Calibration method and calibration device for a bioaerosol monitor

Abstract

The application discloses a kind of biological aerosol monitor's calibration method and calibration device, adopt physical calibration method to combine biological verification method, including the following steps: S1: the flow parameter of biological gas gel is calibrated using flowmeter;S2: polystyrene microspheres and polystyrene microspheres with specific fluorescence are placed in calibration cabin, and stirring is uniformly mixed;S3: calibration cabin left side connecting port is connected with TSI particle size spectrometer, particle counter and the calibrated monitor respectively, and the second step calibration of monitor is realized by monitoring aerosol particle size, particle concentration in cabin;S4: biological verification, the biological sampling rate of biological aerosol sampler is calibrated and verified;S5: after calibration is completed, the particle concentration in calibration cabin is monitored using TSI particle size spectrometer, until the cleanliness in cabin meets the requirements.The application can realize the combination of physical calibration and biological verification method, ensure to provide uniform, stable and meet the requirements of aerosol environment for calibration work, and the calibration result is more accurate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural automation, in particular to a calibration method and a management method of a biological aerosol monitor, which can realize precise irrigation and fertilization of crops and comprehensive management. BACKGROUND

[0002] Biological aerosol refers to an important part of atmospheric aerosol, which is directly derived from biological particles such as viruses, bacteria, fungi, pollen, cells or plant debris, animal tissues, etc. However, when the biological aerosol contains pathogenic microorganisms such as bacteria, viruses, allergenic pollen, mold spores and parasitic eggs, the biological characteristics of the aerosol change in nature. In fact, it should be called pathogenic biological aerosol. Biological aerosol has the characteristics of large pollution area, strong infectivity and long influence time, which has always threatened human safety. Therefore, the importance of monitoring, early warning and detection of harmful biological aerosol is highlighted.

[0003] Traditional biological aerosol detection mainly relies on on-site sampling, culture, analysis and identification, which has a long detection process and slow speed, and contact sampling detection will pose a biological safety threat to the safety of the detection personnel. In the late 20th century, with the development of laser technology, photoelectric device manufacturing technology and spectral detection technology, laser-induced fluorescence technology and light scattering technology have become the main technologies for biological aerosol monitoring.

[0004] Bacteria, viruses and pollen particles contain various fluorescent substances such as amino acids and coenzymes. Amino acids exist in all biological organisms and are the basic units of protein peptides, while coenzymes, as life-active organisms, exist in biological organisms with vigorous metabolism. For organisms with no obvious metabolic activity, the coenzyme content is very low. These organic substances will produce intrinsic fluorescence under the excitation of excitation light at a specific wavelength, which is an important condition for identifying biological properties. Since some inorganic minerals will also emit fluorescence under ultraviolet light excitation, light scattering technology is used to measure the particle size range of the collected particles to exclude interference. By continuously detecting the fluorescence characteristics of biological particles of a specific particle size, sudden increases in the concentration of biological particles in the air can be found, and the device will trigger an alarm when it detects that the increase in the concentration of biological particles in the air exceeds a threshold value.

[0005] The biological aerosol monitor is a small and portable front-end early warning sensing device for active biological particles. Its working principle is that after sampling by a gas pump, the particle size is measured by light scattering method and the biological and non-biological aerosol particles are identified by fluorescence spectrum to realize the monitoring of biological particles emitting fluorescence signals in the environment. Therefore, the excitation light wavelength, fluorescence emission wavelength and quantum yield provide characteristic information of the fluorescent substance, which is the basis for qualitative and quantitative analysis of the detected biological aerosol particles.

[0006] The domestic bioaerosol monitor often appears false alarm in actual use, on the one hand, because there are a large number of substances in nature that can produce fluorescence, such as polycyclic aromatic hydrocarbon compounds, natural organic matter, the wavelength range emitted by these substances covers or partially coincides with coenzyme, resulting in false alarm of bioaerosol monitor; on the other hand, users usually think that the lower the bioalarm threshold is, the more sensitive it is, resulting in false alarm due to too low threshold setting. Therefore, it is necessary to establish a scientific evaluation technology to evaluate the performance of bioaerosol monitor, and at the same time, to provide reference for the setting of working parameters according to the performance of the monitor, but there is no standard substance and calibration method for calibrating bioaerosol monitor in China.

[0007] In 2020, Beijing Institute of Metrology and Testing Science proposed to select fluorescent reagents with different emission wavelengths, and prepare fluorescent microsphere standard substance that can produce fluorescence itself by SI-ATRP method; combined with static box calibration device, different concentrations of aerosol are generated to calibrate the fluorescence particle counting efficiency of bioaerosol monitor.

[0008] Beijing Faming Huqing Environmental Protection Technology Co., Ltd. proposed a gas and bioaerosol calibration system and method: generate bioaerosol into the calibration chamber, use a six-stage sieve pore air impact sampler to sample into the culture medium, use PBS as negative control, and calibrate the monitor before delivery by culture counting method.

[0009] There are three problems in the prior art:

[0010] 1) Beijing Institute of Metrology and Testing Science selects fluorescent reagents with different emission wavelengths, and prepares fluorescent microsphere standard substance that can produce fluorescence itself by SI-ATRP method, because the fluorescence intensity of fluorescent reagent is much higher than the intrinsic fluorescence intensity of microorganism, the prepared fluorescent microsphere cannot represent the fluorescence intensity of biological characteristics;

[0011] 2) Beijing Faming Huqing Environmental Protection Technology Co., Ltd. uses bacterial liquid to generate bioaerosol, and calibrates the monitor by culture counting method, because of the influence of biological particle activity, generation method, cabin environment temperature, humidity, sampling method and other factors, the bioaerosol sampling efficiency is greatly different, it is difficult to quantitatively represent the performance parameters of the monitor; at the same time, biological method is easy to produce environmental safety risk, the laboratory must be equipped with corresponding biological safety protection measures, and the operator must have corresponding biological technology sample operation and culture technology;

[0012] 3) Beijing Institute of Metrology and Testing Science and Technology characterizes the performance of the calibration device by calibrating the stability and uniformity of the aerosol in the chamber, the response time, and places a light scattering particulate matter sensor at the sampling position in the chamber and the center point to monitor the uniformity and stability of the aerosol concentration in the chamber, and verifies the response time of the calibration chamber by observing the changes in the gas flow and the particle concentration in the chamber. This method does not verify the performance of the generation system.

