EV71 severe hand-foot-and-mouth disease prevention method based on quantum dot fluorescence sensing

Abstract

An EV71 severe hand-foot-and-mouth disease prevention method based on quantum dot fluorescence sensing belongs to the technical field of disease prevention, and comprises the following steps: selecting a non-functionalized quantum dot solution, a carboxyl ligand solution and a buffer solution to prepare a quantum dot fluorescence probe for specifically recognizing EV71 virus capsid protein; the method comprises the following steps: collecting a biological sample of a subject, and pretreating the biological sample to obtain a sample solution; the quantum dot fluorescent probe is added into a sample solution, fluorescence is excited after incubation, and fluorescence intensity data is collected. And performing background correction on the fluorescence intensity data, and comparing the fluorescence intensity data with a pre-established health and infection threshold library to output a detection result and a risk level corresponding to the detection result. And generating an early warning report according to the detection result and the risk level, and sending the early warning report to the clinical management terminal. According to the invention, early, sensitive and specific detection of the EV71 virus is realized, and early warning is given out before clinical symptoms are not caused by infection, so that intervention measures are started in time.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease prevention, and more specifically, relates to a method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing. Background Art

[0002] Hand, foot, and mouth disease (HFMD) is an epidemic primarily caused by enterovirus 71 (EV71) or coxsackievirus A16 (COX A16). Clinical manifestations vary, ranging from asymptomatic infection, mild symptoms such as common HFMD and pharyngitis, to severe symptoms such as neurogenic pulmonary edema, respiratory failure, and central nervous system infection. Some severe cases progress rapidly and can even lead to death. HFMD is widespread in my country and around the world, primarily affecting preschool children, with a particularly high incidence in children under three years of age. Compared with HFMD caused by other enteroviruses, HFMD caused by EV71 has a higher incidence of severe cases and a higher mortality rate.

[0003] Currently, the diagnosis of EV71 relies primarily on viral nucleic acid testing and virus isolation and culture, which are not yet available in primary care hospitals and Centers for Disease Control and Prevention. Furthermore, some severe cases do not exhibit typical clinical manifestations of hand, foot, and mouth disease. Consequently, early diagnosis of severe cases during an outbreak is difficult and often leads to pneumonia, delaying optimal treatment. Therefore, achieving early, sensitive, and specific detection of EV71, and issuing early warnings before infection causes clinical symptoms, thereby enabling timely intervention and reducing the incidence of neurological complications, and addressing the limited sensitivity, short detection window, and delayed warnings inherent in existing technologies, has become a pressing technical challenge. Summary of the Invention

[0004] In order to address the deficiencies in the prior art, the purpose of the present invention is to solve the above-mentioned defects and further propose a method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing.

[0005] The present invention adopts the following technical solutions.

[0006] The present invention discloses a method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing, the method comprising: Quantum dot fluorescent probes of capsid proteins; collecting a biological sample from a subject and pre-treating the biological sample to obtain a sample solution; adding the quantum dot fluorescent probe to the sample solution, exciting fluorescence after incubation, and collecting fluorescence intensity data; Performing background correction on the fluorescence intensity data and comparing it with a pre-established health and infection threshold library to output a detection result and a risk level corresponding to the detection result; Generate an early warning report based on the detection results and risk level, and send the early warning report to the clinical management terminal; The non-functionalized quantum dot solution and the carboxyl ligand solution are diluted in the buffer solution according to a preset concentration ratio to prepare a pre-diluted quantum dot-ligand mixed solution; an EDC / NHS mixed solution is added to the quantum dot-ligand mixed solution to prepare an intermediate solution; a specific ligand containing a free amino group is added to the intermediate solution, and BSA is added to block excess NHS esters for incubation and centrifugation to obtain a functionalized quantum probe solution containing the quantum dot fluorescent probe.

[0007] Furthermore, the preparation of a quantum dot fluorescent probe that specifically recognizes EV71 viral capsid protein by selecting a non-functionalized quantum dot solution, a carboxyl ligand, and a buffer solution comprises: Controlling the pipette to measure the non-functionalized quantum dot solution and the carboxyl ligand solution respectively, diluting them in the buffer solution according to a preset concentration ratio and mixing them evenly to obtain a pre-diluted quantum dot-ligand mixed solution; the buffer solution is PBS solution; An equal volume of EDC / NHS mixed solution was added to the quantum dot-ligand mixed solution, and after mixing and reacting uniformly at room temperature, excess EDC / NHS mixed solution was removed by ethanol precipitation to obtain an intermediate solution.

[0008] Furthermore, the method of selecting a non-functionalized quantum dot solution, a carboxyl ligand, and a buffer solution to prepare a quantum dot fluorescent probe that specifically recognizes EV71 viral capsid protein further includes: A specific ligand containing a free amino group is added to the intermediate solution and mixed to react evenly. After the reaction is completed, BSA is added to block excess NHS esters, and the mixture is incubated and centrifuged to remove free ligands and BSA, thereby obtaining a functionalized quantum probe solution. The functionalized quantum probe solution is placed in a dialysis membrane, and small molecule impurities are removed by replacing the PBS solution multiple times to obtain a stock solution of the quantum dot fluorescent probe; The absorption peak position and fluorescence spectrum were determined by UV-Vis, and the hydrodynamic diameter was determined by DLS to ensure that the purity and particle size of the stock solution met the preset requirements.

[0009] Furthermore, the collecting of biological samples from subjects and pre-processing of the biological samples to obtain sample solutions include: Insert a sterile long-stem swab into the subject's pharynx and gently wipe along the pharyngeal wall several times, then place the sterile long-stem swab into a sampling tube containing RNA preservation solution to collect an oropharyngeal swab from the subject; The subject's peripheral blood is placed in a blood collection tube and allowed to stand, and then centrifuged to collect a serum sample from the subject; Wherein, the biological sample of the subject includes the oropharyngeal swab and serum sample.

