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CN-121994681-A - System and method for tracing and calibrating fluorescent channel magnitude of bioaerosol monitor

CN121994681ACN 121994681 ACN121994681 ACN 121994681ACN-121994681-A

Abstract

The invention provides a system and a method for calibrating the value tracing of a fluorescent channel of a biological aerosol monitor, which relate to the technical field of biological aerosol and comprise a biological aerosol generating device, a sampling device, a microscopic counting system and a data processing unit, wherein the units are cooperated to realize the value tracing, the biological aerosol generating device generates dust by adopting an atomization method, is provided with a biological aerosol simulation generating cabin, and adjusts the aerosol flow and the diluent gas flow; the invention establishes a perfect fluorescence channel magnitude tracing chain, solves the problem that the existing method has no complete tracing chain, realizes counting without culturing by adopting a spore-microscopic counting technology, has short time consumption, can directly trace the magnitude to the national measurement standard of spore number concentration, simultaneously avoids the defects of fluorescence screening and microorganism activity destruction of a fluorescent microsphere method, ensures tracing accuracy, ensures that the instrument indication error after calibration is controlled within +/-15 percent, and remarkably improves magnitude accuracy.

Inventors

  • PAN YITING
  • ZHAO HAIBO
  • LIU JIAQI
  • DONG MOU
  • LV CHAO

Assignees

  • 北京市计量检测科学研究院

Dates

Publication Date
20260508
Application Date
20260205

Claims (10)

  1. 1. The fluorescent channel magnitude tracing calibration system of the biological aerosol monitor comprises a biological aerosol generating device, a sampling device, a microscopic counting system and a data processing unit, wherein the biological aerosol generating device adopts an atomization method to generate dust, is provided with a biological aerosol simulation generation cabin, and controls the stability of aerosol in the cabin to be within +/-5% by adjusting the flow of aerosol and the flow of dilution gas so as to generate stable aerosol containing standard biological substances; The system comprises a disposable bacterial counting plate, a binocular fluorescence microscope and an imaging unit, wherein the disposable bacterial counting plate is used for carrying a processed sample and matched with the binocular fluorescence microscope to realize quantitative counting of microorganisms, the binocular fluorescence microscope is provided with a corresponding emission filter by adjusting the multiple of an objective lens and fluorescence excitation wavelength, the imaging unit records particle images, and the data processing unit is connected with a monitor and the microscopic counting system to acquire counting data, calculate counting efficiency, indication errors and pseudo biological particle counting rate, generate a calibration report and store data.
  2. 2. The bioaerosol monitor fluorescence channel magnitude traceability calibration system according to claim 1, wherein the standard biological material comprises at least one of Bacillus subtilis spores, aspergillus niger spores, MS2 phage and bovine serum albumin BSA particles, and wherein the concentration of the Bacillus subtilis spore suspension is controlled to be The parameter adjusting range of the centrifugal equipment of the liquid sampling module is that the rotating speed is 2000-5000rpm, the temperature is 0-4 ℃, and the centrifugal time is 5-15min.
  3. 3. The system for tracing and calibrating the fluorescence channel value of the bioaerosol monitor according to claim 1 is characterized in that the disposable bacteria counting plate is of a disposable structure which is matched with quantitative counting of microorganisms and has a definite counting area and a fixed counting pool volume, the binocular fluorescence microscope is of a type with a fluorescence observation function, the adjustable objective lens multiple range is 10-100 times, fluorescence excitation wavelengths are matched with 375nm, 405nm and 488nm, and an emission filter wave band is arranged according to the corresponding excitation wavelength in a matching mode, so that the clear identification of biological particle fluorescence signals is ensured.
  4. 4. The system for tracing the fluorescent channel value of the bioaerosol monitor according to claim 1, wherein in the liquid sampling module of the sampling device, the collecting liquid is ultrapure water containing 0.01% Tween 80, the initial volume is 5mL, the cleaning liquid is ultrapure water, the single cleaning volume is 2mL, and the volume of the mixed liquid after centrifugal treatment is 1mL.
  5. 5. The bioaerosol monitor fluorescence channel magnitude traceability calibration system of claim 1, wherein the data processing unit sets a false biological particle counting rate judgment standard of less than or equal to 0.3%, the counting efficiency is calculated by the ratio of the monitor fluorescence channel counting result to the microscope counting result, and the indication error allowable range is within +/-15%.
  6. 6. A method for tracing and calibrating the magnitude of a fluorescence channel of a bioaerosol monitor, which is applied to the tracing and calibrating system of the magnitude of the fluorescence channel of the bioaerosol monitor according to any one of the claims 1-5, and is characterized by comprising the following steps: s1, selecting at least one biological standard substance, and preparing a standard substance suspension with stable concentration for later use; S2, adding the standard substance suspension into a biological aerosol generating device, atomizing to generate aerosol, adjusting parameters to ensure that the stability of the aerosol in a generating cabin reaches +/-5%, and sampling after balancing for 30 min; S3, setting a sampling flow of 12.5L/min by adopting a liquid sampling method, controlling the sampling time to be 8-12min, cleaning the collector with ultrapure water for 3 times after sampling is finished, and collecting cleaning liquid and mixing the cleaning liquid with the collecting liquid; S4, putting the mixed solution into centrifugal equipment, centrifuging according to set parameters, discarding supernatant, and re-suspending and precipitating with ultrapure water containing 0.01% Tween 80 and fixing the volume; S5, taking The sample is dripped into a bacteria counting plate and placed under a binocular fluorescence microscope, and the sample is counted according to a set step and converted into the spore number concentration; S6, collecting data through the data processing unit, calculating the counting efficiency and indicating value error, adjusting the sensitivity of the monitor until the error reaches the standard, and simultaneously verifying that the counting rate of the pseudo biological particles is less than or equal to 0.3%.
  7. 7. The method for traceable calibration of the fluorescent channel value of the bioaerosol monitor according to claim 6, wherein in S1, any one or more of bacillus subtilis spores, aspergillus niger mould spores, MS2 phage and bovine serum albumin BSA particles are selected as standard substances, and a standard substance suspension with stable concentration is prepared.
  8. 8. The method for tracing and calibrating the fluorescence channel quantity value of the bioaerosol monitor according to claim 7, wherein in the step S3, the optimal sampling time is 10min, the average number of spores in each middle square lattice of the disposable bacteria counting plate is controlled to be 10-15, errors caused by insufficient statistics and particle overlapping are avoided, when the sampling time is less than or equal to 5min, the average number of spores in the middle square lattice is less than 5, the indication error is more than 30%, and when the sampling time is more than or equal to 15min, the spores overlap, and the indication error is more than 20%.
  9. 9. The method for tracing and calibrating the fluorescence channel value of the bioaerosol monitor according to claim 8 is characterized in that in the step S4, centrifugal parameters are adjusted according to standard substance types, wherein bacillus subtilis spores and aspergillus niger mould spores are subjected to centrifugation for 10min at 4 ℃ and 3000-3500 rpm, MS2 phage is not required to be subjected to centrifugal treatment, bovine serum albumin BSA particles are subjected to centrifugation for 15min at 4 ℃ and 5000 rpm, the volume of the suspended particles is 1mL, and the suspended particles are subjected to microscope counting after being fully and uniformly mixed.
  10. 10. The method for tracing and calibrating the fluorescence channel value of the bioaerosol monitor according to claim 9, wherein in the step S5, 4 middle square grids of a disposable bacteria counting plate are selected, the left and right principle of counting is adopted to count edge particles without counting, the interference of impurities is eliminated, the fluorescence excitation wavelength is 375nm, the multiple of an objective lens is regulated to 40 times, and particle images are recorded through an imaging unit for data rechecking.

