CN114544472A - Method for controlling cross reaction in flow type dot matrix instrument detection - Google Patents
Method for controlling cross reaction in flow type dot matrix instrument detection Download PDFInfo
- Publication number
- CN114544472A CN114544472A CN202210104497.6A CN202210104497A CN114544472A CN 114544472 A CN114544472 A CN 114544472A CN 202210104497 A CN202210104497 A CN 202210104497A CN 114544472 A CN114544472 A CN 114544472A
- Authority
- CN
- China
- Prior art keywords
- microspheres
- sorting
- microsphere
- fluorescence
- fluorescein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000011159 matrix material Substances 0.000 title claims abstract description 22
- 239000004005 microsphere Substances 0.000 claims abstract description 222
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000006185 dispersion Substances 0.000 claims abstract description 14
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 12
- 238000001215 fluorescent labelling Methods 0.000 claims abstract description 12
- 238000000684 flow cytometry Methods 0.000 claims abstract description 6
- 230000037029 cross reaction Effects 0.000 claims description 18
- 239000002245 particle Substances 0.000 claims description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000853 adhesive Substances 0.000 claims description 2
- 230000001070 adhesive effect Effects 0.000 claims description 2
- 230000009260 cross reactivity Effects 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 238000002493 microarray Methods 0.000 claims 1
- 239000000090 biomarker Substances 0.000 abstract 1
- 239000000376 reactant Substances 0.000 description 6
- 230000009977 dual effect Effects 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000027455 binding Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1429—Signal processing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Signal Processing (AREA)
- Engineering & Computer Science (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a method for controlling cross reaction in flow lattice instrument detection, which comprises the following steps: the flow cytometry sorting technology is utilized to perform restrictive sorting on the same group of microspheres with uneven fluorescence labeling levels according to the difference of the fluorescein levels carried by the microspheres, and partial microspheres with uniform fluorescence labeling levels, concentrated distribution and small dispersion are sorted out and combined with other groups of fluorescence microspheres which are also sorted to form a new multiple fluorescence coding microsphere for detection. The practical application value of the invention is that when multiple fluorescent coding microspheres are adopted on a flow-type dot matrix instrument and other similar equipment to simultaneously detect multiple biomarkers, each group of microspheres are subjected to further limited sorting of fluorescence intensity on the flow-type sorting instrument in advance, so that the aim of reducing or avoiding cross reaction between adjacent microspheres caused by overlapping of fluorescent marking levels between the adjacent microspheres is achieved, and the accuracy of multiple index detection is improved.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for controlling cross reaction in detection of a flow-type dot-matrix instrument, in particular to a method for controlling cross reaction of multiple fluorescent coding microspheres adopted in the flow-type dot-matrix instrument in detection.
Background
The fluorescent coding microsphere detection technology adopted in the flow type dot matrix instrument is that one reactant is coated on the surface of the fluorescent coding microsphere according to the principle that two reactants have mutual binding action, and the fluorescent coding microsphere detection technology is used for detecting the other reactant which has the binding action with the reactant. In the multi-index research of the interaction of a plurality of groups of reactants, the microspheres are subjected to combined marking of multiple fluorescein and different fluorescence intensities, so that each microsphere has the unique combination characteristics of fluorescein types and intensities which are different from those of other microspheres, and unique codes are obtained. Different coded microspheres can coat different reactants to form a detection reagent of multiple microspheres for multi-index detection.
The flow-type dot-matrix instrument uses multiple fluorescence coding microspheres to carry out multi-index simultaneous detection, and the core technology of the flow-type dot-matrix instrument lies in the uniformity of the size and the granularity of the microspheres and the uniformity of fluorescein labeling of each microsphere. If the amount of fluorescein marked on each microsphere in the same group of encoded microspheres is different and the uniformity among the microspheres is insufficient, the visible microsphere colony distribution on a flow meter is not concentrated, which causes the dispersion of the fluorescence intensity of different microsphere individuals in the same group of encoded microspheres to be higher, thereby causing the cross-region migration phenomenon of the part of microspheres scattered around the main colony due to the microspheres marked with the fluorescein to be too high or too low, and the part of microspheres falling into other detection microsphere groups coded by the adjacent fluorescein to cause the occurrence of the cross-reaction phenomenon.