[0010] Furthermore, the process of collecting a biological sample from a subject and pre-treating the biological sample to obtain a sample solution further includes: The oropharyngeal swab or serum sample is placed in a centrifuge tube for centrifugation, and the top supernatant is discarded after centrifugation to retain the cell debris pellet and the clear fluid; Filtering the clarified liquid through a syringe filter membrane, and collecting the filtrate in an RNase-free centrifuge tube to obtain a pretreated sample; The pretreated sample and the buffer solution are diluted according to a preset volume ratio to obtain the sample solution.

[0011] Furthermore, the step of adding the quantum dot fluorescent probe to the sample solution and exciting fluorescence after incubation to collect fluorescence intensity data includes: Adding the stock solution of the quantum dot fluorescent probe, the sample solution, and the buffer solution into a centrifuge tube according to a preset volume ratio, mixing them evenly, and then incubating them to allow the quantum dot fluorescent probe to bind to the EV71 capsid protein in the sample solution to obtain a probe-sample mixture; Transferring the probe-sample mixture to a black bottom plate, and scanning the probe-sample mixture using a multifunctional fluorescence microplate reader to collect the fluorescence intensity value of the probe-sample mixture in each well of the black bottom plate; A fluorescence intensity data matrix is constructed based on the fluorescence intensity value of the probe-sample mixture in each well of the black bottom plate.

[0012] Furthermore, the step of adding the quantum dot fluorescent probe to the sample solution, incubating the solution, and exciting the fluorescence to collect fluorescence intensity data further includes: Obtaining a blank control fluorescence intensity matrix; the blank control fluorescence intensity matrix is a fluorescence intensity value matrix constructed from the fluorescence intensity values of each well of a black bottom plate scanned in the same batch as the fluorescence intensity data matrix and containing no quantum dot fluorescent probes or sample solutions; Based on the blank control fluorescence intensity matrix, calculating the average value of all blank control fluorescence intensity values on the black bottom plate at each time point, and performing background correction on each fluorescence intensity value in the fluorescence intensity data matrix based on the average value to obtain a corrected fluorescence intensity matrix; The corrected fluorescence intensity value of the last time point of each sample in the corrected fluorescence intensity matrix is selected as the endpoint fluorescence response value, and the endpoint fluorescence response value is used to estimate the EV71 antigen concentration range in the sample.

[0013] Furthermore, the fluorescence intensity data is background corrected and compared with a pre-established health and infection threshold library to output a detection result and a risk level corresponding to the detection result, including: A scatter plot is made between the concentration of the EV71 capsid protein standard of known concentration and the endpoint fluorescence response value, and a linear regression is performed based on the scatter plot using the least squares method to construct a linear model and determine the standard curve fitting parameters; Substituting the endpoint fluorescence response value of each sample into the linear model to calculate the predicted value of the antigen concentration corresponding to each sample; The predicted antigen concentration value of each sample is threshold-compared according to the pre-established health and infection threshold library to determine the detection result classification of each sample and the risk level corresponding to each detection result.

[0014] Furthermore, the method further comprises: Obtaining the known actual antigen concentration added with the batch and the quality control sample concentration of the predicted antigen concentration, wherein the quality control sample concentration is divided into multiple levels according to the concentration range; Calculate the recovery rate of each quality control sample; the recovery rate is the ratio between the predicted antigen concentration of each quality control sample and the known true antigen concentration; When the recovery rate of any quality control sample exceeds the preset range, the quality control sample is corrected using the correction coefficient, and the recovery rate of the corrected quality control sample is recalculated.

[0015] Furthermore, generating an early warning report based on the detection result and risk level, and sending the early warning report to the clinical management terminal, includes: Traverse all samples and screen out a list of high-risk samples based on the detection result classification and risk level of each sample; the high-risk sample list includes the predicted antigen concentration value of each screened sample; Comparing the predicted antigen concentration value of each sample in the high-risk sample list with a preset level threshold to generate a warning level for each sample in the high-risk sample list; In response to the warning level, the warning report is generated by filling in the template engine, and the warning report is sent to the clinical management terminal.

[0016] The beneficial effects of the present invention are that, compared with the prior art, the present invention has the following advantages: (1) The present invention improves the binding efficiency of target molecules by preparing quantum dot fluorescent probes that can specifically recognize EV71 viral capsid protein, thus overcoming the technical defect of insufficient sensitivity in the prior art. In addition, after collecting oropharyngeal swabs and serum samples from subjects, they are pre-treated by centrifugation, filtration, and standardized dilution, eliminating nonspecific background signals, enhancing detection specificity, and overcoming the risk of false positives.

[0017] (2) The present invention adds functionalized probes to the pretreated sample, excites fluorescence after incubation, and collects fluorescence intensity data, thereby amplifying the virus-specific signal and solving the defect of the existing technology that is insensitive to low viral loads.

[0018] (3) The present invention performs background correction on the collected fluorescence intensity and compares it with a pre-established health and infection threshold library. Through quantifiable threshold judgment, it ensures the accuracy and reliability of the detection results to a certain extent, eliminates subjective judgment errors, and provides effective intervention for disease prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a flow chart of the method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention.

[0020] Figure 2 It is a line graph of the fluorescence response test data provided by the present invention.

[0021] Figure 3 It is a line graph of the verification data of the standard curve provided by the present invention. DETAILED DESCRIPTION

[0022] The present application will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present application.

[0023] like Figure 1 As shown, in one embodiment, a method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing includes the following steps: Step S110 , selecting a non-functionalized quantum dot solution, a carboxyl ligand solution, and a buffer solution to prepare a quantum dot fluorescent probe that specifically recognizes EV71 virus capsid protein.

[0024] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S110 specifically includes the following steps: In step S111, the pipette is controlled to measure the non-functionalized quantum dot solution and the carboxyl ligand solution respectively, and the solution is diluted in a buffer solution according to a preset concentration ratio and mixed evenly to obtain a pre-diluted quantum dot-ligand mixed solution; the buffer solution is a PBS solution.

