes:

[0019] S21, Nanosphere dispersion: Add three-dimensional porous silica nanospheres to a mixed solution of deionized water and ethanol (volume ratio 1:1) in a mass ratio of 1:10, and add 0.1% polyvinylpyrrolidone (PVP) as a dispersant, and disperse it under ultrasonic conditions for 30 minutes to make the nanospheres uniformly suspended;

[0020] S22, First - layer deposition: Immerse the hydroxylated optical - fiber probe into the nanosphere suspension. By using the method of electrostatic self - assembly or dip - coating, make silica nanospheres with a particle size of 50 - 100 nm uniformly adhere to the surface of the optical fiber, and fix them by natural drying or low - temperature drying (50 - 70 °C);

[0021] S23, Multilayer assembly: Adjust the particle - size distribution of the nanosphere suspension in sequence, and use the layer - by - layer self - assembly technique (LBL) or spin - coating method to gradually deposit large - particle - size nanospheres with a particle size of 100 - 300 nm on the basis of the first layer to form a gradient - pore - size structure;

[0022] S24, Surface stabilization: Perform heat treatment (100 - 150 °C) or plasma treatment on the modified optical - fiber probe.

[0023] Optionally, the carboxyl functionalization in S3 includes:

[0024] S31, Preparation of activation solution: Prepare a mixed solution of 3 - aminopropyltriethoxysilane (APTES) and absolute ethanol according to a volume ratio of 1:9, add 0.5% deionized water, and stir for 1 hour at room temperature (25 °C) to make the solution evenly dispersed;

[0025] S32, Silanization treatment: Immerse the optical - fiber probe modified with the gradient - pore - size surface topology into the activation solution, and let it stand and react for 2 hours at 37 °C to form an aminated layer of 3 - aminopropyltriethoxysilane on the surface of the probe;

[0026] S33, Introduction of carboxyl group: Take out the aminated probe, rinse it successively with absolute ethanol and deionized water and dry it, then transfer it to a methyl methacrylate (MAA) ethanol solution with a mass concentration of 10 mg / mL, add 1% 1 - ethyl - 3 - (3 - dimethylaminopropyl) carbodiimide (EDC) and 0.5% N - hydroxysuccinimide (NHS), and react in a buffer solution with pH 5.5 for 4 hours to covalently link methyl methacrylate to the surface of the probe to form a stable carboxyl - activated layer;

[0027] S34, Cleaning and drying: Take out the reacted probe, rinse it successively with deionized water and absolute ethanol to remove the unreacted reagents, and dry it at room temperature for standby.

[0028] Optionally, the aptamer immobilization in S4 includes:

[0029] S41, Preparation of aptamer solution: Dissolve the virus - specific aptamer in phosphate - buffered saline (PBS) with pH 7.4 to make its final concentration 10 - 50 μM, and store it in the dark at 4 °C;

[0030] S42, Activation Pretreatment: Immerse the carboxylated optical fiber probe into the activation solution composed of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS), and react at room temperature (25 °C) for 30 - 60 minutes to activate the carboxyl group into highly reactive NHS ester groups;

[0031] S43, Aptamer Immobilization: Take out the activated probe and immediately place it into the virus-specific aptamer solution, incubate at 4 °C for 2 - 4 hours, and covalently link the aptamer to the probe surface through carboxyl-amino coupling reaction;

[0032] S44, Washing and Stabilization: Rinse the modified probe successively with phosphate buffer solution (PBS) and deionized water to remove the unbound aptamer, and dry it at room temperature to obtain the aptamer-modified layer.

[0033] Optionally, the non-specific site blocking in S5 includes:

[0034] S51, Preparation of Blocking Solution: Prepare a 0.1 M thioacetamide (TAA) solution, add 2 mg / mL polydopamine (PDA) into it, and stir evenly in Tris-HCl buffer solution with pH 8.5 to form a composite blocking solution;

[0035] S52, Blocking Treatment: Immerse the optical fiber SPR probe immobilized with aptamer into the composite blocking solution, and statically incubate at 25 - 30 °C for 2 - 4 hours to chemically crosslink thioacetamide with the remaining active sites on the probe surface, and at the same time, polydopamine self-polymerizes on the probe surface to form a protective layer;

[0036] S53, Washing and Drying: Rinse the blocked probe successively with deionized water and PBS buffer solution to remove the unbound blocking reagent, and dry it under nitrogen gas flow.

