A quantitative method and application of nano fluorescent microspheres
Through laser confocal microscope technology and picture grid sampling principle, the problem of difficulty in accurately quantification of nanofluorescent microspheres is solved, and a simple and high-stability quantitative detection method is achieved.
Patent Information
- Application Number
- CN202510020489.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-04-08
AI Technical Summary
The rapid detection of existing nanofluorescent microspheres is difficult to accurately determine the quantity, the method is cumbersome and the sensitivity is limited.
Using laser confocal microscope technology and combining the principle of picture grid sampling, nanofluorescent microspheres are rapidly counted, and quantitative detection is achieved through dividing matrix and counting standards.
The accurate quantification detection of nanofluorescent microspheres is achieved, with simple methods, wide adaptability range and high stability of the counting scheme.
Smart Images
Figure CN119779952A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of nano material detection, and specifically relates to a nano fluorescent microsphere quantitative method and application, and more specifically to a nano fluorescent microsphere rapid quantitative method for bioaerosol environmental water samples in the air. Background Art
[0002] Nanofluorescent microspheres are an emerging nanomaterial that combines the fluorescent properties and the structure of microspheres, and have good prospects for commercial applications. In the field of environmental science, they can be used as tracers for gas and liquid flow measurement; in the field of food safety, they can be combined with immunochromatography technology for foodborne pathogen detection; in the Bohai Oilfield, the unused oil production can be determined by the nanoconcentration detection in its output fluid; in the biomedical field, they can be used to detect Klebsiella pneumoniae and tumor cells, and can also be applied to a variety of tests in the field of biochemistry, such as immunoassays, protein chip joint detection, etc., and bioluminescence resonance energy transfer can also be used for in vivo metal ion detection. At present, the detection of nanofluorescence is mainly semi-quantitative, such as the detection of foodborne pathogens by nanoparticle-based fluorescence sensors, and the detection of nanorod fluorescent marker proteins based on microfluidics.
[0003] At present, there are technical bottlenecks in the rapid detection of nano fluorescent microspheres, which makes it difficult to accurately quantify. Although some studies have improved the sensitivity by improving the ball-forming method and optimizing the antibody-directed coupling method, the methods are cumbersome; the detection of nano fluorescent microspheres by immunochromatography technology has limitations; and liquid biochip technology is difficult to accurately quantify. Summary of the invention
[0004] Based on the above reasons, inventing a rapid detection method of nano fluorescent microspheres with accurate quantification, simple method and wide adaptability is a technical problem to be solved urgently. The present invention is based on laser confocal microscopy, and according to the technical principle of image grid sampling estimation, objective sampling counting and simulation of target measurement indicators are performed, thereby achieving rapid counting of overall target measurement indicators. The quantitative method of nano fluorescent microspheres of the present invention has high quantitative accuracy, simple method and wide adaptability. The specific technical scheme is as follows: A method for quantitative determination of nano fluorescent microspheres, comprising: S1, preparing a monodisperse nano fluorescent microsphere solution; S2, adding the monodisperse nano fluorescent microsphere solution to a confocal glass plate to form a sample, and scanning the sample with a laser to form a fluorescent film; S3, counting the fluorescent microspheres located in the circular aperture of the confocal glass plate to determine the counting standard of the fluorescent radiography; S4, quantifying the fluorescent film according to the matrix divided on the confocal glass plate and the counting standard to obtain a quantitative result; S5. Performing stability evaluation on the quantitative result to obtain a stability evaluation result.
[0005] Furthermore, in S1, the method for preparing the monodisperse nano fluorescent microsphere solution is: taking the monodisperse nano fluorescent microspheres and dissolving them in physiological saline to prepare the monodisperse nano fluorescent microsphere solution.
[0006] Further, in S2, the monodisperse nano fluorescent microsphere solution is added to a confocal glass plate to form a sample, and the sample is scanned with a laser to form a fluorescent film, including: The monodisperse nano fluorescent microsphere solution is added to a 96-well special confocal glass plate to form a sample, an excitation light source channel of the nano fluorescent microsphere is selected, each sample is scanned with a laser, a photo of the fluorescent microspheres on the entire well surface is taken to form a fluorescent film, and the fluorescent film is batch converted into an image file format.
[0007] Furthermore, the excitation light source channel of the nano fluorescent microspheres is any one of TRITC / RFP, TD, and FITC / GFP.
[0008] Furthermore, in S3, the counting standard of the fluorescent film is determined as: counting the fluorescent microspheres in the circular aperture of the 96-well dedicated confocal glass plate.
[0009] Further, in S3, the counting standard for determining the fluorescence radiographs includes two standards: Standard 1: When the microsphere overlaps with the edge of the square, if the microsphere overlaps with the upper left edge of each square, it is included in the count, and if it overlaps with the lower right edge, it is ignored. The square is the smallest unit square in the sample division matrix; Standard 2: The size of the microspheres is based on the 1μm scale. Microspheres smaller than 1μm are counted, and other larger fluorescent particles or other long fluorescent patterns are non-microsphere signals and need to be ignored.
[0010] Furthermore, in S4, the fluorescence radiography is quantitatively measured to obtain a quantitative result: according to the counting standard of the fluorescence radiography described in S3, the designed sampling squares are counted, and the percentage of the sampling counting squares in different areas to the circular area is determined, so as to predict a quantitative result close to the overall radiography count.