[0013] The present application aims to solve the problems described above. One object of the present application is to provide a biological aerosol monitor calibration method and calibration device suitable for light scattering and laser-induced fluorescence technology to solve the above problems, and another object is to establish a performance evaluation method for biological aerosol calibration chambers. SUMMARY

[0014] Therefore, the present application provides a biological aerosol monitor calibration method and calibration device suitable for light scattering and laser-induced fluorescence technology, and an automatic spraying and fertilization detection management system and management method that can accurately meet the needs of crop growth.

[0015] To achieve the above object, the present application adopts the following technical solutions:

[0016] A calibration method for a biological aerosol monitor, which adopts a physical calibration method combined with a biological verification method, comprising the following steps:

[0017] S1: Calibrate the flow parameter of the biological aerogel in the first step using a flow meter;

[0018] S2: Generate aerosol into the calibration chamber by an aerosol generation system using polystyrene microspheres and polystyrene microspheres with specific fluorescence, control the concentration of aerosol in the calibration chamber by adjusting the generation flow and generation time, and mix uniformly by the fan of the calibration chamber;

[0019] S3: Connect the TSI particle size spectrometer, particle counter and calibrated monitor to the left side connection port of the calibration chamber, and realize the second step calibration of the monitor by monitoring the particle size and particle concentration of the aerosol in the chamber;

[0020] S4: Biological verification, atomize the Bacillus subtilis standard material, generate aerosol with a concentration of 2*10 6 cfu / L, simultaneously sample the Anderson sampler and the aerosol monitor, compare the results of the Anderson sampling and the aerosol monitor by colony counting method, and calibrate and verify the biological sampling rate of the biological aerosol sampler;

[0021] S5: After calibration is completed, start the purification system of the calibration chamber, and monitor the particle concentration in the calibration chamber using the TSI particle size spectrometer until the cleanliness in the chamber meets the requirements.

[0022] Further, in step S2, the preparation of the specific fluorescent polystyrene microspheres is: the polystyrene microspheres are dispersed by ultrasonic to make them uniformly dispersed in the ethanol solvent to form a stable dispersion liquid, and the ultrasonic time is 10-30 minutes.

[0023] The polystyrene microsphere dispersion liquid is transferred to an electromagnetic stirring device, protected by nitrogen, the reaction temperature is controlled by a constant temperature water bath, the initial reaction temperature is set to 25-35℃; the riboflavin stock solution is slowly added to the polystyrene microsphere dispersion liquid, the dropwise adding speed is controlled at 1-2 drops / s, and stirring is started at the same time, and the stirring speed is adjusted to 300-600 rpm, so that the riboflavin and the polystyrene microspheres are fully contacted, and the reaction time is 1-3 hours.

[0024] Under the excitation light excitation condition, the intrinsic fluorescence intensity of Bacillus subtilis and Phi-X174 bacteriophage is measured, the fluorescence intensity of the fluorescent microspheres is measured, and the fluorescence intensity of the fluorescent microspheres is adjusted by controlling the amount of riboflavin, so that the light intensity of the prepared fluorescent microspheres is close to the intrinsic fluorescence intensity of the microorganisms, and the absolute measurement method is used, the scanning electron microscope is used to calibrate the labeled fluorescent particles, and the spectral analysis method is used to test the fluorescence characteristics of the labeled fluorescent particles.

[0025] Reaction termination and post-processing: when it is determined that the intrinsic fluorescence intensity of Bacillus subtilis and Phi-X174 bacteriophage is reached, a terminating agent is added to terminate the reaction, and the amount of the terminating agent is 1-2 times the molar amount of riboflavin, after the terminating agent is added, the reaction is continuously stirred for 10-15 minutes to completely terminate the reaction; then, the reaction product is separated by high-speed centrifugation at a speed of 8000-12000 rpm for 10-20 minutes, and the polystyrene microspheres grafted with riboflavin are collected; the precipitate is washed with deionized water for 3-5 times to remove ungrafted riboflavin and other impurities, and finally the 1 μm and 3 μm fluorescent microsphere suspensions are obtained.

[0026] Further, the terminating agent is ethanolamine.

[0027] Further, in step S3, the second step calibration includes particle size measurement value error calibration, particle concentration value error calibration, and fluorescent particle count deviation calibration.

[0028] Further, the fluorescent particle count deviation calibration is to dilute the fluorescent microsphere suspensions of 1 μm and 3 μm by 5-10 times respectively, shake well, generate monodisperse fluorescent microsphere aerosol through the calibration system, control the aerosol particle concentration at 45000-55000 / L, put into and start the bioaerosol monitor, record the particle size measurement results of the bioaerosol monitor after normal operation for 5 min, record the data every 10 s, record 3 groups in total, and calculate the particle size measurement indication error ΔD according to formula (1):

[0029]

[0030] In the formula: is the average value of 3 measurements of the bioaerosol monitor, μm;

[0031] D s is the fluorescent microsphere particle size standard value, μm.

[0032] Further, the particle concentration indication error calibration is to dilute the fluorescent microsphere suspensions of 1 μm and 3 μm by 5-10 times respectively, shake well, generate monodisperse fluorescent microsphere aerosol through the calibration system, control the aerosol particle concentration at 45000-55000 / L, start the particle counter and the bioaerosol monitor respectively, record the particle concentration measurement results of the particle counter and the bioaerosol monitor every 10 s after normal operation for 5 min, record 10 groups in total, and calculate the particle concentration indication error γ according to formula (2)-(4) d :

[0033]

[0034] In the formula: C si is the particle concentration value measured by the particle counter for the i-th time, pieces / L;

[0035] C di is the particle concentration value measured by the bioaerosol monitor for the i-th time, pieces / L.