[0037] Optionally, the virus detection in S6 includes:

[0038] S61, Sample Injection and Reaction: Slowly inject the sample to be tested into the SPR detection chamber through an injection pump to make it fully contact with the optical fiber SPR probe modified with aptamer, and incubate at room temperature (25 - 30 °C) for 5 - 15 minutes;

[0039] S62, Signal Acquisition and Analysis: Use a phase modulation type SPR detection system to monitor the refractive index change on the probe surface, calculate the virus concentration by real-time recording the SPR phase change curve, and combine the signal analysis algorithm to improve the detection sensitivity, and finally output the quantitative or qualitative detection result.

[0040] Optionally, the sample injection and reaction in S61 include:

[0041] S611, sample preparation: Dilute the sample to be tested to the target concentration with phosphate buffer solution (PBS) at pH 7.4 and filter it using a 0.22 μm filter membrane;

[0042] S612, slow injection: Slowly inject the sample into the SPR detection chamber at a flow rate of 0.5 - 2 μL / min using an injection pump;

[0043] S613, incubation and binding: Keep the sample in full contact with the fiber optic SPR probe at room temperature (25 - 30 °C) and let it stand for incubation for 5 - 15 minutes;

[0044] S614, rinsing to remove non-specific binding: After incubation, rinse the detection chamber with the diluted phosphate buffer solution (PBS) at a flow rate of 0.5 - 1 μL / min.

[0045] Optionally, the signal acquisition and analysis in S62 include:

[0046] S621, SPR signal acquisition: Use a phase modulation type SPR detection system to monitor the refractive index change through the phase change of the reflected light on the surface of the fiber optic SPR probe, collect the SPR response signal in real time, and record the dynamic curve of the phase change over time;

[0047] S622, phase change calculation: According to the SPR phase modulation principle, calculate the local refractive index change Δn on the surface of the fiber optic probe, expressed as:

[0048]

[0049] where Δφ is the SPR phase change, d is the thickness of the nano-modified layer on the probe surface, λ is the central wavelength of the SPR detection light, n eff is the effective refractive index of the modified layer, n 0 is the background refractive index of the sample solution, Δn = n eff -n 0 represents the local refractive index change and is related to the virus concentration;

[0050] S623, virus concentration calculation: Combine the Langmuir adsorption kinetic model to calculate the virus concentration C v , expressed as:

[0051] Δn = K a C v / (1 + K d C v );

[0052] where C v is the virus concentration, Ka is the association constant, K d is the dissociation constant;

[0053] S624, Data analysis and result output: Input the phase change data Δφ(t) into the signal analysis algorithm to optimize the quantitative mapping between the SPR signal and the virus concentration, and finally output the virus detection result, including the quantitative concentration value or the qualitative positive / negative determination.

[0054] Optionally, the data analysis and result output in S624 include:

[0055] S6241, Filtering and noise reduction: Use Savitzky-Golay smoothing filter to smooth the SPR signal curve;

[0056] S6242, Nonlinear fitting: Use the four-parameter Logistic regression model (4PL) for fitting to quantitatively correct the virus concentration and obtain the corrected SPR signal change Δφ ′ ;

[0057] S6243, Result determination: Set the detection threshold If then determine the sample as positive, otherwise determine it as negative.

[0058] Advantages of the present invention:

[0059] In the present invention, through gradient nanostructure modification, carboxyl functionalization and aptamer directed immobilization, efficient functionalization of the probe surface is achieved. By using three-dimensional porous silica nanospheres for modification, the specific surface area and the local electric field enhancement effect are improved, making the SPR signal response more optimized, enhancing the virus capture ability, and thus significantly improving the detection sensitivity. At the same time, the aptamer directed immobilization strategy combined with the EDC / NHS activation method ensures the efficient binding and biological activity of the aptamer, improving the specificity of virus recognition. In addition, the thioacetamide / polydopamine composite blocking strategy effectively reduces non-specific adsorption, reduces background interference, and improves the specificity and stability of detection.