[0025] Step S112, adding an equal volume of EDC / NHS mixed solution to the quantum dot-ligand mixed solution, mixing and reacting uniformly at room temperature, and removing excess EDC / NHS mixed solution by ethanol precipitation to obtain an intermediate solution.

[0026] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S110 specifically further includes the following steps: In step S113, a specific ligand containing a free amino group is added to the intermediate solution and mixed to react evenly. After the reaction is completed, BSA is added to block excess NHS ester, and the mixture is incubated and centrifuged to remove free ligand and BSA to obtain a functionalized quantum probe solution.

[0027] Step S114 , placing the functionalized quantum probe solution in a dialysis membrane, and removing small molecule impurities by replacing the PBS solution multiple times to obtain a stock solution of the quantum dot fluorescent probe.

[0028] In step S115 , the absorption peak position and fluorescence spectrum are measured by UV-Vis, and the hydrodynamic diameter is measured by DLS to ensure that the purity and particle size of the stock solution meet the preset requirements.

[0029] In a specific embodiment, the method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention includes steps 1 to 5: Step 1: Functional preparation of quantum dot probes.

[0030] Step 1 is used to prepare a quantum dot fluorescent probe that can specifically identify the EV71 virus capsid protein to improve the binding efficiency to the target molecule and solve the problem of insufficient sensitivity in the existing technology.

[0031] The following steps are involved: Step 1.1, material preparation before quantum dot surface selection.

[0032] Specifically, use a pipette to measure the non-functionalized quantum dot solution (concentration C QD , unit: μM) and hydroxy ligand solution (such as mercaptopolyethylene glycol-COOH, concentration C L , unit: mM), diluted in PBS buffer (PBS with pH = 7.4) according to a preset concentration ratio R, and gently mixed to obtain a pre-diluted quantum dot-ligand mixed solution.

[0033] Among them, the concentration ratio R=C L / C QD , the reasonable range is 100-500.

[0034] Step 1.1 can determine the molar ratio of ligand to quantum dots, ensuring a balance between subsequent coupling efficiency and fluorescence performance, and avoiding steric hindrance or fluorescence quenching caused by excessive ligand.

[0035] Step 1.2, quantum dot surface activation.

[0036] Specifically, an equal volume of an EDC / NHS mixture (EDC / NHS activation reagent, concentration: 10mM EDC + 10mM NHS) was added to the pre-diluted quantum dot-ligand mixture. The mixture was gently inverted to mix thoroughly at room temperature. After reacting for 15 minutes, excess EDC / NHS was removed by ethanol precipitation. The specific process was as follows: add 4 volumes of ethanol, centrifuge at 4°C (10,000g, 10 minutes), suspend, and resuspend in PBS. This resulted in an NHS-activated intermediate quantum dot solution (short-lived, requiring immediate use), also known as the intermediate solution.

[0037] Step 1.2 activates the carboxyl terminus to an NHS ester, which improves the subsequent coupling efficiency with the amino ligand and removes residual activation reagent to avoid nonspecific binding.

[0038] Step 1.3, ligand coupling and directed modification.

[0039] Specifically, a specific ligand containing free amino groups (such as an anti-EV71 monoclonal antibody or peptide, at a concentration of 0.1-1 mg / mL) is added to the intermediate solution, gently inverted to mix, and allowed to react for 2 hours (at 4°C, with gentle shaking). After the reaction, 1% BSA is added to block excess NHS esters, followed by a 30-minute incubation. Free ligands and BSA are then removed by centrifugation (10,000 g, 10 minutes), and the solution is resuspended in storage buffer (0.1% BSA in PBS) to obtain a functionalized quantum dot probe solution.

[0040] In step 1.3, the ligand that recognizes the EV71 capsid protein is directed to the quantum dot surface to achieve highly specific recognition, and BSA blocking is used to reduce nonspecific adsorption.

[0041] Step 1.4, purification and characterization.

[0042] Specifically, the functionalized quantum dot probe solution obtained in step 1.3 was placed in a dialysis membrane (molecular weight cutoff: 100 kDa), and the PBS solution was replaced twice (2 h each) at 4°C to remove small molecule impurities. The absorption peak position and fluorescence spectrum were measured using UV-Vis (ultraviolet-visible spectrometer) to confirm that there was no obvious drift in the peak position; the hydrodynamic diameter was measured using DLS (dynamic light scattering) to confirm that the particle size was in the range of 10-20 nm and the polydispersity index PDI was <0.2.

[0043] In step 1.4, the chemical purity, structural stability, and biological activity of the probe are ensured through dialysis and spectral / particle size characterization, laying the foundation for subsequent sensitive and reproducible fluorescence detection.

[0044] Step S120 : collecting a biological sample from a subject and pre-processing the biological sample to obtain a sample solution.

[0045] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S120 specifically includes the following steps: Step S121: insert a sterile long-stem swab into the subject's pharynx, gently wipe along the pharyngeal wall several times, and then place the sterile long-stem swab into a sampling tube containing RNA preservation solution to collect the subject's oropharyngeal swab.

[0046] In step S122, the subject's peripheral blood is placed in a blood collection tube and allowed to stand, and then centrifuged to collect a serum sample from the subject.

[0047] Among them, the subjects' biological samples include oropharyngeal swabs and serum samples.

[0048] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S120 specifically further includes the following steps: In step S123, the oropharyngeal swab or serum sample is placed in a centrifuge tube for centrifugation, and the top supernatant is discarded after centrifugation to retain the cell debris precipitate and the clear solution.

[0049] In step S124 , the clarified liquid is filtered through a syringe filter membrane, and the filtrate is collected in an RNase-free centrifuge tube to obtain a pretreated sample.

[0050] Step S125 , diluting the pretreated sample and the buffer solution according to a preset volume ratio to obtain a sample solution.

[0051] In a specific embodiment, the present invention provides a method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing. Step 2 involves clinical sample collection and pretreatment. This step involves collecting an oropharyngeal swab or serum sample from the subject, centrifuging it, filtering it, and performing standardized dilution to remove nonspecific background signals, enhance detection specificity, and mitigate the risk of false positives.