[0036] Further, the fluorescent particle count deviation calibration is to dilute the fluorescent microsphere suspensions of 1 μm and 3 μm by 5-10 times respectively, shake well, start the bioaerosol monitor and the AGI sampler respectively, sample for 5 min, record the fluorescent particle concentration and the total particle concentration measured by the bioaerosol monitor; add the AGI sampler collection liquid into the flow cytometer, measure the fluorescent particle concentration and the total particle concentration in the collection liquid; and calculate the fluorescent particle count deviation according to formula (5)

[0037]

[0038] In the formula: C M is the fluorescent particle count measured by the bioaerosol monitor, pieces;

[0039] C MT is the total particle number measured by the bioaerosol monitor, pieces;

[0040] C S is the fluorescent particle number in the aerosol sample solution, pieces;

[0041] C ST is the total particle number in the aerosol sample solution, pieces.

[0042] A calibration device of a bioaerosol monitor, the calibration device of the bioaerosol monitor is calibrated by the calibration method of the bioaerosol monitor, and at least comprises an aerosol generation and sampling module, a constant temperature and humidity module, a stirring and uniform mixing module, a disinfection module, a purification module and the bioaerosol monitor.

[0043] Further, the aerosol generation and sampling module comprises a pump, a generator, an AGI sampler, a TSI particle size spectrometer, a particle counter and an Anderson impactor; one end of the pump is connected with the generator, and the other end is connected with the bioaerosol monitor cabin; the AGI sampler, the TSI particle size spectrometer, the particle counter and the Anderson impactor are all connected with the bioaerosol monitor cabin.

[0044] Further, the stirring and uniform mixing module is arranged at the top and the bottom of the bioaerosol monitor cabin.

[0045] Further, the disinfection module comprises a disinfectant box and a peristaltic pump, one end of the peristaltic pump is connected with the disinfectant box, and the other end is connected with the generator.

[0046] The purification module is arranged at the top and the bottom of the bioaerosol monitor cabin, the gas in the bioaerosol monitor cabin is discharged through the side wall bottom high-efficiency filter, and the supplemented gas enters the cabin through the top high-efficiency filter, so that the gas in the cabin is purified in circulation.

[0047] Further, the constant temperature and humidity module is arranged in the bioaerosol monitor cabin.

[0048] The constant temperature and humidity module arranged in the cabin can control the temperature and humidity in the cabin, so that a stable experimental environment is obtained.

[0049] Further, the stirring and uniform mixing module comprises at least one large fan arranged at the top of the bioaerosol monitor cabin and at least two small fans arranged at the bottom of the bioaerosol monitor cabin.

[0050] The small fan at the cabin bottom is combined with the large fan at the cabin top, vortexes are formed between the small fans, and the large fan blows away, so that biological aerosols can be effectively stirred and mixed.

[0051] The fluorescence value of the prepared fluorescent polystyrene microspheres is close to the intrinsic fluorescence of microorganisms, and a physical calibration method instead of a biological sampling and monitoring method can be realized, biological sampling and monitoring processes can be avoided, and the differences in biological particle activity, biological aerosol generation efficiency and sampling efficiency and other factors can be avoided to affect the calibration results; the "fluorescent particle counting deviation" calibration is proposed in the calibration method, which can evaluate the fluorescent particle counting efficiency of the biological aerosol monitor, and verify the anti-interference ability of the fluorescent monitoring system of the monitor; the comprehensive evaluation method for the working performance of the aerosol calibration cabin is established, which can provide a basis for setting the working parameters of the calibration cabin to ensure that a uniform, stable and required aerosol environment is provided for calibration work. BRIEF DESCRIPTION OF DRAWINGS

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0053] Figure 1 Biological aerosol monitor calibration technical route map;

[0054] Figure 2 Fluorescent polystyrene microsphere preparation technical route;

[0055] Figure 3 Calibration system performance evaluation technical route map. DETAILED DESCRIPTION

[0056] The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0057] The present embodiment provides a calibration method and management method for a biological aerosol monitor.

[0058] As Figure 1The biological aerosol monitor calibration method is shown: the physical calibration method is combined with the biological verification method. The physical calibration method is that the polystyrene microspheres with specific fluorescence are generated into aerosols by an aerosol generation system into a calibration cabin, the concentration of the aerosols in the calibration cabin is controlled by adjusting the generation flow and the generation time, the uniformity in the cabin is ensured by stirring and mixing by a large fan at the top and four small fans at the bottom, the left side of the calibration cabin is connected with a TSI particle size spectrometer, a particle counter and a calibrated monitor, the physical calibration of the monitor is realized by monitoring the particle size and the particle concentration of the aerosols in the cabin, and the specific calibration method is as follows:

[0059] 1) Particle size measurement value error. The 1 μm and 3 μm fluorescent microsphere suspensions are diluted, fully shaken, and then monodisperse fluorescent microsphere aerosols are generated by the calibration system, the aerosol particle concentration is controlled to be between (45000-55000) pieces / L or within the range specified by the manufacturer, the biological aerosol monitor is started, and after 5 min of normal operation, the particle size measurement results of the biological aerosol monitor are recorded, and the data is recorded every 10 s, a total of 3 groups, and the particle size measurement value error ΔD is calculated according to formula (1):

[0060]

[0061] In the formula: is the average value of 3 measurements of the biological aerosol monitor, μm;

[0062] D s is the standard value of the fluorescent microsphere particle size, μm.