[0060] In the present invention, by introducing a precisely controlled sample injection and signal acquisition and analysis strategy, using a low-flow injection pump to control the slow flow of the sample, optimizing the binding conditions between the virus and the aptamer, ensuring sufficient interaction between the virus particles and the probe surface, enhancing the detection repeatability and reliability. During the signal acquisition process, a phase modulation type SPR detection technology is used to record the phase change in real time, and the virus concentration is calculated in combination with the Langmuir adsorption kinetic model. Based on the non-linear relationship between the SPR signal change and the virus concentration, a four-parameter Logistic regression standard curve is constructed to achieve precise fitting and quantitative correction of the virus concentration, improving the stability and accuracy of detection.

[0061] In the present invention, through Savitzky-Golay filtering for noise reduction, wavelet transform and non-linear fitting, the experimental noise interference is reduced, the signal stability is enhanced, and combined with the standard curve for quantitative analysis of virus concentration. The detection result adopts the dynamic threshold determination method, calculates the detection threshold based on the mean and standard deviation of negative samples, effectively distinguishes negative and positive samples, and improves the sensitivity and accuracy of the detection system. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0063] Figure 1 It is a schematic flowchart of the method for detecting the sensitivity of the embodiment of the present invention;

[0064] Figure 2 It is a schematic diagram of the gradient nanostructure modification of the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0065] The present invention will be described in detail below with reference to the drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; and the drawings are only for more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0066] It should be pointed out that in the specification, when referring to "an embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc., it indicates that the described embodiment may include specific features, structures or characteristics, but not necessarily every embodiment includes the specific features, structures or characteristics. In addition, when combining an embodiment to describe a specific feature, structure or characteristic, implementing such a feature, structure or characteristic in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0067] Generally, the terms can be understood at least in part from their use in the context. For example, at least in part depending on the context, the term "one or more" used herein can be used to describe any feature, structure or characteristic in a singular sense, or can be used to describe a combination of features, structures or characteristics in a plural sense. In addition, the term "based on" can be understood as not necessarily intended to convey a set of exclusive factors, but instead, at least in part depending on the context, allowing the existence of other factors that are not necessarily explicitly described.

[0068] As Figure 1 - Figure 2 shown, a method for enhancing the detection sensitivity of animal viruses by surface modification of an optical fiber SPR probe includes the following steps:

[0069] S1, surface pretreatment of the optical fiber SPR probe: Immerse the etched section of the optical fiber in piranha solution and perform ultrasonic treatment to obtain a hydroxylated surface;

[0070] S2, gradient nanostructure modification: Layer-by-layer modify three-dimensional porous silica nanospheres on the hydroxylated surface to form a gradient pore-size surface topology;

[0071] S3, carboxyl functionalization: Based on the probe modified with the gradient pore-size surface topology, immerse it in a mixed solution of 3-aminopropyltriethoxysilane / methacrylic acid to construct a carboxyl activation layer on the surface;

[0072] S4, aptamer immobilization: Pass the virus-specific aptamer solution into the surface carboxyl activation layer to achieve the directional immobilization of the aptamer through the EDC / NHS-mediated carboxyl-amine coupling reaction;

[0073] S5, non-specific site blocking: Crosslink and block the unbound sites with a thioacetamide / polydopamine composite solution;

[0074] S6, virus detection: Inject the sample to be tested into the detection chamber and obtain the change in surface refractive index through a phase-modulated SPR detection system;

[0075] Through the above content, efficient functionalization of the probe surface is achieved, significantly improving the virus capture ability and detection sensitivity. The modification with three-dimensional porous silica nanospheres increases the specific surface area and the local electric field enhancement effect, thereby optimizing the SPR signal response. The strategies of directional immobilization of aptamers and blocking of non-specific sites effectively reduce background interference and improve the specificity and stability of detection.

[0076] The surface pretreatment of the optical fiber SPR probe in S1 includes:

[0077] S11, solution preparation: Prepare a mixed solution of 98% sulfuric acid and 30% hydrogen peroxide according to a volume ratio of 3:1 to form piranha solution, and let it stand at room temperature to release oxidizing gases;

[0078] S12, optical fiber cleaning: Place the etched section of the optical fiber in deionized water and ultrasonically clean it for 10 minutes. After removing surface contaminants, rinse it successively with absolute ethanol and acetone, and dry it under a nitrogen gas stream;

[0079] S13, Hydroxylation treatment: Immerse the cleaned etched-section optical fiber completely into the pre-prepared piranha solution, and perform ultrasonic treatment for 3 - 5 minutes at room temperature or 50 °C to form a uniform hydroxylation layer on the fiber surface;

[0080] S14, Post-treatment: Take out the optical fiber after ultrasonic treatment, rinse it successively with deionized water and absolute ethanol to remove the residual piranha solution, and finally dry it under a nitrogen stream for standby.