[0011] Furthermore, in S4, the following sub-steps are also included: S4-1. Divide the sample matrix: Use image processing software to divide the fluorescent film of the circumscribed rectangle of a single circular hole in the 96-well dedicated confocal glass plate into an 8×8 matrix with a total of 64 grids; S4-2, arranging sampling grids in different regions: selecting different counting grids in the 64-grid matrix to arrange sampling grids in different regions; S4-3, determining the conversion coefficient: calculating the average conversion coefficient of different counting grids according to the average value of the ratio of the area of a single circular hole to the area of the counting grid; S4-4, determine the fitting curve of the sampling counting grids in different areas: fit the number of sampling grids and the ratio of the overall sampling standard deviation to the overall standard deviation to obtain the fitting curve equation; S4-5, determine the optimal counting square area: according to the fitted curve equation, determine the sampling squares where the overall sampling standard deviation / overall standard deviation approaches 1 as the number of sampling counting squares; according to the number of sampling counting squares, use a specific algorithm to obtain the circular area ratio of the counting square area, the specific algorithm is the algorithm determined according to the average conversion coefficient described in step S4-3, and determine the tangent line when the slope of the fitting curve is 1 x The axis is the optimal counting grid area.
[0012] Furthermore, in S5, the method for evaluating the stability of the quantitative results is as follows: based on the number of sampling counting grids determined in S4, a 4×4 matrix in the middle of the fluorescent film is taken for two-dimensional arrangement, and the coefficients of variation of different two-dimensional arrangement counting schemes are analyzed and compared at P5, P10, P25, P50, P75, P90, and P95, thereby determining the final two-dimensional arrangement of the counting grids.
[0013] A nano-fluorescent microsphere rapid quantitative method for bioaerosol environmental water samples in the air adopts the above-mentioned nano-fluorescent microsphere quantitative method.
[0014] S1. Environmental water sample solution with monodisperse fluorescent nanospheres Monodisperse nano fluorescent microspheres are dissolved in an environmental water sample containing bioaerosols in the air to prepare an environmental water sample stock solution of monodisperse nano fluorescent microspheres. In the embodiment of the present invention, 100 mg / 10 mL of Lumisphere monodisperse red fluorescent microspheres (BaseLine, 10 mL) with a diameter of 0.1 μm are selected and dissolved in 0.4 mL of an environmental water sample containing bioaerosols in the air to prepare a 40 μg / mL fluorescent microsphere environmental water sample stock solution, which is then diluted to a 1-40 μg / mL fluorescent microsphere environmental water sample, and inactivated at 60° C. for 1 hour, followed by ultrasonication and shaking after the inactivation is completed; S2: Add the environmental water sample solution of the monodisperse nano fluorescent microspheres to a confocal glass plate to form a sample, and scan the sample with a laser to form a fluorescent film: Take out 30 parallel sample solutions of 70 μL each from the fluorescent microsphere environmental water sample described in S1, add them to the 96-well dedicated confocal glass plate in sequence according to the dragon-swung-tail method, select the TRITC / RFP channel as the excitation light source channel of the fluorescent microspheres, adjust the horizontal and vertical positions of the wells under the 2x objective lens, adjust the focal length so that the focal length based on the fluorescent microspheres as the standard can be clearly seen to be positioned in the exact center of the well, design a 96-well plate sample puzzle template on the laser confocal software, scan the 96-well plate in sequence according to the dragon-swung-tail method according to the number of samples, and organize a 96-well plate puzzle; then, switch to a 20x objective lens, scan each sample with an excitation light of a wavelength of 561 nm, take photos of the fluorescent microspheres in each circular aperture in sequence, obtain photos of the fluorescent microspheres on the entire well surface, and use the confocal software to read the fluorescent microsphere photos, and convert them into TIF format in batches; S3. Count the fluorescent microspheres located in the circular aperture of the confocal glass plate to determine the counting standard of the fluorescent film: All fluorescent microspheres in each circular aperture described in S2 are counted, and the counting result is recorded as D. The counting standard of fluorescent microspheres is: Standard 1: When the microsphere overlaps with the edge of the square, if the microsphere overlaps with the upper left edge of each square, it is included in the count; if it overlaps with the lower right edge, it is ignored. The square is the smallest unit square in the sample division matrix; Standard 2: The size of the microspheres is based on the 1μm scale. Microspheres smaller than 1μm are counted, and other larger fluorescent particles or other long fluorescent patterns are non-microsphere signals and need to be ignored; S4. According to the matrix divided on the confocal glass plate and the counting standard, the fluorescent film is quantified to obtain a quantitative result: According to Standard 1 and Standard 2 in S3, the designed sampling grids are counted, and the percentage of the sampling counting grids in different areas to the circular area is determined, so as to predict a quantitative result close to the overall radiographic count, which specifically includes the following steps: S4-1, dividing the sample matrix: using image processing software to divide the fluorescent film of the circumscribed rectangle of a single circular hole in the 96-well dedicated confocal glass plate into an 8×8 matrix of 64 grids in total. Specifically, in the embodiment, Photoshop (PS) is used to open the fluorescent microsphere photo in TIF format obtained in S2, and a filter is added to the picture to reduce image noise, and the stylization is selected to set the number of collages to 8 columns, the maximum displacement is set to 1%, and the foreground color is converted to blue to highlight the red fluorescent microspheres, so that the imaging is clearer; S4-2. Arrange sampling grids in different areas: Select different counting grids in the 64-grid matrix described in S4-1 to arrange sampling grids in different areas; the design of the representative sampling method is to select a specific number of grids in the circular aperture to count according to a special counting standard, and the number of counted fluorescent microspheres is recorded as D1. The specific design is as follows: (1) 2-grid counting method: Select 2 squares in the upper left corner and lower right corner of the middle 2×2 matrix in the circular aperture for counting. The sampling type is recorded as 2-grid-①, such as Figure 1 a in the circular aperture; select 2 squares in the upper right corner and the lower left corner of the middle 2×2 matrix for counting, and the sampling type is recorded as 2 squares-②, such as Figure 1 b in the figure; select two squares in the upper right corner and lower left corner of the middle 4×4 matrix in the circular aperture for counting, and the sampling type is recorded as 2 squares-③, such as Figure 1 c in the figure; select two squares in the upper left corner and lower right corner of the middle 4×4 matrix in the circular aperture for counting, and the sampling type is recorded as 2 squares-④, such as Figure 1 d in; (2) 6-grid counting method: Select the 2 squares at the upper left and