[0063] 2) Particle concentration value error. The 1 μm and 3 μm fluorescent microsphere suspensions are respectively diluted, fully shaken, and then monodisperse fluorescent microsphere aerosols are generated by the calibration system, the aerosol particle concentration is controlled to be between (45000-55000) pieces / L; the particle counter and the biological aerosol monitor are respectively started, and after 5 min of normal operation, the particle concentration measurement results of the particle counter and the biological aerosol monitor are respectively recorded every 10 s, a total of 10 groups, and the particle concentration value error γ is calculated according to formula (2)-(4) d :

[0064]

[0065] In the formula: C si is the particle concentration value measured by the particle counter for the i-th time, L pieces / L;

[0066] C di is the particle concentration value measured by the biological aerosol monitor for the i-th time, pieces / L;

[0067] 3) Fluorescent particle counting bias. The 1 μm and 3 μm fluorescent microspheres suspensions and non-fluorescent microspheres suspensions after rinsing are diluted at the same ratio, the mixture is passed through the calibration system to generate a monodisperse aerosol, the bioaerosol monitor and the AGI sampler are turned on at the same time, and sampling is performed for 5 minutes. The fluorescent particle concentration and total particle concentration measured by the bioaerosol monitor are recorded. The AGI sampler collection solution is added to the flow cytometer, and the fluorescent particle concentration and total particle concentration in the collection solution are measured. The fluorescent particle counting bias is calculated according to formula (5)

[0068]

[0069] In the formula: C M is the number of fluorescent particles measured by the bioaerosol monitor, pieces;

[0070] C MT is the number of total particles measured by the bioaerosol monitor, pieces;

[0071] C S is the number of fluorescent particles in the aerosol sample solution, pieces;

[0072] C ST is the number of total particles in the aerosol sample solution, pieces.

[0073] After calibration is completed, the calibration cabin purification module is turned on, and the TSI particle size spectrometer is used to monitor the particle concentration in the calibration cabin. When the particle concentration in the calibration cabin is not greater than the ISO 5 level requirement specified in GB / T 25915.1-2010, the cleanliness in the cabin meets the requirements.

[0074] As shown in Figure 2 : the preparation of the specific fluorescent polystyrene microspheres is: the polystyrene microspheres are dispersed by ultrasonic dispersion, so that they are uniformly dispersed in an ethanol solvent to form a stable dispersion liquid, and the ultrasonic time is 10-30 minutes;

[0075] The polystyrene microsphere dispersion liquid is transferred to an electromagnetic stirring device, nitrogen protection is performed, the reaction temperature is controlled by a constant temperature water bath, and the initial reaction temperature is set to 25-35°C. The riboflavin stock solution is slowly added to the polystyrene microsphere dispersion liquid, the addition speed is controlled at 1-2 drops per second, stirring is started at the same time, the stirring speed is adjusted to 300-600 rpm, the riboflavin is fully contacted with the polystyrene microspheres, and the reaction time is 1-3 hours;

[0076] Under the condition of excitation light excitation, by measuring the intrinsic fluorescence intensity of Bacillus subtilis and Phi-X174 bacteriophage in the microorganism, the fluorescence intensity of the fluorescent microspheres, and by controlling the amount of riboflavin to adjust the fluorescence intensity of the fluorescent microspheres, the light intensity of the prepared fluorescent microspheres is close to the intrinsic fluorescence intensity of the microorganism, and the absolute measurement method is adopted, the scanning electron microscope is used to calibrate the labeled fluorescent particles, and the spectral analysis method is used to test the fluorescence characteristics of the labeled fluorescent particles;

[0077] Reaction termination and post-processing: when it is determined that the intrinsic fluorescence intensity of Bacillus subtilis and Phi-X174 bacteriophage in the microorganism is the same, a terminating agent is added to terminate the reaction, and the amount is 1-2 times the molar amount of riboflavin. After adding the terminating agent, continue stirring for 10-15 minutes to completely terminate the reaction. Then, the reaction product is separated by high-speed centrifugation at a speed of 8000-12000 rpm for 10-20 minutes, and the precipitate of polystyrene microspheres grafted with riboflavin is collected. The precipitate is washed repeatedly with deionized water for 3-5 times to remove ungrafted riboflavin and other impurities, and finally 1 μm and 3 μm fluorescent microsphere suspensions are obtained.

[0078] Further, the terminating agent is ethanolamine.

[0079] The calibration device is composed of an aerosol generation and sampling module, a constant temperature and humidity module, a stirring and mixing module, a sterilization module, and a purification module.

[0080] 1) Generation and sampling module: The generation and sampling module includes a generation module and a sampling module, including a pump, a generator, an AGI sampler, a TSI particle size spectrometer, a particle counter, and an Anderson impactor. The pump provides power for the generator or the left side connection port is connected to the Collisin generator for aerosol generation. The pump can be connected to the AGI sampler, TSI particle size spectrometer, particle counter, Anderson impactor, and aerosol monitor through the sampling port or left side external port for aerosol collection.

[0081] 2) Constant temperature and humidity module: The constant temperature and humidity module in the cabin can control the temperature and humidity in the cabin to obtain a stable experimental environment.

[0082] 3) Stirring and mixing module: The aerosol is generated in the cabin, which can be stirred and mixed by the top large fan and the bottom four small fans to ensure the uniformity in the cabin.

[0083] 4) Sterilization module: After the test is completed, the generator generates a disinfectant into the cabin body through the peristaltic pump to kill the microorganisms in the cabin. The top ultraviolet lamp can also be turned on for disinfection in the cabin.

[0084] 5) Purification module: before the test starts and after the test ends, the cabin gas is purified, the cabin gas is discharged through the high-efficiency filter at the bottom of the side wall, and the supplemented gas enters the cabin through the high-efficiency filter at the top, thereby circulating and purifying.

[0085] As Figure 3 described, the present application can evaluate the performance of the biological aerosol calibration cabin, specifically including:

[0086] The performance evaluation of the generation module is more specific. Known particle size particles are generated, and TSI measures the particle size distribution. If the geometric standard deviation of the particle size distribution is less than 3%, it is determined to be monodisperse. Particle concentration repeatability is generated. Under repeated conditions, known particle size particles are generated, and TSI measures the particle concentration value. A total of 6 groups are measured, and the repeatability is calculated using the Bessel formula. The repeatability is not greater than 5%.

[0087] The performance evaluation of the sampling module is more specific. Flow deviation: the sampling flow of the flowmeter calibration cabin is set to 5 minutes, and the set flow of the calibration cabin is compared.

[0088] The performance evaluation of the stirring and mixing is more specific. The aerosol distribution uniformity in the calibration cabin is generated. Known particle size particles are generated, and 6 measurement points are uniformly distributed in the sampling plane. TSI particle size spectrometer is used to measure the particle concentration value of the 6 measurement points, and the aerosol distribution uniformity in the calibration cabin is calculated by the range method. The uniformity is not greater than 5%.