[0052] The following steps are involved: Step 2.1, sample type selection and collection.

[0053] Specifically, a sterile, long-stem swab is inserted into the subject's pharynx, gently rubbed along the pharyngeal wall 3-5 times, and then quickly placed in a sampling tube containing RNA preservation solution to complete the oropharyngeal swab collection. In addition to oropharyngeal swabs, serum samples can also be collected. During the serum collection process, 5 mL of peripheral blood is collected in a clean blood collection tube, allowed to stand at 4°C for 30 minutes, and then centrifuged at 3000g for 10 minutes. The supernatant is aspirated as the serum sample.

[0054] Step 2.1 obtains the original sample representing the EV71 infection status in the body, laying the foundation for subsequent accurate detection.

[0055] Step 2.2: Centrifuge and enrich, and retain the supernatant.

[0056] Specifically, place the oropharyngeal swab digestion fluid or serum sample in a 1.5 mL centrifuge tube, centrifuge at 5000 g for 10 minutes at 4°C, carefully discard the upper 10% volume of the supernatant (containing a small amount of cell debris), and retain 90% of the clarified supernatant in a new tube to obtain the cell debris precipitate and clarified supernatant (i.e., clarified fluid).

[0057] Step 2.2 removes cell debris and macromolecular impurities, reduces the scattering background in fluorescence detection, and improves the signal-to-noise ratio.

[0058] Step 2.3, membrane filtration.

[0059] Specifically, the clarified supernatant (i.e., clarified liquid) obtained in step 2.2 is slowly filtered through a 0.22 μm syringe filter to avoid generating bubbles, and the filtrate is collected in an RNase-free centrifuge tube and immediately placed on ice to obtain a sterile pretreated sample free of macromolecular aggregates.

[0060] Step 2.3 completely removes particles that may clog the capillary or produce nonspecific adsorption, achieving sample stability and detection repeatability.

[0061] Step 2.4, standardization dilution and concentration correction.

[0062] Specifically, according to the detection requirements, the pretreated sample obtained in step 2.3 is diluted with dilution buffer (0.1% BSA in PBS) according to the volume ratio to the final sample concentration D, which is expressed as:

[0063] Where, is the original sample volume (unit: μL), The volume of buffer added to the sample (unit: μL) should be such that the final sample concentration D is maintained in the range of 0.5-0.8 to ensure that the fluorescence signal is in equilibrium with the sample matrix.

[0064] After the pre-treated sample is mixed with the dilution buffer, 10 μL is pipetted for residual detection to ensure that the dilution accuracy error is <5%.

[0065] Step 2.4 standardizes the matrix concentration in the samples that may affect fluorescence, eliminates detection bias caused by differences in concentration between individual samples, and ensures reliable comparison between samples.

[0066] In step S130 , the quantum dot fluorescent probe is added to the sample solution, and fluorescence is excited after incubation to collect fluorescence intensity data.

[0067] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S130 specifically includes the following steps: In step S131, a stock solution of quantum dot fluorescent probe, a sample solution and a buffer solution are added to a centrifuge tube according to a preset volume ratio, mixed evenly and then incubated to allow the quantum dot fluorescent probe to bind to the EV71 capsid protein in the sample solution to obtain a probe-sample mixture.

[0068] Step S132 , transferring the probe-sample mixture to a black bottom plate, and scanning the probe-sample mixture with a multifunctional fluorescence microplate reader to collect the fluorescence intensity value of the probe-sample mixture in each well of the black bottom plate.

[0069] Step S133 : constructing a fluorescence intensity data matrix based on the fluorescence intensity value of the probe-sample mixture in each well of the black bottom plate.

[0070] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S130 specifically further includes the following steps: Step S134 , obtaining a blank control fluorescence intensity matrix; the blank control fluorescence intensity matrix is a fluorescence intensity value matrix constructed from the fluorescence intensity values of each well of a black bottom plate scanned in the same batch as the fluorescence intensity data matrix and containing no quantum dot fluorescent probes or sample solutions.

[0071] Step S135 , based on the blank control fluorescence intensity matrix, calculating the average value of all blank control fluorescence intensity values on the black background plate at each time point, and performing background correction on each fluorescence intensity value in the fluorescence intensity data matrix based on the average value to obtain a corrected fluorescence intensity matrix.

[0072] Step S136 , selecting the corrected fluorescence intensity value of the last time point of each sample in the corrected fluorescence intensity matrix as the endpoint fluorescence response value, and the endpoint fluorescence response value is used to estimate the EV71 antigen concentration range in the sample.

[0073] In a specific embodiment, the present invention provides a method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing. Step 3, fluorescence sensing reaction. In step 3, functionalized probes are added to the pretreated sample, and after incubation, fluorescence is excited and fluorescence intensity data is collected. This achieves virus-specific signal amplification, addressing the drawback of existing tests that are insensitive to low viral loads.

[0074] The following steps are involved: Step 3.1: Mix and pre-incubate the probe with the sample.

[0075] Specifically, add the functionalized quantum dot probe solution (μM), pretreated sample solution (i.e., the supernatant at dilution factor D), and reaction buffer (PBS, pH 7.4, containing 0.1% BSA) to a 1.5 mL microcentrifuge tube at a volume ratio of 0.5:1:0.5. For example, add 100 μL of the functionalized quantum dot probe solution, 200 μL of the pretreated sample solution, and 100 μL of the reaction buffer. Gently invert the tube 10 times to mix thoroughly. Incubate at 37°C for 10 minutes to allow the quantum dot probes to fully bind to the EV71 capsid protein in the sample, resulting in a probe-sample mixture. Avoid vigorous shaking during incubation to prevent quantum dot aggregation or probe detachment.

[0076] Step 3.1: Pre-incubate to allow the probe to fully bind to the target antigen and form a stable complex, providing a basis for subsequent fluorescence signal amplification.