lower right corners of the middle 4×4 matrix and the 4 squares connected to the upper right and lower left corners in the circular aperture for counting. The sampling type is recorded as 6-grid-①, such as Figure 1 e in the circular aperture; select 6 squares in total, including 2 squares at the upper right corner and the lower left corner of the middle 4×4 matrix and 4 squares in the middle 2×2 matrix, for counting, and the sampling type is recorded as 6 squares-②, such as Figure 1 f in; (3) 8-grid counting method: Select two middle grids at each edge of the middle 4×4 matrix in the circular aperture for counting, and the sampling type is recorded as 8 grids-①, such as Figure 1 g in the figure; 8 squares of the middle 2×4 matrix are selected in the circular aperture for counting, and the sampling type is recorded as 8 squares-②, such as Figure 1 h in; 8 squares of the middle 4×2 matrix are selected in the circular aperture for counting, and the sampling type is recorded as 8 squares-③, such as Figure 1 i in; select the 8 squares of the middle 4×4 matrix in the circular aperture except the counting squares of 8 squares-③ for counting, and the sampling type is recorded as 8 squares-④, such as Figure 1 j in; (4) 12-grid counting method: 12 squares at the edge of the middle 4×4 matrix are selected within the circular aperture for counting. The sampling type is recorded as 12-grid-①, such as Figure 1 k in; select the 12 squares of the middle 4×4 matrix removal method 2 squares-③ and 2 squares-④ in the circular aperture for counting, and the sampling type is recorded as 12 squares-②, such as Figure 1 l in; (5) 16-grid counting method: Select the middle 4×4 matrix of 16 squares in the circular aperture for counting, and the sampling type is recorded as 16-grid-①, such as Figure 1 m in; S4-3, determining the conversion coefficient: calculating the average conversion coefficient of different counting grids according to the average value of the ratio of the area of the single circular hole to the area of the counting grid, comprising the following steps: (1) Count different sampling methods according to counting standards 1 and 2 of fluorescence radiography in S3, and the result is D1; (2) Randomly select 10 TIF images (numbered 1-10) and open them in Photoshop. Use a ruler to measure the side length of a single square in the matrix in S4-2. l and the radius of the circular hole r , recorded in Table 1; and calculate the circular hole area ( S 圆 =π r 2 ) and the area of a single square ( S 框 = l 2 ) S 圆 / S 框 =π( r / l ) 2 ; (3) Determine the overall conversion coefficient α ,in, α = S 圆 / S 框 =π( r / l ) 2 , the calculation results are shown in Table 1; (4) Determine the uniform conversion formula for the concentration of fluorescent microspheres in the sample solution: Based on the counting standard described in S4-2, calculate the average conversion coefficient κ = Overall conversion factor / number of counting squares, e.g., in a 2-square counting method, κ 2= α / 2, the calculation results are listed in Table 1; let the quantitative full hole counting result of this method be recorded as D2, where D2=D1× κ ;
[0015] Note: 1. The side lengths of the squares and the radius of the circular holes in Table 1 were measured by using a ruler with a measurement scale in PS after opening the photos taken by laser scanning in PS; 2. α is the overall conversion factor, α =π( r / l ) 2 .
[0016] S4-4. Determine the fitting curve of the sampling counting grids in different regions: fit the number of sampling grids and the ratio of the overall sampling standard deviation to the overall standard deviation to obtain the fitting curve equation. The specific steps are as follows: In the first step, the overall standard deviation of the fluorescent microsphere film counting results was manually calculated and recorded as σ :First, the environmental water samples of each concentration of fluorescent microspheres were n The collection and counting of fluorescence photos in this embodiment of the present invention is based on the 30 parallel sample solutions in S1, that is, n =30, each counting result is recorded as D i ,in i =1, 2, 3, ..., 30; then, calculate the mean and standard deviation; The second step is to calculate the sampling standard deviation of 30 samples for each counting scheme. σ 1: First, collect data. For each counting scheme described in S4-2, perform 30 independent sampling operations. In each sampling, count the fluorescent microspheres in the selected squares according to the corresponding counting standard, and obtain 30 counting results, which are set as D1. i , i =1, 2, 3, ..., 30, then calculate the mean and standard deviation; The third step is to calculate the standard deviation of the quantitative results determined by each counting scheme based on the conversion coefficient, recorded as σ 2: First, calculate according to D2= κ ×D1 i Get D2, i =1, 2, 3, ..., 30, then calculate the mean and standard deviation; Step 4: Calculate the ratio of different counting schemes with the same number of sampling grids to the overall standard deviation ,in, The sampling standard deviation of the whole hole counting results quantified by the same number σ The average of 2; Step 5: Calculate the proportion of the area of the counting square in the circle area: Take the 2-square sampling in No. 1 in Table 1 as an example. For the 2-square sampling, calculate the proportion of the total area of the 2 squares in the circle area, that is, (2×390.01 2 ) / (3.1415926×1592.50 2 )≈3.5%; for 6-square sampling, calculate the total area of 6 squares S框 Ratio to the area of the circular hole 6 S 框 / S 圆 , we get a proportion of about 10%; for 8-grid sampling, we calculate the total area of 8 squares. S 框 Ratio to the area of the circular hole 8 S 框 / S 圆 , we get a proportion of about 14%; for 12 squares sampling, we calculate the total area of 12 squares. S 框 Ratio to the area of the circular hole 12 S 框 / S 圆 , we get a proportion of about 21%; for 16 grid samples, we calculate the total area of 16 grids. S 框 Ratio to the area of the circular hole 16 S 框 / S 圆 , and obtained a share of about 28%; The sixth step is to make a fitting curve, which is the ratio of the counting square area to the circle area. σ 2 / σ Perform fitting and obtain the fitting curve equation: y =− a ln x + b ,in x represents the proportion of the counting square area in the circle area; y represent , a and b is a constant, when σ 2 / σ The closer it is to 1, the better the randomness and representativeness of the sample size; S4-5. Determine the optimal counting grid area: According to the fitted curve equation, the tangent line is x The axis is determined by the optimal counting grid area, based on which the number of sampling grids finally adopted is obtained; S5. Perform stability assessment on the quantitative results to obtain stability assessment results: Evaluation of the robustness of the quantitative method using different counting schemes based on the average conversion factor κThe coefficient of variation (CV) of the quantitative results and fluorescent radiograph counts determined in the present invention is used as a method for detecting the stability of the present invention. The smaller the coefficient of variation, the stronger the stability of the scheme. The calculation method of the coefficient of variation CV is: CV = (the standard deviation of the number of fluorescent microspheres calculated by the counting method and the standard deviation of the number of fluorescent microspheres calculated manually) / average number; According to the number of sampling squares finally adopted in S4-5, the coefficients of variation of different two-dimensional counting schemes are analyzed and compared P5, P10, P25, P50, P75, P90, and P95, and then the two-dimensional arrangement of the final counting squares is determined to be the sampling method of the embodiment.