Description

System and method for tracing and calibrating fluorescent channel magnitude of bioaerosol monitor Technical Field The invention relates to the technical field of bioaerosols, in particular to a system and a method for tracing and calibrating the magnitude of a fluorescence channel of a bioaerosol monitor. Background With the promotion of respiratory disease transmission prevention and control and limited space biosafety requirements, bioaerosol monitoring becomes a research hot spot in the field of environmental sanitation, and laser-induced fluorescence spectroscopy (LIF) technology is widely applied to a core detection module of a bioaerosol monitor due to the rapid detection advantage of the LIF technology. The existing bioaerosol monitor mainly recognizes and counts biological particles through a fluorescent channel, measures particle size and total particle number through a scattered light channel, and the accuracy of the counting efficiency of the fluorescent channel directly determines the reliability of a monitoring result. The related standards at present mainly comprise GB/T38517-2020 general rules of particle biological aerosol sampling and analysis and group standards T/CSP 7-2020 technical requirements of particle technology ultraviolet fluorescence biological aerosol monitors, wherein the former standardizes the sampling mode, the latter makes clear the technical index of the monitor, and meanwhile, the existing calibration method is mainly divided into two categories, namely, a precise particle counter calibration method based on fluorescent microsphere atomization, which is convenient to operate and has low requirements on laboratory level, and the other category is a sampling counting method based on microorganism culture, and calibration is realized by culturing counting after being acquired by a microorganism sampler; The prior art has the following defects, so that the demand of tracing the magnitude of a fluorescence channel of the bioaerosol monitor cannot be met. On one hand, the fluorescent microsphere calibration method lacks the capability of discriminating fluorescence performance, fluorescent-free small liquid drops are easy to generate in the atomization process, the counting result is disturbed, the fluorescent microsphere can only trace the source from the particle size and cannot realize quantitative tracing of fluorescence performance, and on the other hand, the microbial culture method is easy to damage the microbial structure, influence the activity and the culture property, so that the counting result is smaller, the time consumption is longer and the operation is complex. In addition, in the existing sampling method, the problems of autofluorescence interference and tiny fiber particles generated after dissolution exist in the soluble film sampling, the calibration accuracy cannot be guaranteed due to the fact that the lattice filter film sampling has fluorescence quenching phenomena on biological particles, and the unified calibration system and the standardized operation flow are lacked, so that the quantity value consistency of different monitors is poor, and the corresponding metering technical specification support industry development does not exist, therefore, the invention provides a system and a method for tracing the quantity value of a fluorescent channel of a biological aerosol monitor to solve the problems in the prior art. Disclosure of Invention Aiming at the problems, the invention provides a system and a method for tracing and calibrating the quantity value of a fluorescent channel of a bioaerosol monitor, which establish a perfect tracing chain of the quantity value of the fluorescent channel, solve the problem of poor tracing performance of the traditional method, realize counting without culturing by adopting a spore-microscopic counting technology, consume short time, directly trace the quantity value to the national measuring standard of the quantity concentration of spores, avoid the defects of non-fluorescent screening of a fluorescent microsphere method and damage to the activity of microorganisms by a culturing method, ensure tracing accuracy, ensure that the error of the indication value of the calibrated instrument is controlled within +/-15 percent, and remarkably improve the accuracy of the quantity value. The invention aims at realizing the aim by the following technical scheme that the fluorescent channel magnitude traceability calibration system of the biological aerosol monitor comprises a biological aerosol generating device, a sampling device, a microscopic counting system and a data processing unit, wherein the biological aerosol generating device adopts an atomization method to generate dust, is provided with a biological aerosol simulation generating cabin, and is used for generating stable aerosol containing standard biological substances by adjusting aerosol flow and diluent gas flow, the sampling device takes a liquid sampling mo