[0089] The aerosol distribution stability of the calibration cabin per unit time. Known particle size particles are generated, and TSI particle size spectrometer is used for continuous sampling for 10 minutes. Every 1 minute, a group of data is recorded, a total of 10 groups, and the aerosol concentration stability in the calibration cabin is obtained by the range method, which is not greater than 5%.

[0090] The performance evaluation of the purification module is more specific. Self-purification time: generate a specified concentration of aerosol, start the calibration self-purification mode, and use TSI to monitor the concentration of aerosol in the calibration cabin in real time. When the particle concentration value meets the requirement of IS05 level cleanliness.

[0091] The calibration fluorescent polystyrene microspheres are prepared by using chemical grafting method to realize the fluorescence of polystyrene microspheres with specific particle size by selecting riboflavin. According to the measurement of the intrinsic fluorescence intensity of two representative microorganisms (Bacillus subtilis and Phi-X174 bacteriophage) in different growth cycles, four concentrations and two forms (liquid and aerosol) under three excitation light conditions, the fluorescence intensity of the fluorescent microspheres is adjusted by controlling the amount of riboflavin, so that the light intensity of the prepared fluorescent microspheres is as close as possible to the intrinsic fluorescence intensity of the microorganisms. Absolute measurement method is used, and scanning electron microscope is used to set the value of the labeled fluorescent particles, and spectral analysis method is used to test the fluorescence characteristics of the labeled fluorescent particles.

[0092] The fluorescence spectrum of the fluorescent microsphere solution provided by the application is obviously weaker than the intensity of the commercial fluorescent microsphere after exciting the fluorescent microsphere solution, commercial fluorescent microspheres and two representative microbial liquid with 405nm excitation light on the same optical detection platform; the fluorescence intensity of the fluorescent microsphere solution provided by the application is 2.0*10 6 The fluorescence intensity of the phage liquid in the exponential phase is pfu / mL.

[0093] The performance evaluation method of the calibration device established by the application is that polystyrene microspheres with a known particle size are atomized to generate aerosols in the calibration cabin, the performance of the generation and sampling module of the calibration cabin, the performance of the stirring and mixing module and the performance of the purification module are verified by measuring the particle size distribution and particle concentration, the performance of the generation and sampling module of the calibration cabin is verified by using a flowmeter, and the performance of the constant temperature and humidity module of the calibration cabin during work is verified by using a temperature and humidity meter, so that the working performance of the calibration device can be comprehensively evaluated.

[0094] The innovation points of the application are as follows: 1) the fluorescence particle counting deviation of the biological aerosol monitor is calibrated by using a physical method, and the specific method is as follows: the fluorescent microsphere suspension and the non-fluorescent microsphere suspension which are diluted at the same ratio are atomized to generate monodisperse aerosols in the calibration cabin, the aerosols are uniformly distributed after mixing, the biological aerosol monitor and the AGI sampler are started respectively, and the fluorescence particle concentration and the total particle concentration measured by the biological aerosol monitor are recorded; the sampling liquid of the AGI sampler is added into the flow cytometer, the fluorescence particle concentration and the total particle concentration in the sampling are measured, and the fluorescence particle counting deviation of the biological aerosol monitor is obtained by using the deviation of the ratio of the fluorescence particle counting result and the total particle counting result of the biological aerosol monitor to the ratio of the actual fluorescence particle number and the total particle number in the generated aerosol.

[0095] 2) the polystyrene microspheres are used to generate aerosols through the generation module of the calibration cabin, the monodispersity, the aerosol concentration repeatability, the aerosol concentration uniformity and the aerosol concentration stability of the generation module of the calibration cabin are verified by measuring the aerodynamic particle diameter of the generated aerosol particles and the particle concentration by using the TSI particle size spectrometer.

[0096] 3) the polystyrene fluorescent microspheres with specific particle sizes are prepared by using the epoxy method, the biological intrinsic fluorescence spectra of Bacillus subtilis and phage in different growth cycles, four gradient concentrations in the state of the bacterial liquid and the state of the aerosol are measured respectively, the fluorescence value of the fluorescent microspheres is adjusted, and the standard microspheres close to the intrinsic fluorescence of the microorganisms are prepared.

[0097] Example 1

[0098] Preparation work

[0099] Calibration device preparation:

[0100] Ensure that the calibration cabin is intact, with good sealing performance and smooth inner walls. Check the aerosol generation and sampling module to ensure that the cabin pump is tightly connected to the generator, and that the sampling pump is securely connected to the external equipment (AGI sampler, TSI particle size spectrometer, particle counter, Anderson impactor sampler, and aerosol monitor) without blockage. Confirm that the temperature sensor and humidity sensor of the constant temperature and humidity module are working normally, and that the temperature and humidity control module functions are intact, with the heat exchanger, ultrasonic atomization or condensation dehumidification equipment operating normally. The top large fan and four small fans of the stirring and mixing module can rotate normally, and the blades are not damaged. The peristaltic pump, generator, and top ultraviolet lamp of the disinfection module can work normally, the flexible pipeline has no leakage, and the disinfectant storage tank has enough disinfectant. The high-efficiency filters at the inlet and outlet of the purification module are installed correctly and have no damage.

[0101] Preparation of calibration fluorescent polystyrene microspheres:

[0102] In the spectral laboratory, high-precision spectrometers were used to measure Bacillus subtilis and Phi-X174 bacteriophages. The excitation light wavelengths were selected as 350 nm, 450 nm, and 550 nm, respectively. The intrinsic fluorescence intensity of the microorganisms was measured in liquid and aerosol forms at four concentration levels of 10 3 , 10 4 , 10 5 , and 10 6 / mL in the logarithmic growth phase, stationary phase, and decline phase of the two microorganisms. The photodetector sensitivity of the spectrometer reached 0.01 mV, and the grating resolution element was 0.1 nm. Accurate data were obtained and stored through complex signal processing algorithms.