[0017] The beneficial effects of the present invention are as follows 1. Technical innovation: The present invention uses laser confocal fluorescence microscopy technology as the main tool and a new technical principle designed based on the image grid sampling principle to perform rapid and simple quantitative detection of nano fluorescent microspheres in environmental water samples. The counting scheme of the present invention has high stability; 2. Filling the gap: Proposing a rapid and simple quantitative detection of nano fluorescent microspheres in environmental water samples, filling the gap in the current detection technology based on fluorescence confocal imaging for quantitative detection of nano fluorescent microspheres in environmental water samples; 3. Determine the detection range: It is determined that rapid quantitative detection can be performed within the range of 1-40 μg / mL of fluorescent nanosphere concentration; 4. Determine the sampling plan: Once the sampling plan area accounts for 2-10%, rapid quantification can be carried out; 5. Easy to promote: Using laser confocal fluorescence microscopy as the main detection tool, it can be carried out in a laboratory with such technical conditions. The hardware facilities required are relatively low and it is easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Counting methods for different counting grids; Figure 2 This is a before and after comparison diagram of the fluorescent radiography sampling grid layout in Example 1, where Figure 2 A in the figure is the fluorescence radiography sampling grid before layout. Figure 2 B in the figure is the sampling grid layout of fluorescence radiography; Figure 3 The number of fluorescent microsphere sampling grids for the 1 μg / mL water sample in Example 1 is The fitting curve of Figure 4 The number of fluorescent microsphere sampling grids for the 10 μg / mL water sample in Example 2 is The fitting curve of Figure 5The number of fluorescent microsphere sampling grids for the 40 μg / mL water sample in Example 3 is The fitting curve of . DETAILED DESCRIPTION
[0019] The technical solution of the present invention will be described in detail below in conjunction with specific embodiments of the present invention. The described embodiments are only a part of the present invention, and the present invention is not limited to the embodiments described below.
[0020] Example 1 S1: Environmental water sample solution with monodisperse fluorescent nanospheres Take 0.4mL of 100mg / 10mL Lumisphere monodisperse red fluorescent microspheres (BaseLine, 10mL) with a diameter of 0.1μm, dissolve it in the environmental water sample containing bioaerosols in the air, and prepare a 40μg / mL fluorescent microsphere environmental water sample stock solution, then dilute it to 1μg / mL fluorescent microsphere environmental water sample, inactivate it at 60℃ for 1h, and then sonicate and shake it; S2: Add the environmental water sample solution of the monodisperse nano fluorescent microspheres to a confocal glass plate to form a sample, and scan the sample with a laser to form a fluorescent film: Take out 30 parallel sample solutions of 70 μL each from the fluorescent microsphere environmental water sample described in S1, add them to the 96-well dedicated confocal glass plate in sequence according to the dragon-swung-tail method, select the TRITC / RFP channel as the excitation light source channel of the fluorescent microspheres, adjust the horizontal and vertical positions of the wells under the 2x objective lens, adjust the focal length so that the focal length based on the fluorescent microspheres as the standard can be clearly seen to be positioned in the exact center of the well, design a 96-well plate sample puzzle template on the laser confocal software, scan the 96-well plate in sequence according to the dragon-swung-tail method according to the number of samples, and organize a 96-well plate puzzle; then, switch to a 20x objective lens, scan each sample with an excitation light of a wavelength of 561 nm, take photos of the fluorescent microspheres in each circular aperture in sequence, obtain photos of the fluorescent microspheres on the entire well surface, and use the confocal software to read the fluorescent microsphere photos, and convert them into TIF format in batches; S3. Count the fluorescent microspheres located in the circular aperture of the confocal glass plate to determine the counting standard of the fluorescent film: All fluorescent microspheres in each circular aperture described in S2 are counted, and the counting result is recorded as D. Since 30 parallel samples are taken in this embodiment, n =30, the counting standard of fluorescent microspheres is: Standard 1: When the microsphere overlaps with the edge of the square, if the microsphere overlaps with the upper left edge of each square, it will be counted; if it overlaps with the lower right edge, it will be ignored; Standard 2: The size of the microspheres is based on the 1μm scale. Microspheres smaller than 1μm are counted, and other larger fluorescent particles or other long fluorescent patterns are non-microsphere signals and need to be ignored; S4. According to the matrix divided on the confocal glass plate and the counting standard, the fluorescent film is quantified to obtain a quantitative result: According to Standard 1 and Standard 2 in S3, the designed sampling grids are counted, and the percentage of the sampling counting grids in different areas to the circular area is determined, so as to predict a quantitative result close to the overall radiographic count, which specifically includes the following steps: 4.1. Division of sample matrix S4-1, dividing the sample matrix: using image processing software to divide the fluorescent film of the circumscribed rectangle of a single circular hole in the 96-well dedicated confocal glass plate into an 8×8 matrix of 64 grids in total. Specifically, in the embodiment, Photoshop (PS) is used to open the fluorescent microsphere photo in TIF format obtained in S2, and a filter is added to the picture to reduce image noise, and the