[0077] Step 3.2: Fluorescence excitation and signal acquisition.

[0078] Specifically, the probe-sample mixture obtained in step 3.2 was transferred to a 96-well black-bottom plate, 50 μL was taken from each well, and a multifunctional fluorescence microplate reader was used with the excitation wavelength set to 360 nm, the emission wavelength set to 620 nm, and the gain set to 200. Time scanning was performed, and each well was measured continuously for 5 min with a time interval of 30 s, for a total of 10 time points. The fluorescence intensity values were collected, and the original fluorescence intensity data matrix can be constructed based on the fluorescence intensity values of each well on the 96-well black-bottom plate at each time point.

[0079] In step 3.2, by setting appropriate excitation / emission parameters and time scanning, a dynamic fluorescence response curve of the probe-sample mixture is obtained to reflect the real-time characteristics of the binding reaction.

[0080] In this example, the following experimental scheme was used to verify the sensitivity and dynamic range of fluorescence excitation and signal acquisition: (1) Antigen gradient preparation: Prepare EV71 capsid protein standards of known concentrations: 0, 1, 5, 10, and 20 ng / mL, at least 5 concentration points, and perform 10 replicate wells for each concentration point.

[0081] (2) Probe binding and fluorescence measurement: According to the pre-incubation conditions in sub-step 3.1, the functionalized quantum dot probes were mixed with samples of various concentrations, and time scans were performed in a multifunctional microplate reader (excitation 360 nm, emission 620 nm). The fluorescence intensity value at the end of 5 minutes was taken as the endpoint response.

[0082] (3) Data recording and analysis: The endpoint fluorescence intensity of each well was collected, the background-corrected signal was processed, the concentration-response curve was drawn, and the limit of detection (LOD) and limit of quantification (LOQ) were calculated.

[0083] Figure 2 This is a line graph of fluorescence response test data. Based on the fluorescence sensing reaction experiment, the endpoint fluorescence intensity of 10 replicate wells of different antigen gradients (0–20 ng / mL) was measured using a multifunctional microplate reader (excitation 360 nm / emission 620 nm). Figure 2 In each group of 10 replicate wells, endpoint fluorescence intensity (unit: AU) at a gradient concentration from 0 to 20 ng / mL is calculated. This data can be used to perform standard curve fitting and LOD / LOQ calculations. Sample_ID represents the sample name, Concentration_ng_per_mL is the actual concentration of EV71 capsid protein (antigen) in the sample, in nanograms per milliliter (ng / mL). Fluorescence_Intensity_AU is the fluorescence signal intensity measured at the specified excitation / emission wavelength for the corresponding sample, expressed in Arbitrary Fluorescence Units (AU), which reflects the change in fluorescence intensity resulting from probe-antigen binding.

[0084] Step 3.3, background correction and photobleaching verification.

[0085] Specifically, a blank control well fluorescence matrix is established, i.e., a matrix of fluorescence intensity values from a blank control group scanned from the same batch without probe solution or sample antigen. For each time point, the average value of the blank control is calculated, i.e., the mean fluorescence intensity of the blank control groups across all wells of a 96-well black-bottom plate. This is the ratio of the sum of the fluorescence intensity values of all wells to the total number of wells. The difference between the true value of each blank control well and the mean is then divided by the mean. This result is then used to perform background correction on all sample data, resulting in a corrected fluorescence intensity matrix to ensure dimensionless corrected data. The trend of the corrected data over time is examined. If a signal decreases by more than 20% over time, it is considered quantum dot bleaching, requiring re-incubation or a reduction in excitation intensity.

[0086] Step 3.3 eliminates instrument background and nonspecific fluorescence, improving detection accuracy; at the same time, the photostability of quantum dots is monitored to ensure data reliability.

[0087] Step 3.4: Endpoint extraction and preliminary quantification of fluorescence signals.

[0088] Specifically, for each sample in the corrected fluorescence intensity matrix obtained in step 3.3, the corrected fluorescence intensity value at the last time point is taken as the endpoint fluorescence response value. The endpoint fluorescence response value can be compared with the fluorescence-antigen concentration relationship established in advance in the standard curve to preliminarily estimate the EV71 antigen concentration range in the sample.

[0089] Step 3.4 extracts the stable endpoint response to simplify data processing and quickly obtain the relative fluorescence changes of each sample, providing a basis for subsequent precise quantification and threshold judgment.

[0090] In step S140 , background correction is performed on the fluorescence intensity data, and the data is compared with a pre-established healthy and infected threshold library to output a detection result and a risk level corresponding to the detection result.

[0091] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S140 specifically includes the following steps: In step S141 , a scatter plot is made between the concentration of the EV71 capsid protein standard with known concentration and the end-point fluorescence response value, and a linear regression is performed based on the scatter plot using the least squares method to construct a linear model and determine the standard curve fitting parameters.

[0092] In step S142 , the endpoint fluorescence response value of each sample is brought into the linear model to calculate the predicted value of the antigen concentration corresponding to each sample.

[0093] In step S143, threshold comparison is performed on the predicted antigen concentration value of each sample according to the pre-established health and infection threshold library to determine the detection result classification of each sample and the risk level corresponding to each detection result.

[0094] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention further includes the following steps: Step S210 , obtaining the quality control sample concentration of the known actual antigen concentration added with the batch and the predicted antigen concentration value, and the quality control sample concentration is divided into multiple levels according to the concentration range.

[0095] Step S220 , calculating the recovery rate of each quality control sample; the recovery rate is the ratio between the predicted antigen concentration of each quality control sample and the known true antigen concentration.

[0096] In step S230 , when the recovery rate of any quality control sample exceeds a preset range, the quality control sample is corrected using a correction coefficient, and the recovery rate of the corrected quality control sample is recalculated.

[0097] In a specific embodiment, the present invention provides a method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing. Step 4, data analysis and threshold determination, involves background correction of the collected fluorescence intensity and comparison with a pre-established library of healthy and infected thresholds. This quantifiable threshold determination ensures accurate and reliable results, eliminating subjective judgment errors.