[0103] In the chemical reaction kettle, according to the above measurement data, an electronic balance with an accuracy of 0.0001 g was used to accurately weigh the riboflavin, and a pipette was used to slowly drop the riboflavin solution into the polystyrene microsphere dispersion liquid with a particle size of 1 μm and 3 μm, while a magnetic stirrer was used to stir at a speed of 300 r / min. During the reaction process, the temperature was controlled at 25℃±0.5℃, and the pH value was controlled at 7.0±0.1, which was monitored in real time by temperature sensor and pH sensor, and the heating device and acid-base regulator were adjusted.

[0104] After preparation, take a small amount of fluorescent particle sample under a scanning electron microscope, the microscope magnification is 50000 times, the image acquisition pixel is 10 million, and the particle size and morphology are valued. Then place the fluorescent particles in a spectrum analyzer, use 488 nm excitation light to irradiate, collect the fluorescence spectrum, analyze the fluorescence characteristics, and ensure that it meets the calibration requirements. Record the amount of riboflavin added during preparation (such as 0.5 mg for 1 μm microspheres and 1.0 mg for 3 μm microspheres), reaction time (3 hours), reaction temperature and other parameters to form a quality traceability file.

[0105] Calibration of measurement error of particle size

[0106] Accurately take 5 mL of 1 μm and 3 μm fluorescent microsphere suspensions from the storage container with a calibrated pipette, and transfer them to a clean beaker. Add an appropriate amount of solvent and use a vortex oscillator to shake at a speed of 2000 r / min for 5 minutes to fully disperse the microspheres.

[0107] Slowly inject the diluted and shaken fluorescent microsphere suspension into the aerosol generator inlet of the calibration device. The calibration device uses microfluidic chip technology and ultrasonic dispersion technology to generate monodisperse fluorescent microsphere aerosols in the cabin under the condition that the pump provides stable energy to the generator. The high-precision concentration control device based on the principle of laser scattering monitors the particle concentration in real time and stabilizes it in the range of (45000-55000) / L.

[0108] Turn on the biological aerosol monitor, the instrument starts to preheat, and after 5 minutes it enters a stable running state. At this time, the connected computer data acquisition system automatically starts to record particle size measurement data at intervals of 10 seconds, a total of 3 groups of data, for example, the measured values are 1.02 μm, 1.03 μm, 0.98 μm (1 μm microspheres) and 3.05 μm, 3.03 μm, 3.02 μm (3 μm microspheres). Calculate the measurement error of particle size according to the formula, the average value of 1 μm microspheres is 1.01 μm, the standard value is 1 μm, and the error is (1.01-1) / 1 x 100% = 1%; the average value of 3 μm microspheres is 3.03 μm, the standard value is 3 μm, and the error is (3.03-3) / 3 x 100% = 1%.

[0109] Calibration of particle concentration measurement error

[0110] Dilute the 1 μm and 3 μm fluorescent microsphere suspensions on the clean bench. Use an electronic balance with a precision of 0.0001 g to weigh 0.1 g of microsphere suspension, and use a pipette to accurately take 9.9 mL of solvent, and shake it with a vortex oscillator.

[0111] The diluted solution is introduced into the calibration device to generate a monodisperse fluorescent microsphere aerosol by high-pressure gas injection and Venturi atomization technology. The concentration monitoring and control components control the particle concentration to (45000-55000) per liter.

[0112] The particle counter and the bioaerosol monitor are simultaneously turned on, and after 5 minutes, the data acquisition line transmits the measurement signals of both to the central data processing unit. The particle concentration measured by the particle counter and the bioaerosol monitor is recorded every 10 seconds for 10 groups. For example, the particle concentration values measured by the particle counter are 48000 per liter, 47500 per liter, 48200 per liter, etc. (1 μm microspheres), and the values measured by the bioaerosol monitor are 46500 per liter, 47000 per liter, 47800 per liter, etc. (1 μm microspheres). The particle concentration indication error is calculated according to the formula, and the particle concentration indication error of 1 μm microspheres is calculated to be 2% (assuming the calculation result is 2%). Similarly, the particle concentration indication error of 3 μm microspheres is calculated to be 1.5% (assuming the result is 1.5%).

[0113] Fluorescent particle count bias calibration

[0114] The 1 μm and 3 μm fluorescent microsphere suspensions and the non-fluorescent microsphere suspension are diluted in a 1:1 ratio with the assistance of a high-precision balance. A magnetic stirrer is used to stir at a speed of 500 r / min for 10 minutes to ensure uniform mixing.

[0115] The diluted mixture is converted into a monodisperse aerosol by the piezoelectric ceramic-driven micro-spray device of the calibration device. The bioaerosol monitor and the AGI sampler are simultaneously turned on, the sampling ports of the two are opposite and the distance is set to 10 cm, and the sampling is synchronized for 5 minutes. During sampling, the bioaerosol monitor displays the fluorescent particle concentration and the total particle concentration in real time, and the operator records, for example, the fluorescent particle concentration is 2000 per mL, and the total particle concentration is 5000 per mL (1 μm microspheres).

[0116] After sampling is completed, the AGI sampler collection liquid is carefully injected into the flow cytometer sample chamber. The flow cytometer uses 488 nm laser excitation, fluorescence detection, and signal amplification functions to accurately measure the fluorescent particle concentration and the total particle concentration in the collection liquid. Assuming that the measured fluorescent particle concentration is 1800 per mL and the total particle concentration is 4800 per mL (1 μm microspheres), the fluorescent particle count bias is calculated according to the formula, and the fluorescent particle count bias of 1 μm microspheres is 10% (assuming the calculation result is 10%). Similarly, the fluorescent particle count bias of 3 μm microspheres is calculated to be 8% (assuming the result is 8%).

[0117] Purification of the calibration chamber after calibration

[0118] Press the calibration cabin purification module start button, the ventilation duct valve switches to the purification mode, and the air circulation pump runs at high speed. The TSI particle size spectrometer is connected to the cabin gas through the sampling tube, and the particle concentration is monitored in real time. When the particle concentration is not greater than the ISO 5 level standard specified in GB / T 25915.1-2010 (assuming 1000 / m 3 ) is monitored, the purification control system issues a prompt sound, and the control panel displays "cabin cleanliness meets the standard", the purification operation is completed, and the next round of calibration or other operations is prepared.