stylization is selected to set the number of collages to 8 columns, the maximum displacement is set to 1%, and the foreground color is converted to blue to highlight the red fluorescent microspheres, so that the imaging is clearer; S4-2. Arrangement of sampling grids in different areas: Different counting grids are selected from the 64-grid matrix described in S4-1 to arrange sampling grids in different areas; the design of the representative sampling method is to select a specific number of grids within the circular aperture to count according to a special counting standard, and the number of counted fluorescent microspheres is recorded as D1. The counting results are shown in Table 2. The specific design is as follows: (1) 2-grid counting method: Select 2 squares in the upper left corner and lower right corner of the middle 2×2 matrix in the circular aperture for counting. The sampling type is recorded as 2-grid-①, such as Figure 1 a in the circular aperture; select 2 squares in the upper right corner and the lower left corner of the middle 2×2 matrix for counting, and the sampling type is recorded as 2 squares-②, such as Figure 1 b in the figure; select two squares in the upper right corner and lower left corner of the middle 4×4 matrix in the circular aperture for counting, and the sampling type is recorded as 2 squares-③, such as Figure 1 c in the figure; select two squares in the upper left corner and lower right corner of the middle 4×4 matrix in the circular aperture for counting, and the sampling type is recorded as 2 squares-④, such as Figure 1 d in; (2) 6-grid counting method: Select the 2 squares at the upper left and lower right corners of the middle 4×4 matrix and the 4 squares connected to the upper right and lower left corners in the circular aperture for counting. The sampling type is recorded as 6-grid-①, such as Figure 1e in the circular aperture; select 6 squares in total, including 2 squares at the upper right corner and the lower left corner of the middle 4×4 matrix and 4 squares in the middle 2×2 matrix, for counting, and the sampling type is recorded as 6 squares-②, such as Figure 1 f in; (3) 8-grid counting method: Select two middle grids at each edge of the middle 4×4 matrix in the circular aperture for counting, and the sampling type is recorded as 8 grids-①, such as Figure 1 g in the figure; 8 squares of the middle 2×4 matrix are selected in the circular aperture for counting, and the sampling type is recorded as 8 squares-②, such as Figure 1 h in; 8 squares of the middle 4×2 matrix are selected in the circular aperture for counting, and the sampling type is recorded as 8 squares-③, such as Figure 1 i in; 8 squares of the middle 4×4 matrix removing counting squares of method three are selected in the circular aperture for counting, and the sampling type is recorded as 8 squares -④, such as Figure 1 j in; (4) 12-grid counting method: 12 squares at the edge of the middle 4×4 matrix are selected within the circular aperture for counting. The sampling type is recorded as 12-grid-①, such as Figure 1 k in; select the middle 4×4 matrix in the circular aperture to remove the 12 squares of method 2 for counting, and the sampling type is recorded as 12 squares -②, such as Figure 1 l in; (5) 16-grid counting method: Select the middle 4×4 matrix of 16 squares in the circular aperture for counting, and the sampling type is recorded as 16-grid-①, such as Figure 1 The m in.
[0021]
[0022] S4-3, determining the conversion coefficient: calculating the average conversion coefficient of different counting grids according to the average value of the ratio of the area of the single circular hole to the area of the counting grid, comprising the following steps: (1) Count different sampling methods according to the counting standards 1 and 2 of fluorescence radiography in S3, i.e. D1; (2) Randomly select 10 TIF images (numbered 1-10) and open them in PS. Use a ruler to measure the side lengths of the squares in the matrix described in S4-2. l and the radius of the circular hole r , recorded in Table 1; and calculate the circular hole area ( S 圆 =π r 2 ) and the grid area ( S 框 = l 2 ) S圆 / S 框 =π( r / l ) 2 ; (3) Determine the overall conversion coefficient α ,in, α = S 圆 / S 框 =π( r / l ) 2 , calculation see Table 1; (4) Determine the uniform conversion formula for the concentration of fluorescent microspheres in the sample solution: Based on the counting standard described in S4-2, calculate the average conversion coefficient κ = Overall conversion factor / number of counting squares, for example, in a 2-square counting method, κ 2= α / 2, the calculation results are listed in Table 1; let the quantitative full hole counting result of this method be recorded as D2, where D2=D1× κ , the calculation results of D2 are shown in Table 3;
[0023] S4-4. Determine the fitting curve of the sampling counting grids in different regions: fit the number of sampling grids and the ratio of the overall sampling standard deviation to the overall standard deviation to obtain the fitting curve equation. The specific steps are as follows: The first step is to calculate the overall standard deviation of all fluorescent microsphere imaging results within each circular aperture. σ , the method is: (1) (2) The calculation results are listed in Table 4; In this embodiment n =30, i =1,2,3,..., n ; The results are listed in Table 4; The second step is to calculate the sampling standard deviation of 30 samples for each counting scheme. σ 1. The method is: (3) (4) The calculation results are listed in Table 4; The third step is to calculate the sampling standard deviation of the full-hole count results quantified by this method. σ 2. The method is: (5) (6)