[0081] The gradient nanostructure modification in S2 includes:

[0082] S21, Nanosphere dispersion: Add three-dimensional porous silica nanospheres to the mixed solution of deionized water and ethanol (volume ratio 1:1) according to a mass ratio of 1:10, and add 0.1% polyvinylpyrrolidone (PVP) as a dispersant, and disperse it under ultrasonic conditions for 30 minutes to make the nanospheres uniformly suspended;

[0083] S22, First-layer deposition: Immerse the hydroxylated optical fiber probe into the nanosphere suspension, and use the method of electrostatic self-assembly or dip coating to make silica nanospheres with a particle size of 50 - 100 nm uniformly adhere to the fiber surface, and fix them by natural drying or low-temperature drying (50 - 70 °C);

[0084] S23, Multilayer assembly: Adjust the particle size distribution of the nanosphere suspension in turn, and use the layer-by-layer self-assembly technique (LBL) or spin coating method to gradually deposit large-sized nanospheres with a particle size of 100 - 300 nm on the basis of the first layer to form a gradient pore structure;

[0085] S24, Surface stabilization: Perform heat treatment (100 - 150 °C) or plasma treatment on the modified optical fiber probe to enhance the stability of the nanostructure and ensure its structural integrity during the subsequent functionalization process;

[0086] Through the above content, the specific surface area and the light field local enhancement effect are improved. Small-sized nanospheres provide high-density binding sites, and large-sized nanospheres optimize the light propagation path, making the SPR signal more sensitive. This structure not only enhances the virus capture ability and improves the detection sensitivity, but also ensures the stability and reproducibility of the probe surface, providing an excellent solution for highly efficient and low-background-interference biosensing detection.

[0087] The carboxyl functionalization in S3 includes:

[0088] S31, Activation solution preparation: Prepare a mixed solution of 3-aminopropyltriethoxysilane (APTES) and absolute ethanol according to a volume ratio of 1:9, and add 0.5% deionized water, and stir for 1 hour at room temperature (25 °C) to make the solution uniformly dispersed;

[0089] S32, Silanization treatment: Immerse the optical fiber probe modified with gradient pore surface topology into the activation solution, and let it stand and react for 2 hours at 37°C to form an aminated layer of 3-aminopropyltriethoxysilane on the probe surface;

[0090] S33, Carboxyl group introduction: Take out the aminated probe, rinse it successively with absolute ethanol and deionized water and dry it, then transfer it to a methyl methacrylate (MAA) ethanol solution with a mass concentration of 10 mg / mL, and add 1% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.5% N-hydroxysuccinimide (NHS), and react in a buffer solution with pH 5.5 for 4 hours to covalently link methyl methacrylate to the probe surface to form a stable carboxyl activation layer;

[0091] S34, Cleaning and drying: Take out the reacted probe, rinse it successively with deionized water and absolute ethanol to remove the unreacted reagents, and dry it at room temperature for standby;

[0092] Through the above content, a highly controllable chemical activation layer construction is achieved. The amination treatment provides stable grafting sites to ensure the efficient introduction of subsequent carboxyl groups, and the EDC / NHS activation strategy further enhances the stability and reactivity of the carboxyl layer, guarantees the directional immobilization of aptamers, improves the specificity of virus recognition, and at the same time enhances the biocompatibility and reusability of the probe surface, providing reliable technical support for highly sensitive and low-background-interference virus detection.