[0098] The following steps are involved: Step 4.1, standard curve construction.

[0099] Specifically, first, a series of EV71 capsid protein standards with known concentrations (unit: ng / mL, range 0-100 ng / mL) and their corresponding endpoint fluorescence response values were determined, as shown in , is the concentration of the kth standard, is the endpoint fluorescence response value after calibration of the kth standard, and K is the total number of samples. End point fluorescence response Make a scatter plot and use the least squares method for linear regression. The linear model expression is:

[0100] Where, It represents the slope of the curve, reflecting the sensitivity of the fluorescence response to concentration. Represents the offset of the zero concentration point after background correction, Indicates concentration, Indicates the end-point fluorescence response value.

[0101] Calculate the coefficient of determination ,Require If not, it is necessary to re-prepare the standard or optimize the probe concentration.

[0102] Step 4.1 quantitatively maps the fluorescence response to the antigen concentration to achieve accurate estimation of subsequent sample concentrations. The high coefficient of determination ensures the accuracy of model fitting and reduces systematic errors.

[0103] In this example, to verify the accuracy and reproducibility of the linear fit constructed by the standard curve, the following experimental scheme was used: (1) Preparation and measurement of standards: Prepare five EV71 capsid protein standards of known concentrations: 0, 1, 5, 10, and 20 ng / mL. Set up 10 replicate wells for each concentration, for a total of 50 data points. Perform fluorescence measurement according to step 3.2 and record the endpoint-corrected fluorescence intensity (Fluorescence_AU) of each well.

[0104] (2) Data display: Figure 3This is a line graph of the standard curve construction validation data, which is plotted using the fluorescence response data of 10 replicate wells of known concentrations of standards (0, 1, 5, 10, and 20 ng / mL) measured using the same microplate reader. Figure 3 Including concentration (Conc_ng_per_mL), replicate number (Replicate) and fluorescence intensity (Fluorescence_AU): (3) Linear regression analysis: Fluorescence intensity was used as the independent variable R and concentration as the dependent variable C, and the least squares method was used to fit the linear model. The fitting results were: slope: a = 0.196 (ng / mL per AU); intercept: b = −9.31 ng / mL; determination coefficient: R 2 =0.992 (≥0.98, meeting the precision requirement). Three independent experiments were repeated, and the coefficients of variation (CV) of the slope and intercept were <5%, demonstrating good reproducibility of the method.

[0105] Step 4.2, calculation of sample concentration.

[0106] Based on the linear model expression obtained in step 4.1, substitute the endpoint fluorescence response value of each sample into the linear model expression:

[0107] Where, represents the concentration of the i-th sample (unit: ng / mL), Represents the endpoint fluorescence response value of the i-th sample. , it is considered “not detected”, if If the concentration exceeds the upper limit of the standard curve, it will be marked as "greater than 100 ng / mL" and it is recommended to dilute and retest.

[0108] Step 4.2 converts the fluorescence signal into quantitative concentration to estimate the amount of EV71 antigen in each clinical sample, facilitating subsequent threshold determination.

[0109] Step 4.3: Threshold determination and risk grading.

[0110] Specifically, for each Perform threshold comparison (negative threshold: , positive threshold: ): like , the detection result is judged to be “negative”; like , the test result is judged as "suspicious", and retesting or combined with clinical symptom assessment is recommended; like , the detection result is judged as "positive" and timely intervention is recommended.

[0111] Finally, the detection results of each sample are statistically classified and a bar chart is drawn to evaluate the overall risk distribution of this batch of samples.

[0112] Step 4.3 uses clear quantitative thresholds to achieve rapid and reproducible result judgment, and proposes secondary testing recommendations for "suspicious" samples to reduce the risk of misjudgment.

[0113] Step 4.4, quality control sample verification and bias correction.

[0114] Specifically, first, quality control sample concentrations (low, medium, and high, with known true and predicted concentrations) are added to each batch, and the recovery rate of each quality control sample is calculated, that is, predicted concentration / true concentration × 100%, with the recovery rate required to be within the range of 90%-110%. If the recovery rate of a certain level exceeds the required range, a correction factor is calculated, that is, the sum of true concentrations / the sum of predicted concentrations, and the concentrations of all samples are converted to the correct value. Multiply the calculated calibration factor to achieve uniform calibration. Recalculate the corrected recovery to confirm whether the calibration is effective.

[0115] In step 4.4, quality control samples are introduced to verify the accuracy of the test, and batch deviations are promptly detected and corrected to ensure stability and comparability in long-term applications.

[0116] Step S150: Generate an early warning report based on the detection results and risk level, and send the early warning report to the clinical management terminal.

[0117] In some embodiments, the method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing provided by the present invention, step S150 specifically includes the following steps: Step S151 traverses all samples and selects a high-risk sample list based on the detection result classification and risk level of each sample; the high-risk sample list includes the predicted antigen concentration value of each selected sample.

[0118] Step S152 : Compare the predicted antigen concentration value of each sample in the high-risk sample list with a preset level threshold to generate a warning level for each sample in the high-risk sample list.

[0119] Step S153: In response to the warning level, a warning report is generated by filling in the template engine, and the warning report is sent to the clinical management terminal.

[0120] In a specific embodiment, the present invention provides a method for preventing severe EV71 hand, foot and mouth disease based on quantum dot fluorescence sensing. Step 5, "Emergency Warning Triggering and Intervention Recommendations," automatically generates an early warning report based on the risk stratification results and sends it to the clinical management system, recommending antiviral or immunomodulatory interventions. This initiates preventive measures before clinical symptoms appear, addressing the issue of delayed prevention of severe HFMD.

[0121] The following steps are involved: Step 5.1, high-risk sample identification.

[0122] Specifically, all samples are traversed, and the sample indexes classified as "positive" are screened out. The indexes and their concentration values are stored in a high-risk list, and samples that require immediate intervention are quickly located, providing objects for the next step of reporting and intervention strategy formulation.