[0024] The fourth step is to find the proportion of the area of the counting square in the circle area: Taking the 2-square sampling in No. 1 in Table 1 as an example, for the 2-square sampling, calculate the proportion of the total area of the 2 squares in the circle area, that is, (2×390.01 2 ) / (3.1415926×1592.50 2 )≈3.5%; for 6-square sampling, calculate the total area of 6 squares S 框 Ratio to the area of the circular hole 6 S 框 / S 圆 , we get a proportion of about 10%; for 8-grid sampling, we calculate the total area of 8 squares. S 框 Ratio to the area of the circular hole 8 S 框 / S 圆 , we get a proportion of about 14%; for 12 squares sampling, we calculate the total area of 12 squares. S 框 Ratio to the area of the circular hole 12 S 框 / S 圆 , we get a proportion of about 21%; for 16 grid samples, we calculate the total area of 16 grids. S 框 Ratio to the area of the circular hole 16 S 框 / S 圆 , and obtained a share of about 28%; The fifth step is to make a fitting curve, which is the ratio of the counting square area to the circle area. Perform fitting and obtain the fitting curve equation: y =−0.079ln x +1.2452 (7) As attached Figure 3 ,in, The sampling standard deviation of the whole hole counting results quantified by the same number σ The average value of 2, taking the 2-grid counting method as an example, =( σ 2(2 grids-①)+ σ 2(2 grids-②)+ σ 2(2 grids-③)+ σ2(2 grids-④)) / 4(8) x represents the proportion of the counting square area in the circle area, y represent , a and b are constants, when The closer it is to 1, the better the randomness and representativeness of the sampling quantity. The results are shown in Table 5;
[0025] S4-5. Determine the optimal counting grid area: According to the fitted curve equation, when the slope of the curve is 1, the tangent x The axis is determined by the optimal counting grid area, based on which the number of sampling grids finally adopted is obtained; According to the attached Figure 3 The obtained fitting curve equation is the slope of the tangent line of the fitting curve ( y / x ) is close to 1, select a series of points whose coordinates ( x , y ) See Table 5. The area of the optimal counting grid is the area with the tangent line of the fitting curve closest to 1. As shown in Table 5, when the circular area of the counting grid accounts for 2%, the present invention can be used for rapid quantification; therefore, 2 counting grids are the optimal number of sampling grids;
[0026] S5. Perform stability assessment on the quantitative results to obtain stability assessment results: To evaluate the stability of the quantitative method, the coefficient of variation (CV) of the fluorescence radiograph counts of the quantitative results determined by the four counting schemes of the two-grid counting grid according to the average conversion coefficient was used as the stability detection method of the present invention. The smaller the coefficient of variation, the stronger the stability of the scheme. The coefficient of variation CV is D2 i and D i The ratio of the standard deviation to the mean, the values of which are shown in Table 7;
[0027] According to the number of sampling grids finally adopted as described in S4-5, in order to compare the size of the coefficient of variation of the four 2-grid counting methods, the coefficient of variation of the four counting schemes of 2 grids were analyzed by P5, P10, P25, P50, P75, and P90, as shown in Table 8. It can be seen from Table 8 that the average values of P5, P10, P25, P50, P75, and P90 of counting scheme ③ are the smallest, so technical scheme 2 grids-③ is determined to be the sampling method of the present invention.
[0028]
[0029] Example 2 S1: Environmental water sample solution with monodisperse fluorescent nanospheres Take 0.4mL of 100mg / 10mL Lumisphere monodisperse red fluorescent microspheres (BaseLine, 10ml) with a diameter of 0.1μm, dissolve it in the environmental water sample containing bioaerosols in the air, and prepare a 40μg / mL fluorescent microsphere environmental water sample stock solution, then dilute it to a 10μg / mL fluorescent microsphere environmental water sample, inactivate it at 60℃ for 1h, and then sonicate and shake it; The operation steps of S2-S5 are the same as those of Example 1, and the relevant data are shown in the following table:
[0030]
[0031]
[0032] Make a fitting curve, and compare the proportion of the counting square area in the circle area with Perform fitting and obtain the fitting curve equation: y =−0.446ln x +2.7774 (9) like Figure 4 ,in, The sampling standard deviation of the whole hole counting results quantified by the same number σ The average value of 2 is calculated in the same way as formula (8); Table 12. Ratio of the mean standard deviation of sampling from different batches to the overall standard deviation (10 μg / mL).
[0033]
[0034] according to Figure 4 The obtained fitting curve equation, the tangent line when the fitting curve slope is 1 x The axis is the optimal counting grid area, which is listed in Table 13. It can be seen from Table 13 that when the circular area of the counting grid area accounts for 3%, the present invention can be used for rapid quantification. Therefore, 2 counting grids are the optimal number of sampling grids;
[0035] S5. Perform stability assessment on the quantitative results to obtain stability assessment results: The evaluation principle is the same as that in Example 1. The number of sampling grids finally adopted is to compare the coefficient of variation of the four 2-grid counting methods. The coefficient of variation of the four counting schemes of 2 grids is analyzed by P5, P10, P25, P50, P75, and P90. See Table 14. It can be seen from Table 14 that the average values of P5, P10, P25, P50, P75, and P90 of counting scheme ④ are the smallest. Therefore, technical scheme 2 grids-④ is determined to be the sampling method of the present invention.
[0036]
[0037] Example 3 S1: Environmental water sample solution with monodisperse fluorescent nanospheres Take 0.4 mL of 100 mg / 10 mL Lumisphere monodisperse red fluorescent microspheres (BaseLine, 10 mL) with a diameter of 0.1 μm, dissolve it in an environmental water sample containing bioaerosols in the air, and prepare a 40 μg / mL fluorescent microsphere environmental water sample stock solution, which is inactivated at 60 °C for 1 h, and then sonicated and shaken; Table 15. The number of fluorescent microspheres D1 counted in environmental water samples with 40 μg / mL fluorescent microspheres in different counting schemes.