[0093] The aptamer immobilization in S4 includes:

[0094] S41, Aptamer solution preparation: Dissolve the virus-specific aptamer in a phosphate buffer solution (PBS) with pH 7.4 to make its final concentration 10 - 50 μM, and store it in the dark at 4°C;

[0095] S42, Activation pretreatment: Immerse the carboxylated optical fiber probe into the activation solution composed of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS), and react at room temperature (25°C) for 30 - 60 minutes to activate the carboxyl group into a highly reactive NHS ester group;

[0096] S43, Aptamer immobilization: Take out the activated probe and immediately place it in the virus-specific aptamer solution, and incubate it at 4°C for 2 - 4 hours to covalently link the aptamer to the probe surface through carboxyl-amino coupling reaction;

[0097] S44, Washing and Stabilization: The modified probe was rinsed successively with phosphate buffered saline (PBS) buffer and deionized water to remove unbound aptamers, and dried at room temperature to obtain a stable aptamer-modified layer;

[0098] Through the above, the directional immobilization of virus-specific aptamers on the surface of the fiber optic SPR probe was achieved, ensuring the biological activity and efficient binding of the aptamers. Through the activation of NHS ester groups, the problem of non-specific binding caused by traditional physical adsorption was avoided, improving the stability and binding efficiency of the aptamers. In addition, the optimized aptamer immobilization conditions (suitable buffer system, temperature and reaction time) further enhanced the detection sensitivity and specificity of the probe, providing a more reliable molecular recognition interface for the SPR detection system, and contributing to improving the accuracy and repeatability of virus detection.

[0099] The non-specific site blocking in S5 includes:

[0100] S51, Preparation of Blocking Solution: Prepare a 0.1 M thioacetamide (TAA) solution, and add 2 mg / mL polydopamine (PDA) to it, and stir evenly in a Tris-HCl buffer at pH 8.5 to form a stable composite blocking solution;

[0101] S52, Blocking Treatment: Immerse the fiber optic SPR probe immobilized with aptamers in the composite blocking solution, and incubate it statically at 25 - 30 °C for 2 - 4 hours, so that thioacetamide chemically cross-links with the remaining active sites on the probe surface, and at the same time polydopamine self-polymerizes on the probe surface to form a protective layer;

[0102] S53, Washing and Drying: Rinse the blocked probe successively with deionized water and PBS buffer to remove unbound blocking reagents, and dry it under a nitrogen stream to ensure uniform and stable blocking on the probe surface;

[0103] Through the above, the unbound active sites on the surface of the fiber optic SPR probe were effectively blocked, reducing non-specific adsorption, thereby improving the specificity and accuracy of detection. Thioacetamide blocks the remaining active sites through chemical cross-linking to prevent interference from non-specific molecules, while polydopamine self-polymerizes on the probe surface to form a stable protective layer, further enhancing the chemical stability and biocompatibility of the probe. It not only optimizes the SPR signal response, reduces background noise, but also improves the reproducibility and long-term stability of the probe, providing a reliable guarantee for highly sensitive virus detection.

[0104] The virus detection in S6 includes:

[0105] S61, Sample injection and reaction: Slowly inject the sample to be tested into the SPR detection chamber through an injection pump, so that it is in full contact with the optical fiber SPR probe modified with the aptamer, and incubate for 5 - 15 minutes at room temperature (25 - 30 °C) to ensure that the specific binding reaction between the virus and the aptamer reaches equilibrium;

[0106] S62, Signal acquisition and analysis: Use a phase modulation type SPR detection system to monitor the refractive index change on the probe surface, calculate the virus concentration by recording the SPR phase change curve in real time, and combine signal analysis algorithms to improve the detection sensitivity, and finally output quantitative or qualitative detection results.

[0107] The sample injection and reaction in S61 include:

[0108] S611, Sample preparation: Dilute the sample to be tested to the target concentration with phosphate buffer solution (PBS) at pH 7.4, and filter it with a 0.22 μm filter membrane to remove particles and impurities that affect the detection and ensure sample uniformity;

[0109] S612, Slow injection: Use an injection pump to slowly inject the sample into the SPR detection chamber at a flow rate of 0.5 - 2 μL / min, so that the sample is evenly distributed in the flowing state and reduce the noise interference caused by non-specific adsorption;

[0110] S613, Incubation and binding: Keep the sample in full contact with the optical fiber SPR probe at room temperature (25 - 30 °C) and incubate statically for 5 - 15 minutes to ensure that the specific binding reaction between virus particles and aptamers reaches equilibrium;

[0111] S614, Flushing to remove non-specific binding: After incubation, use the diluted phosphate buffer solution (PBS) to flush the detection chamber at a flow rate of 0.5 - 1 μL / min to remove unbound virus particles and other interfering substances, and ensure the stability of the SPR signal and the high specificity of the detection;

[0112] Through the above content, ensure that the sample is evenly distributed on the surface of the optical fiber SPR probe, and reduce non-specific adsorption and signal drift caused by too fast flow rate. The constant temperature incubation further optimizes the binding conditions between the virus and the aptamer, enables them to react fully, and ensures high sensitivity and high specificity of the detection. In addition, the precise flushing step of the PBS buffer effectively removes unbound impurities, reduces background noise, improves signal stability, and provides a more reliable and accurate virus detection ability for the SPR system.