[0119] Example Two

[0120] Preparation

[0121] Calibration device preparation: the same as the calibration device preparation steps of example one, ensure that each system and equipment is working properly.

[0122] Preparation of fluorescent polystyrene microspheres for calibration:

[0123] In the spectral measurement link, the excitation light wavelength is selected as 360nm, 460nm and 560nm, and the measurement is carried out for Bacillus subtilis and Phi-X174 bacteriophage in different growth cycles and concentration levels, and in two forms. The spectrometer parameters are the same as in example one, and the data is obtained and stored.

[0124] In the chemical synthesis reaction, according to the measurement data, adjust the amount of riboflavin added (0.6mg for 1μm microspheres, 1.2mg for 3μm microspheres), other reaction conditions such as temperature control at 26℃±0.5℃, pH value control at 7.2±0.1, stirring speed at 350r / min, reaction time at 3.5 hours.

[0125] The quality detection steps are similar to example one, the fluorescent particles are detected by scanning electron microscope and spectrometer to ensure the quality meets the requirements, the preparation parameters are recorded to form the file.

[0126] Calibration of indication error of particle size measurement

[0127] Take 6mL of 1μm and 3μm fluorescent microsphere suspension respectively, add different solvents for dilution, and shake well in vortex oscillator for 6 minutes.

[0128] The injection aerosol generating device is fed into the inlet, and a monodisperse fluorescent microsphere aerosol is generated, and the particle concentration is controlled at (45000-55000) per / L. After the biological aerosol monitor is turned on and preheated for 5 minutes, it enters stable operation, and 3 sets of particle size measurement data are recorded, such as 1.01 μm, 1.04 μm, 0.99 μm for 1 μm microspheres, and 3.04 μm, 3.02 μm, 3.06 μm for 3 μm microspheres. Calculate the particle size measurement error, the error of 1 μm microspheres is (1.013-1) / 1*100%=1.3%, and the error of 3 μm microspheres is (3.04-3) / 3*100%=1.33%.

[0129] Particle concentration indication error calibration

[0130] Dilute 1 μm and 3 μm fluorescent microsphere suspensions, weigh 0.12 g of microsphere suspension, and remove 9.88 mL of solvent, and shake well.

[0131] Generate aerosol and control concentration, turn on particle counter and biological aerosol monitor, and record 10 sets of particle concentration measurement results after 5 minutes, such as 47800 per / L, 48500 per / L, etc. for 1 μm microsphere particle concentration measured by the particle counter, and 46800 per / L, 47300 per / L, etc. for the biological aerosol monitor. Calculate the particle concentration indication error, the error of 1 μm microspheres is (assuming the calculation result is) 2.2%, and the error of 3 μm microspheres is (assuming the result is) 1.8%.

[0132] Fluorescent particle counting deviation calibration

[0133] Dilute the fluorescent microsphere and non-fluorescent microsphere suspension at a ratio of 1:1.2, and stir with a magnetic stirrer for 12 minutes.

[0134] After conversion into aerosol, turn on the biological aerosol monitor and the AGI sampler, set the sampling port distance to 12 cm, and simultaneously sample for 5 minutes. Record the monitor display data, such as fluorescent particle concentration of 2200 per / mL and total particle concentration of 5500 per / mL (1 μm microspheres).

[0135] Sample liquid is injected into the flow cytometer for measurement. Assuming that the fluorescent particle concentration measured is 1900 per / mL, and the total particle concentration is 5000 per / mL (1 μm microspheres). Calculate the fluorescent particle counting deviation, the deviation of 1 μm microspheres is (assuming the calculation result is) 13.6%, and the deviation of 3 μm microspheres is (assuming the result is) 10.5%.

[0136] Purify the calibration cabin after calibration

[0137] Start the purification module, when the TSI particle size spectrometer monitors that the particle concentration reaches the standard (not more than ISO 5 level standard), complete the purification, determine that the cleanliness of the cabin reaches the standard, and the subsequent operation can be carried out.

[0138] From the above examples, it can be seen that the calibration method and device of the biological aerosol monitor can effectively calibrate the monitor, ensure the accuracy and reliability of the measurement, and achieve good calibration effect under different operating conditions and parameter settings. At the same time, the preparation process of the fluorescent polystyrene microspheres for calibration is strict and traceable, providing high-quality standard substances for calibration work.

[0139] The above description of disclosed embodiments enables those skilled in the art to implement or use the embodiments. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the embodiments.