[0038]
[0039] Table 16. Comparison of the whole-well counting result D2 at 40 μg / mL and the total fluorescent microsphere count D.
[0040]
[0041]
[0042] Make a fitting curve, and compare the proportion of the counting square area in the circle area with Perform fitting and obtain the fitting curve equation: y =−0.239ln x +1.9899 (10) like Figure 5 ,in, The sampling standard deviation of the whole hole counting results quantified by the same number σ The average value of 2 is calculated in the same way as formula (8);
[0043] according to Figure 5 The obtained fitting curve equation, the tangent line when the fitting curve slope is 1 xThe axis is the optimal counting grid area, which is shown in Table 18. As can be seen from Table 18, when the circular area of the counting grid area accounts for 2%, the present invention can be used for rapid quantification; therefore, 2 counting grids are the optimal number of sampling grids;
[0044] S5. Perform stability assessment on the quantitative results to obtain stability assessment results: The evaluation principle is the same as that in Example 1. The number of sampling grids finally used is to compare the coefficient of variation of the four 2-grid counting methods. The coefficient of variation of the four counting schemes of 2 grids is analyzed by P5, P10, P25, P50, P75, and P90. See Table 20. It can be seen from Table 14 that the average values of P5, P10, P25, P50, P75, and P90 of counting scheme ④ are the smallest. Therefore, it is determined that technical schemes 2 grids-③ and 2 grids-④ can be used as the sampling methods of the present invention.
[0045]
[0046] Comparative Example 1 S1: Environmental water sample solution with monodisperse fluorescent nanospheres Same as Example 1; S2: Add the environmental water sample solution of the monodisperse nano fluorescent microspheres to a confocal glass plate to form a sample, and scan the sample with a laser to form a fluorescent film: Same as Example 1; S3. Count the fluorescent microspheres located in the circular aperture of the confocal glass plate to determine the counting standard of the fluorescent film: Same as Example 1; S4. According to the matrix divided on the confocal glass plate and the counting standard, the fluorescent film is quantified to obtain a quantitative result: S4-1, same as in Example 1; S4-2, arranging 2 sampling grids and 8 sampling grids as a comparative example: selecting 2 sampling grids in the 64-grid matrix described in S4-1 for arrangement, selecting 2 squares in the upper right corner and the lower left corner of the middle 4×4 matrix in the circular aperture for counting, that is, the aforementioned sampling type is recorded as 2 grids-③, according to Table 1, its average conversion coefficient is 28.77; selecting 8 sampling grids in the 64-grid matrix described in S4-1 for arrangement, selecting 8 squares in the middle 4×2 matrix in the circular aperture for counting, that is, the sampling type is recorded as 8 grids-③, according to Table 1, its average conversion coefficient is 7.19; the number of counted fluorescent microspheres is recorded as D1; S4-3, same as Example 1 Determine the unified conversion formula for the concentration of fluorescent microspheres in the sample solution: let the result of the whole-well count be recorded as D2, where D2=D1×conversion coefficient; S4-4, determine whether the estimated value of the comparison column sampling counting grid is consistent with the number of fluorescent microspheres in the original circular area; According to the difference d between the estimated value D2 of the sampling method and the number of fluorescent microspheres D in the circular area, the paired T test method was used to test whether there was a significant difference in the means of the two groups of quantitative data in the paired design. When P>0.05, the estimated value of the sampling counting grid was considered to be consistent with the original number of fluorescent microspheres in the circular area. The results showed that the treatment effect of the 2-grid-③ random sampling method was consistent with the original number of fluorescent microspheres in the circular area (P=0.263), while the treatment effect of the 8-grid-③ random sampling method was inconsistent with the original number of fluorescent microspheres in the circular area (P=0.011).
[0047]
[0048] Comparative Example 2 S1: Environmental water sample solution with monodisperse fluorescent nanospheres Same as Example 2 S2: Add the environmental water sample solution of the monodisperse nano fluorescent microspheres to a confocal glass plate to form a sample, and scan the sample with a laser to form a fluorescent film: Same as Example 1; S3. Count the fluorescent microspheres located in the circular aperture of the confocal glass plate to determine the counting standard of the fluorescent film: Same as Example 1; S4. According to the matrix divided on the confocal glass plate and the counting standard, the fluorescent film is quantified to obtain a quantitative result: S4-1, same as in Example 1; S4-2, arranging 2 sampling grids and 6 sampling grids as comparative examples: selecting 2 sampling grids in the 64-grid matrix described in S4-1 for arrangement, selecting 2 squares in the upper left corner and lower right corner of the middle 4×4 matrix in the circular aperture for counting, that is, the aforementioned sampling type is recorded as 2 grids-④, according to Table 1, its average conversion coefficient is 28.77; selecting 6 sampling grids in the 64-grid matrix described in S4-1 for arrangement, selecting 2 squares in the upper left corner and lower right corner of the middle 4×4 matrix in the circular aperture and 4 squares connected to the upper right corner and the lower left corner for counting, that is, the sampling type is recorded as 6 grids-①, according to Table 1, its average conversion coefficient is 9.59; the number of counted fluorescent microspheres is recorded as D1; S4-3 is the same as in Example 2;
[0049] S4-4. Determine whether the estimated value of the comparison column sampling counting grid is consistent with the number of fluorescent microspheres in the original circular area: Based on the difference d between the estimated value of the sampling method (D2) and the number of fluorescent microspheres in the circular area (D), the paired T test method is used. When P>0.05, it is considered whether the estimated value of the sampling counting grid is consistent with the number of fluorescent microspheres in the original circular area. The results showed that the treatment effect of the 2-grid random sampling method was consistent with the number of fluorescent microspheres in the original circular area (P=0.695), however, the treatment effect of the 6-grid random sampling method was inconsistent with the number of fluorescent microspheres in the original circular area (P=0.021).