[0113] The signal acquisition and analysis in S62 include:

[0114] S621, SPR signal acquisition: Using a phase-modulated SPR detection system, monitor the refractive index change by the phase change of the reflected light on the surface of the fiber optic SPR probe, collect the SPR response signal in real time, and record the dynamic curve of the phase change over time;

[0115] S622, Phase change calculation: According to the SPR phase modulation principle, calculate the local refractive index change Δn on the surface of the fiber optic probe, expressed as:

[0116]

[0117] where Δφ is the SPR phase change, d is the thickness of the nano-modified layer on the probe surface, λ is the central wavelength of the SPR detection light, n eff is the effective refractive index of the modified layer, n 0 is the background refractive index of the sample solution, Δn = n eff -n 0 represents the local refractive index change, which is related to the virus concentration;

[0118] S623, Virus concentration calculation: Combining the Langmuir adsorption kinetic model, calculate the virus concentration C v , expressed as:

[0119] Δn = K a C v / (1 + K d C v );

[0120] where C v is the virus concentration, K a is the binding constant, K d is the dissociation constant;

[0121] S624, Data analysis and result output: Input the phase change data Δφ(t) into the signal analysis algorithm, optimize the quantitative mapping between the SPR signal and the virus concentration, and finally output the virus detection result, including the quantitative concentration value or the qualitative positive / negative determination.

[0122] The data analysis and result output in S624 include:

[0123] S6241, Filtering and noise reduction: Use Savitzky-Golay smoothing filtering to smooth the SPR signal curve to reduce experimental noise, expressed as:

[0124]

[0125] where S(i) is the smoothed SPR signal value, Y(i) is the original SPR signal value, C j is the filtering coefficient, and m is half the width of the filtering window;

[0126] S6242, Nonlinear fitting: The four-parameter Logistic regression model (4PL) is used for fitting to quantitatively correct the virus concentration and obtain the change amount Δφ of the SPR signal after correction. ′ , which is expressed as:

[0127]

[0128] where y is the change amount Δφ of the SPR signal ′ , x is the virus concentration C v , A and B are the lowest and highest signal values respectively, C is the half-maximal effective concentration (EC50), that is, the virus concentration corresponding to the 50% saturation point, and D is the slope factor;

[0129] S6243, Result determination: Set the detection threshold If then the sample is determined to be positive, otherwise it is determined to be negative;

[0130] The detection threshold is set specifically as follows:

[0131] Background signal measurement: In the environment of a virus-free negative sample (such as PBS buffer or non-specific protein solution), SPR signal detection is carried out, and the phase change amounts of multiple measurements are recorded. Through N repeated measurements (N≥10), its mean value μ neg and standard deviation σ neg are obtained to calculate the background signal range;

[0132] Threshold calculation: Based on the mean value μ neg and standard deviation σ neg , the threshold for SPR detection is set, which is expressed as:

[0133]

[0134] where k is the sensitivity adjustment factor, taking 2 - 3, that is, the 95% or 99% confidence interval.

[0135] The present invention covers any substitutions, modifications, equivalent methods and solutions made on the essence and scope of the present invention. For the public to have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, and those skilled in the art can fully understand the present invention without the description of these details. In addition, well-known methods, processes, procedures, elements and circuits, etc. are not described in detail to avoid unnecessary confusion to the essence of the present invention.