[0123] Step 5.2: Classification of warning levels.

[0124] Specifically, for each sample in the high-risk list obtained in step 5.1, the concentration of each sample Perform level threshold comparison: like , the level is classified as mild warning; like , the level is classified as moderate warning; like , the level is classified as high warning.

[0125] Finally, based on the above warning level classification results, a sample-level mapping table is generated for subsequent notifications. According to the viral load classification warning, medical resources and intervention intensity are accurately allocated.

[0126] Step 5.3: Generate and push the early warning report.

[0127] Specifically, based on the sample-level mapping table obtained in step 5.2, a template engine is used to fill in the report content: including sample ID, detection time, concentration value, warning level, and recommended measures. The report is automatically pushed to the corresponding medical staff or monitoring system interface through the hospital information system or SMS platform, and the timestamp and receipt confirmation mark of each push are recorded to achieve instant transmission of warning information and ensure that the clinical team can obtain and respond in the first time.

[0128] Step 5.4: Follow-up and effect evaluation.

[0129] Specifically, the pushed early warning records and medical feedback (such as the implementation of intervention measures and secondary test results) are regularly recorded. For example, medical feedback is automatically summarized every 24 hours: including actual medication, changes in secondary sampling concentrations, comparison of concentration differences before and after intervention, calculation of the percentage of concentration decrease, that is, the ratio of the difference between the first test concentration and the second test concentration to the first test concentration multiplied by 100%. The intervention effect is evaluated based on the calculated percentage of concentration decrease, and an effect evaluation report is generated, realizing closed-loop management of early warning and intervention, verifying the actual effectiveness of detection and suggestions, and continuously optimizing thresholds and intervention strategies.

[0130] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of such features. "Multiple" means two or more, unless otherwise specifically defined.

[0131] In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "connect," "fixed," etc. should be understood broadly. For example, they may refer to fixed connection, detachable connection, or integration; mechanical connection or electrical connection; direct connection or indirect connection through an intermediate medium; internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0132] In the present invention, unless otherwise expressly specified or limited, when a first feature is "above" or "below" a second feature, it may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediary. Furthermore, when a first feature is "above," "above," or "above" a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is "below," "below," or "below" a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0133] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0134] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code comprising one or more executable instructions for implementing the steps of a specific logical function or process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may be performed out of the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present invention pertain.

[0135] The logic and / or steps represented in a flowchart or otherwise described herein, for example, can be considered a sequenced list of executable instructions for implementing the logical functions, and can be embodied in any computer-readable medium for use by, or in conjunction with, an instruction execution system, apparatus, or device (e.g., a computer-based system, a system including a processor, or other system that can fetch and execute instructions from an instruction execution system, apparatus, or device). For purposes of this specification, a "computer-readable medium" can be any device that can contain, store, communicate, propagate, or transport a program for use by, or in conjunction with, an instruction execution system, apparatus, or device. More specific examples (not exhaustive) of computer-readable media include: an electrical connection with one or more wires (electronic devices), a portable computer disk cartridge (magnetic device), random access memory (RAM), read-only memory (ROM), erasable and programmable read-only memory (EPROM or flash memory), fiber optic devices, and a portable compact disc read-only memory (CDROM). Furthermore, the computer-readable medium may even be paper or other suitable medium on which the program is printed, since the program may be obtained electronically, for example, by optically scanning the paper or other medium and then editing, interpreting or processing it in another suitable manner if necessary, and then storing it in a computer memory.

[0136] It should be understood that various components of the present invention may be implemented using hardware, software, firmware, or a combination thereof. In the above-described embodiments, multiple steps or methods may be implemented using software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented using hardware, as in another embodiment, any one of the following technologies known in the art or a combination thereof may be used: a discrete logic circuit having logic gate circuits for implementing logic functions on data signals, an application-specific integrated circuit having suitable combinational logic gate circuits, a programmable gate array (PGA), a field-programmable gate array (FPGA), etc.

[0137] Those skilled in the art will understand that all or part of the steps in the method of the above embodiment can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. When the program is executed, it includes one or a combination of the steps of the method embodiment.

[0138] In addition, the functional units in the various embodiments of the present invention may be integrated into a single processing module, or each unit may exist physically separately, or two or more units may be integrated into a single module. The aforementioned integrated modules may be implemented in the form of hardware or in the form of software functional modules. If the integrated modules are implemented in the form of software functional modules and sold or used as independent products, they may also be stored in a computer-readable storage medium.

[0139] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing, characterized in that: The method comprises: A non-functionalized quantum dot solution, a carboxyl ligand solution, and a buffer solution were selected to prepare a quantum dot fluorescent probe that specifically recognizes EV71 viral capsid protein. collecting a biological sample from a subject and pre-treating the biological sample to obtain a sample solution; adding the quantum dot fluorescent probe to the sample solution, exciting fluorescence after incubation, and collecting fluorescence intensity data; Performing background correction on the fluorescence intensity data and comparing it with a pre-established health and infection threshold library to output a detection result and a risk level corresponding to the detection result; Generate an early warning report based on the detection results and risk level, and send the early warning report to the clinical management terminal; The non-functionalized quantum dot solution and the carboxyl ligand solution are diluted in the buffer solution according to a preset concentration ratio to prepare a pre-diluted quantum dot-ligand mixed solution; an EDC / NHS mixed solution is added to the quantum dot-ligand mixed solution to prepare an intermediate solution; a specific ligand containing a free amino group is added to the intermediate solution, and BSA is added to block excess NHS esters for incubation and centrifugation to obtain a functionalized quantum probe solution containing the quantum dot fluorescent probe.

2. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 1, characterized in that: The method of selecting a non-functionalized quantum dot solution, a carboxyl ligand, and a buffer solution to prepare a quantum dot fluorescent probe that specifically recognizes EV71 viral capsid protein comprises: Controlling the pipette to measure the non-functionalized quantum dot solution and the carboxyl ligand solution respectively, diluting them in the buffer solution according to a preset concentration ratio and mixing them evenly to obtain a pre-diluted quantum dot-ligand mixed solution; the buffer solution is PBS solution; An equal volume of EDC / NHS mixed solution was added to the quantum dot-ligand mixed solution, and after mixing and reacting uniformly at room temperature, excess EDC / NHS mixed solution was removed by ethanol precipitation to obtain an intermediate solution.

3. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 2, characterized in that: The method of selecting a non-functional quantum dot solution, a carboxyl ligand, and a buffer solution to prepare a quantum dot fluorescent probe that specifically recognizes EV71 virus capsid protein further includes: A specific ligand containing a free amino group is added to the intermediate solution and mixed to react evenly. After the reaction is completed, BSA is added to block excess NHS esters, and the mixture is incubated and centrifuged to remove free ligands and BSA, thereby obtaining a functionalized quantum probe solution. The functionalized quantum probe solution is placed in a dialysis membrane, and small molecule impurities are removed by replacing the PBS solution multiple times to obtain a stock solution of the quantum dot fluorescent probe; The absorption peak position and fluorescence spectrum were determined by UV-Vis, and the hydrodynamic diameter was determined by DLS to ensure that the purity and particle size of the stock solution met the preset requirements.

4. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 3, characterized in that: The step of collecting a biological sample from a subject and pre-treating the biological sample to obtain a sample solution comprises: Insert a sterile long-stem swab into the subject's pharynx and gently wipe along the pharyngeal wall several times, then place the sterile long-stem swab into a sampling tube containing RNA preservation solution to collect an oropharyngeal swab from the subject; The subject's peripheral blood is placed in a blood collection tube and allowed to stand, and then centrifuged to collect a serum sample from the subject; Wherein, the biological sample of the subject includes the oropharyngeal swab and serum sample.

5. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 4, characterized in that: The method of collecting a biological sample from a subject and pre-treating the biological sample to obtain a sample solution further includes: The oropharyngeal swab or serum sample is placed in a centrifuge tube for centrifugation, and the top supernatant is discarded after centrifugation to retain the cell debris pellet and the clear fluid; Filtering the clarified liquid through a syringe filter membrane, and collecting the filtrate in an RNase-free centrifuge tube to obtain a pretreated sample; The pretreated sample and the buffer solution are diluted according to a preset volume ratio to obtain the sample solution.

6. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 5, characterized in that: The step of adding the quantum dot fluorescent probe to the sample solution and exciting fluorescence after incubation to collect fluorescence intensity data includes: Adding the stock solution of the quantum dot fluorescent probe, the sample solution, and the buffer solution into a centrifuge tube according to a preset volume ratio, mixing them evenly, and then incubating them to allow the quantum dot fluorescent probe to bind to the EV71 capsid protein in the sample solution to obtain a probe-sample mixture; Transferring the probe-sample mixture to a black bottom plate, and scanning the probe-sample mixture using a multifunctional fluorescence microplate reader to collect the fluorescence intensity value of the probe-sample mixture in each well of the black bottom plate; A fluorescence intensity data matrix is constructed based on the fluorescence intensity value of the probe-sample mixture in each well of the black bottom plate.

7. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 6, characterized in that: The step of adding the quantum dot fluorescent probe to the sample solution and exciting fluorescence after incubation to collect fluorescence intensity data further includes: Obtaining a blank control fluorescence intensity matrix; the blank control fluorescence intensity matrix is a fluorescence intensity value matrix constructed from the fluorescence intensity values of each well of a black bottom plate scanned in the same batch as the fluorescence intensity data matrix and containing no quantum dot fluorescent probes or sample solutions; Based on the blank control fluorescence intensity matrix, calculating the average value of all blank control fluorescence intensity values on the black bottom plate at each time point, and performing background correction on each fluorescence intensity value in the fluorescence intensity data matrix based on the average value to obtain a corrected fluorescence intensity matrix; The corrected fluorescence intensity value of the last time point of each sample in the corrected fluorescence intensity matrix is selected as the endpoint fluorescence response value, and the endpoint fluorescence response value is used to estimate the EV71 antigen concentration range in the sample.

8. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 7, characterized in that: The fluorescence intensity data is subjected to background correction and compared with a pre-established health and infection threshold library to output a detection result and a risk level corresponding to the detection result, including: A scatter plot is made between the concentration of the EV71 capsid protein standard of known concentration and the endpoint fluorescence response value, and a linear regression is performed based on the scatter plot using the least squares method to construct a linear model and determine the standard curve fitting parameters; Substituting the endpoint fluorescence response value of each sample into the linear model to calculate the predicted value of the antigen concentration corresponding to each sample; The predicted antigen concentration value of each sample is threshold-compared according to the pre-established health and infection threshold library to determine the detection result classification of each sample and the risk level corresponding to each detection result.

9. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 8, characterized in that: The method further comprises: Obtaining the known actual antigen concentration added with the batch and the quality control sample concentration of the predicted antigen concentration, wherein the quality control sample concentration is divided into multiple levels according to the concentration range; Calculate the recovery rate of each quality control sample; the recovery rate is the ratio between the predicted antigen concentration of each quality control sample and the known true antigen concentration; When the recovery rate of any quality control sample exceeds the preset range, the quality control sample is corrected using the correction coefficient, and the recovery rate of the corrected quality control sample is recalculated.

10. The method for preventing EV71 severe hand, foot and mouth disease based on quantum dot fluorescence sensing according to claim 9, characterized in that: Generating an early warning report according to the detection result and risk level, and sending the early warning report to the clinical management terminal, includes: Traverse all samples and screen out a list of high-risk samples based on the detection result classification and risk level of each sample; the high-risk sample list includes the predicted antigen concentration value of each screened sample; Comparing the predicted antigen concentration value of each sample in the high-risk sample list with a preset level threshold to generate a warning level for each sample in the high-risk sample list; In response to the warning level, the warning report is generated by filling in the template engine, and the warning report is sent to the clinical management terminal.

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