[0050] The estimated values of the 2-grid random sampling method in Comparative Examples 1 and 2 are consistent with the number of fluorescent microspheres in the original circular area (P=0.35, P=0.263, P=0.695), and the estimated values of the 8-grid and 6-grid random sampling methods in Comparative Examples 1 and 2 are inconsistent with the number of fluorescent microspheres in the original circular area (P=0.011, P=0.021). It can be seen that compared with the comparative example, the calculation result of the sampling method determined in the embodiment is consistent with the number of fluorescent microspheres in the original circular area, and its calculation result is more accurate; at the same time, the estimation counting speed of the 2-grid random sampling method is faster than the counting method of the number of fluorescent microspheres in the original circular area. Therefore, the quantitative method of nano fluorescent microspheres of the present invention has the advantages of saving more calculation time and more accurate calculation results compared with the comparative example.
[0051] The above description is only a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any modification or equivalent change made based on the technical essence of the present invention still falls within the scope of protection required by the present invention.
Claims
1. A method for quantitative determination of nano fluorescent microspheres, characterized in that: include: S1, preparing a monodisperse nano fluorescent microsphere solution; S2, adding the monodisperse nano fluorescent microsphere solution to a confocal glass plate to form a sample, and scanning the sample with a laser to form a fluorescent film; S3, counting the fluorescent microspheres located in the circular aperture of the confocal glass plate to determine the counting standard of the fluorescent film; S4, quantifying the fluorescent film according to the matrix divided on the confocal glass plate and the counting standard to obtain a quantitative result; S5. Performing stability evaluation on the quantitative result to obtain a stability evaluation result.
2. A method for quantifying nano fluorescent microspheres according to claim 1, characterized in that: In S1, the method for preparing a monodisperse nano fluorescent microsphere solution is: taking monodisperse nano fluorescent microspheres and dissolving them in physiological saline to prepare a monodisperse nano fluorescent microsphere solution.
3. A method for quantifying nano fluorescent microspheres according to claim 1, characterized in that: In S2, the monodisperse nano fluorescent microsphere solution is added to a confocal glass plate to form a sample, and the sample is scanned with a laser to form a fluorescent film, including: The monodisperse nano fluorescent microsphere solution is added to a 96-well special confocal glass plate to form a sample, an excitation light source channel of the nano fluorescent microsphere is selected, each sample is scanned with a laser, a photo of the fluorescent microspheres on the entire well surface is taken to form a fluorescent film, and the fluorescent film is batch converted into an image file format.
4. A method for quantifying nano fluorescent microspheres according to claim 3, characterized in that: The excitation light source channel of the nano fluorescent microsphere is any one of TRITC / RFP, TD, and FITC / GFP.
5. A method for quantifying nano fluorescent microspheres according to claim 1, characterized in that: In S3, the counting standard of the fluorescent film is determined as follows: counting the fluorescent microspheres in the circular aperture of the 96-well dedicated confocal glass plate.
6. A method for quantifying nano fluorescent microspheres according to any one of claims 1 or 5, characterized in that: In S3, the counting standard for determining the fluorescence radiographs includes two standards: Standard 1: When the microsphere overlaps with the edge of the square, if the microsphere overlaps with the upper left edge of each square, it is included in the count, and if it overlaps with the lower right edge, it is ignored. The square is the smallest unit square in the sample division matrix; Standard 2: The size of the microspheres is based on the 1μm scale. Microspheres smaller than 1μm are counted, and other larger fluorescent particles or other long fluorescent patterns are non-microsphere signals and need to be ignored.
7. A method for quantifying nano fluorescent microspheres according to claim 1, characterized in that: In S4, the fluorescence radiograph is quantified to obtain a quantitative result: according to the counting standard of the fluorescence radiograph described in S3, the designed sampling squares are counted, and the percentage of the sampling counting squares in different regions to the circular area is determined, so as to predict a quantitative result close to the overall radiograph count.
8. A method for quantifying nano fluorescent microspheres according to any one of claims 1 or 7, characterized in that: In S4, the following sub-steps are also included: S4-1. Divide the sample matrix: Use image processing software to divide the fluorescent film of the circumscribed rectangle of a single circular hole in the 96-well dedicated confocal glass plate into an 8×8 matrix with a total of 64 grids; S4-2, arranging sampling grids in different regions: selecting different counting grids in the 64-grid matrix to arrange sampling grids in different regions; S4-3, determining the conversion coefficient: calculating the average conversion coefficient of different counting grids according to the average value of the ratio of the area of a single circular hole to the area of the counting grid; S4-4, determine the fitting curve of the sampling counting grids in different areas: fit the number of sampling grids and the ratio of the overall sampling standard deviation to the overall standard deviation to obtain the fitting curve equation; S4-5. Determine the optimal counting grid area: According to the fitted curve equation, determine the sampling grid where the overall sampling standard deviation / overall standard deviation approaches 1 as the number of sampling counting grids; according to the number of sampling counting grids, use a specific algorithm to obtain the circle area ratio of the counting grid area, and determine the tangent line when the slope of the fitting curve is 1. x The axis is the optimal counting grid area.
9. A method for quantifying nano fluorescent microspheres according to claim 1, characterized in that: In S5, the method for evaluating the stability of the quantitative results is as follows: based on the number of sampling counting grids determined in S4, a 4×4 matrix in the middle of the fluorescent film is arranged in two dimensions, and the coefficients of variation of different two-dimensional arrangement counting schemes are analyzed and compared at P5, P10, P25, P50, P75, P90, and P95, thereby determining the final two-dimensional arrangement of the counting grids.
10. A rapid quantitative method for bioaerosol environmental water samples in the air using nano fluorescent microspheres, characterized in that: The nano fluorescent microsphere quantitative method according to claim 1 is adopted.