[0136] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probes, characterized in that: The following steps are involved: S1, Surface pretreatment of the optical fiber SPR probe: immerse the etched optical fiber in a piranha solution for ultrasonic treatment to obtain a hydroxylated surface; S2, gradient nanostructure modification: three-dimensional porous silica nanospheres are modified layer by layer on the hydroxylated surface to form a gradient pore size surface topology; S3, carboxyl functionalization: Based on the modified gradient pore surface topology, the probe is immersed in a 3-aminopropyltriethoxysilane / methacrylic acid mixed solution to construct a carboxyl activation layer on the surface; S4, aptamer immobilization: the virus-specific aptamer solution is passed into the surface carboxyl activation layer, and the aptamer is fixed in a directional manner through the EDC / NHS-mediated carboxyl-amino coupling reaction; S5, nonspecific site blocking: thioacetamide / polydopamine complex solution was used to cross-link and block the unbound sites; S6, virus detection: inject the sample to be tested into the detection chamber, and obtain the surface refractive index change through the phase modulation SPR detection system.

2. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 1, characterized in that: The surface pretreatment of the optical fiber SPR probe in S1 includes: S11, solution preparation: prepare a mixture of 98% sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1 to form a piranha solution, and let it stand at room temperature to release oxidizing gas; S12, optical fiber cleaning: the etched optical fiber is placed in deionized water for ultrasonic cleaning for 10 minutes to remove surface contaminants, then rinsed with anhydrous ethanol and acetone in sequence, and dried under a nitrogen flow; S13, hydroxylation treatment: completely immerse the cleaned etched section of optical fiber in a pre-prepared piranha solution, and perform ultrasonic treatment at room temperature or 50° C. for 3-5 minutes to form a uniform hydroxylation layer on the surface of the optical fiber; S14, post-processing: taking out the optical fiber after ultrasonic treatment, rinsing it with deionized water and anhydrous ethanol in turn to remove the residual piranha solution, and finally drying it under nitrogen flow for later use.

3. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 2, characterized in that: The gradient nanostructure modification in S2 includes: S21, nanosphere dispersion: three-dimensional porous silica nanospheres were added to a mixed solution of deionized water and ethanol at a mass ratio of 1:10, and 0.1% polyvinyl pyrrolidone was added as a dispersant, and dispersed under ultrasonic conditions for 30 minutes to make the nanospheres uniformly suspended; S22, first layer deposition: immersing the hydroxylated optical fiber probe into the nanosphere suspension, using electrostatic self-assembly or dip coating to make silica nanospheres with a particle size of 50-100 nm uniformly adhere to the optical fiber surface, and fixing by natural drying or low-temperature drying; S23, multi-layer assembly: sequentially adjusting the particle size distribution of the nanosphere suspension, and using layer-by-layer self-assembly technology or spin coating to gradually deposit large-size nanospheres with a particle size of 100-300 nm on the basis of the first layer to form a gradient pore size structure; S24, surface stabilization: heat treatment or plasma treatment of the modified optical fiber probe.

4. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 3, characterized in that: The carboxyl functionalization in S3 includes: S31, preparation of activation solution: prepare a mixed solution of 3-aminopropyltriethoxysilane and anhydrous ethanol in a volume ratio of 1:9, add 0.5% deionized water, and stir at room temperature for 1 hour to make the solution uniformly dispersed; S32, silanization treatment: immersing the optical fiber probe modified with the gradient aperture surface topology into an activation solution, and allowing it to react at 37°C for 2 hours, so that 3-aminopropyltriethoxysilane forms an amino layer on the probe surface; S33, carboxyl group introduction: the amino probe was taken out, rinsed with anhydrous ethanol and deionized water in sequence and dried, and then transferred to a 10 mg / mL methacrylic acid ethanol solution, and 1% carbodiimide and 0.5% N-hydroxysuccinimide were added, and reacted in a pH 5.5 buffer solution for 4 hours to covalently connect methacrylic acid to the probe surface to form a stable carboxyl activation layer; S34, cleaning and drying: the probe after the reaction is taken out, rinsed with deionized water and anhydrous ethanol in turn to remove unreacted reagents, and dried at room temperature for later use.

5. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 4, characterized in that: The aptamer fixation in S4 comprises: S41, preparation of aptamer solution: dissolving the virus-specific aptamer in a phosphate buffer solution at pH 7.4 to a final concentration of 10-50 μM, and storing at 4°C in the dark; S42, activation pretreatment: immersing the carboxylated optical fiber probe in an activation solution consisting of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.05 M N-hydroxysuccinimide, reacting at room temperature for 30-60 minutes to activate the carboxyl group into a highly reactive NHS ester group; S43, aptamer fixation: the activated probe is taken out and immediately placed in a virus-specific aptamer solution, incubated at 4°C for 2-4 hours, and the aptamer is covalently attached to the probe surface through a carboxyl-amino coupling reaction; S44, washing and stabilization: The modified probe is rinsed with phosphate buffer solution and deionized water in sequence to remove unbound aptamers, and dried at room temperature to obtain an aptamer modification layer.

6. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 5, characterized in that: The non-specific site blocking in S5 includes: S51, preparation of blocking solution: prepare 0.1 M thioacetamide solution, add 2 mg / mL polydopamine thereto, and stir evenly in Tris-HCl buffer at pH 8.5 to form a composite blocking solution; S52, blocking treatment: immerse the aptamer-fixed optical fiber SPR probe in a composite blocking solution and incubate it at 25-30°C for 2-4 hours to allow thioacetamide to chemically cross-link with the residual active sites on the probe surface, and polydopamine to self-polymerize on the probe surface to form a protective layer; S53, washing and drying: the blocked probe is rinsed with deionized water and PBS buffer in sequence to remove unbound blocking reagent, and then blown dry under a nitrogen flow.

7. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 6, characterized in that: The virus detection in S6 includes: S61, sample injection and reaction: inject the sample to be tested into the SPR detection chamber through a syringe pump, so that it is fully in contact with the optical fiber SPR probe modified with the aptamer, and incubate at room temperature for 5-15 minutes; S62, signal acquisition and analysis: Use a phase-modulated SPR detection system to monitor the refractive index changes on the probe surface, calculate the virus concentration by real-time recording of the SPR phase change curve, and combine the signal analysis algorithm to improve the detection sensitivity, and finally output quantitative or qualitative detection results.

8. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 7, characterized in that: The sample injection and reaction in S61 include: S611, sample preparation: dilute the sample to be tested with pH 7.4 phosphate buffer solution to the target concentration and filter using a 0.22 μm filter membrane; S612, slow injection: inject the sample into the SPR detection chamber at a flow rate of 0.5-2 μL / min using a syringe pump; S613, incubation and binding: keep the sample in full contact with the fiber-optic SPR probe at room temperature and incubate for 5-15 minutes; S614, flushing to remove non-specific binding: After the incubation is completed, flush the detection chamber with diluted phosphate buffer solution at a flow rate of 0.5-1 μL / min.

9. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 8, characterized in that: The signal acquisition and analysis in S62 includes: S621, SPR signal acquisition: using a phase-modulated SPR detection system, the refractive index change is monitored by the phase change of the reflected light on the surface of the optical fiber SPR probe, the SPR response signal is collected in real time, and the dynamic curve of the phase change over time is recorded; S622, phase change calculation: According to the SPR phase modulation principle, calculate the local refractive index change Δn on the surface of the optical fiber probe, expressed as: Where Δφ is the SPR phase change, d is the thickness of the nano-modified layer on the probe surface, λ is the central wavelength of the SPR detection light, and n eff is the effective refractive index of the modified layer, n0 is the background refractive index of the sample solution, Δn = n eff -n0 represents the local refractive index change, which is related to the virus concentration; S623, virus concentration calculation: Combined with the Langmuir adsorption kinetic model, calculate the virus concentration C v , expressed as: Δn=K a C v / (1+K d C v ); Among them, C v is the virus concentration, K a is the binding constant, K d is the dissociation constant; S624, data analysis and result output: input the phase change data Δφ(t) into the signal analysis algorithm, optimize the quantitative mapping of SPR signal and virus concentration, and finally output the virus detection results, including quantitative concentration value or qualitative positive / negative judgment.

10. The method for enhancing the sensitivity of animal virus detection by surface modification of optical fiber SPR probe according to claim 9, characterized in that: The data analysis and result output in S624 include: S6241, filtering and noise reduction: Savitzky-Golay smoothing filter is used to smooth the SPR signal curve; S6242, nonlinear fitting: a four-parameter logistic regression model was used for fitting, and the virus concentration was quantitatively corrected to obtain the corrected SPR signal change Δφ ′ ; S6243, Result determination: Setting the detection threshold like The sample is judged as positive, otherwise it is judged as negative.

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