[0140] Therefore, the embodiments will not be limited to the examples shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method of calibrating a bioaerosol monitor, the method comprising: The physical calibration method is combined with the biological verification method, and includes the following steps: S1: using a flowmeter to perform first-step calibration on the flow parameter of the biogas aerogel; S2: using polystyrene microspheres and polystyrene microspheres with specific fluorescence to generate aerosols in the calibration cabin through an aerosol generation system, adjusting the generation flow and generation time to control the concentration of the aerosols in the calibration cabin, and stirring and mixing the aerosols in the calibration cabin through a fan; S3: connecting the TSI particle size spectrometer, the particle counter and the calibrated monitor to the left side of the calibration cabin, respectively, and performing second-step calibration on the monitor by monitoring the particle size and particle concentration of the aerosols in the cabin; S4: biological verification, the standard material of bacillus subtilis is atomized, the concentration of aerosol is 2*10 6 cfu / L, the anderson sampler and aerosol monitor are sampled at the same time, the results of anderson sampling are compared with the results of aerosol monitor by colony counting method, and the biological sampling rate of biological aerosol sampler is calibrated and verified; S5: after the calibration is completed, starting the purification module of the calibration cabin, and monitoring the particle concentration in the calibration cabin by using the TSI particle size spectrometer until the cleanliness in the cabin meets the requirements; In step S2, the preparation of the polystyrene microspheres with specific fluorescence is as follows: dispersing the polystyrene microspheres by ultrasonic dispersion to uniformly disperse them in an ethanol solvent to form a stable dispersion liquid, and the ultrasonic time is 10-30 minutes; transferring the polystyrene microsphere dispersion liquid into an electromagnetic stirring device, protecting by nitrogen, controlling the reaction temperature by a constant-temperature water bath, and setting the initial reaction temperature at 25-35 DEG C; slowly dropping a riboflavin stock solution into the polystyrene microsphere dispersion liquid, controlling the dropping speed at 1-2 drops per second, and starting stirring at the same time, adjusting the stirring speed to 300-600 rpm, so that the riboflavin fully contacts the polystyrene microspheres, and the reaction time is 1-3 hours; under the excitation of excitation light, measuring the intrinsic fluorescence intensity of Bacillus subtilis and Phi-X174 bacteriophage, and the fluorescence intensity of the fluorescent microspheres, adjusting the fluorescence intensity of the fluorescent microspheres by controlling the amount of riboflavin, so that the light intensity of the prepared fluorescent microspheres is close to the intrinsic fluorescence intensity of the microorganisms, and using the absolute measurement method, using a scanning electron microscope to calibrate the labeled fluorescent particles, and using a spectral analysis method to test the fluorescence characteristics of the labeled fluorescent particles; reaction termination and post-treatment: when it is determined that the intrinsic fluorescence intensity of Bacillus subtilis and Phi-X174 bacteriophage is reached, adding a termination agent to terminate the reaction, and the amount of the termination agent is 1-2 times the molar amount of riboflavin, after the termination agent is added, the reaction is continuously stirred for 10-15 minutes to completely terminate the reaction; then, the reaction product is separated by high-speed centrifugation at a speed of 8000-12000 rpm for 10-20 minutes, and the polystyrene microspheres grafted with riboflavin are collected; the precipitate is washed repeatedly with deionized water for 3-5 times to remove ungrafted riboflavin and other impurities, and finally 1-micron and 3-micron fluorescent microsphere suspensions are obtained.

2. The method of claim 1, wherein the method further comprises: The termination agent is ethanolamine.

3. The method of claim 2, wherein the method further comprises: In step S3, the second-step calibration includes particle size measurement indication error calibration, particle concentration indication error calibration and fluorescent particle count deviation calibration.

4. The method of claim 3, wherein the method further comprises: The fluorescent particle count bias calibration is to dilute the fluorescent microsphere suspensions of 1 μm and 3 μm by 5-10 times respectively, shake well, then generate monodisperse fluorescent microsphere aerosol through the calibration system, control the aerosol particle concentration at 45000-55000 / L, put into and start the bioaerosol monitor, record the particle size measurement results of the bioaerosol monitor after 5 minutes of normal operation, record the data every 10 seconds, a total of 3 groups, and calculate the particle size measurement indication error ΔD according to formula (1): wherein: Average of 3 measurements with a biological aerosol monitor, pm; D s For fluorescent microspheres, the diameter standard value, μm.

5. The method of claim 3, wherein the method further comprises: The particle concentration indication error calibration is to dilute the 1 μm and 3 μm fluorescent microsphere suspensions by 5-10 times respectively, shake well, and then generate monodisperse fluorescent microsphere aerosol through the calibration system, with the aerosol particle concentration controlled at 45000-55000 per L, the particle counter and the bioaerosol monitor are turned on, and after 5 minutes of normal operation, every 10 seconds, the particle concentration measurement results of the particle counter and the bioaerosol monitor are recorded respectively, 10 groups are recorded continuously, and the particle concentration indication error γ is calculated according to formula (2)-(4) d : In the formula: C si is the particle concentration value measured by the particle counter for the i time, pieces / L; C di Pi is the particle concentration value measured by the bioaerosol monitor for the i-th time, μ / L.

6. The method of claim 3, wherein the method further comprises: The fluorescent particle count bias calibration is to dilute the fluorescent microsphere suspensions of 1 μm and 3 μm by 5-10 times respectively, shake well, then start the bioaerosol monitor and the AGI sampler respectively, sample for 5 minutes, record the fluorescent particle concentration and the total particle concentration measured by the bioaerosol monitor; add the AGI sampler collection liquid into the flow cytometer to measure the fluorescent particle concentration and the total particle concentration in the collection liquid; calculate the fluorescent particle count bias according to formula (5) In the formula: C M Fluorescent particles measured by a bioaerosol monitor, pieces; C MT Total particle counts were measured for the bioaerosol monitor, in units of particles. C S N is the number of fluorescent particles in the aerosol sample solution, pieces; C ST Total particle number in the aerosol sample solution, in units of #.

7. A calibration device for a bioaerosol monitor, characterized in that, The calibration device of the bioaerosol monitor is calibrated by the calibration method of the bioaerosol monitor based on laser-induced biointrinsic fluorescence and light scattering principle according to any one of claims 1-6, and at least includes an aerosol generation and sampling module, a constant temperature and humidity module, a stirring and mixing module, a sterilization module, a purification module and a bioaerosol monitor; The aerosol generation and sampling module includes a pump, a generator, an AGI sampler, a TSI particle size spectrometer, a particle counter and an Anderson impactor; one end of the pump is connected with the generator, and the other end is connected with the bioaerosol monitor cabin; the AGI sampler, the TSI particle size spectrometer, the particle counter and the Anderson impactor are all connected with the bioaerosol monitor cabin; The stirring and mixing module is arranged at the top and bottom of the bioaerosol monitor cabin; The sterilization module includes a disinfectant box and a peristaltic pump, one end of the peristaltic pump is connected with the disinfectant box, and the other end is connected with the generator; The purification module is arranged at the top and bottom of the bioaerosol monitor cabin, the gas in the bioaerosol monitor cabin is discharged through the side wall bottom high-efficiency filter, and the supplemented gas enters the cabin through the top high-efficiency filter, and is circulated and purified.

8. The calibration device for a bioaerosol monitor according to claim 7, wherein, The constant temperature and humidity module is arranged in the bioaerosol monitor cabin.

9. The calibration device for a bioaerosol monitor of claim 7, wherein, The stirring and mixing module includes at least one large fan arranged at the top of the bioaerosol monitor cabin and at least two small fans arranged at the bottom of the bioaerosol monitor cabin.

Images