Disclosure of Invention
The technical core of the flow-type dot-matrix instrument for multi-index detection of the multiple fluorescence-encoded microspheres lies in the uniformity of the sizes and the granularity of the microspheres and the uniformity of the fluorescein labeling of each group of microspheres. However, as found in the commercial microsphere experiments adopted in the examples of the present application, in the preparation process of fluorescent microspheres, when we adopt different groups of microspheres and combine them into a detection reagent of multiple fluorescent microspheres, the dispersion of the fluorescent signal of a certain group of microspheres is often large, so that the fluorescent signal of the group of microspheres drifts to the fluorescent signal detection region of an adjacent group of microspheres, thereby causing a cross reaction between the fluorescent microspheres, due to the reason that the microspheres have different sizes, different particle sizes, and different fluorescein labels of the same group of microspheres.
In order to solve the technical problems, the invention provides a method for controlling the occurrence of cross reaction in the detection of a flow type dot-matrix instrument. By carrying out further limited sorting on the fluorescence intensity of each group of fluorescent microspheres, the aim of reducing or avoiding cross reaction between adjacent microspheres caused by overlapping of fluorescence labeling levels between the adjacent microspheres is fulfilled, and the accuracy of multi-index detection is improved.
In order to realize the technical purpose of the invention, the invention adopts the technical scheme that:
a method for controlling cross-reactions in a flow-cytometer comprising: the flow cytometry sorting technology is utilized to perform restrictive sorting on the same group of microspheres with uneven fluorescence labeling level according to the difference of the fluorescein level carried by each microsphere, and partial microspheres with even fluorescence labeling level, concentrated fluorescence intensity distribution and small dispersion and other groups of fluorescent microspheres which are also sorted are sorted and combined to form new multiple fluorescence coding microspheres for detection.
Preferably, the limiting sorting comprises sorting according to data on the distribution of the microspheres in the forward scattered light FSC and/or the side scattered light SSC.
More preferably, if the microspheres are not distributed in the quadrants in a centralized manner and part of the microspheres are scattered around the main colony, a sorting gate is established according to the FSC data to sort out the microsphere colony centralized in the colony central area, and the microspheres scattered around the colony and two or more microsphere adherends with diameters smaller than or larger than a set standard size are not selected.
More preferably, if the microspheres are not distributed in the quadrant and part of the microspheres are scattered around the main colony, the microspheres with the same particle density and internal complexity concentrated in the center of the colony are sorted according to the distribution of SSC data, and the microspheres with large internal property difference scattered around the colony are not selected.
Preferably, the restricted sorting further comprises selecting a detection channel suitable for the wavelength according to the type of the microsphere-labeled fluorescein.
More preferably, according to the distribution of the microspheres on the FSC/SSC channel and the detection channels corresponding to different fluorescein, the size and granularity of the microspheres and the tendency of relatively uniform and concentrated fluorescence intensity of fluorescein labels are comprehensively considered, and the area near the center of the microsphere colony is selected as a sorting target according to the number of the microspheres for the required purpose.
Preferably, the multiple fluorescence labeled microspheres are selected in a mode of multiple fluorescein combined sorting, and are combined or not combined with the sorting of FSC/SSC, and finally, a group of microspheres with fluorescein, microsphere size and microsphere particle property more uniform than those before sorting are selected selectively and used as a detection reagent source for preparing the multiple microspheres.
Preferably, the method further comprises the step of performing effectiveness detection on the microspheres obtained after the restrictive sorting, wherein the effectiveness detection comprises the following steps:
1) uniformly mixing the sorted microspheres, and detecting on a flow type dot matrix instrument again;
2) observing the distribution of the fluorescence intensity of different microspheres on one or more fluorescein detection channels marked by the sorted microspheres, comparing the dispersion of the sorted microspheres in different fluorescence channels before and after sorting, and specifically analyzing whether the microspheres subjected to fluorescent signal restriction sorting have cross-region drift to a fluorescent signal region of an adjacent microsphere or not so as to cause cross reaction with the adjacent microsphere.
The invention utilizes the flow cytometry sorting technology to perform restricted sorting on the same group of microspheres with unequal fluorescein labeling quantity (microsphere communities are not distributed in a centralized way and partial microspheres are distributed sporadically around the main community on a flow meter), according to the difference of the fluorescein level carried by each microsphere of the same group, partial microspheres with uniform fluorescein labeling level are sorted out and combined with other groups of fluorescent microspheres which are also sorted into the multi-microsphere detection reagent. In the limiting sorting of microspheres for different levels of fluorescent labeling, sorting in combination with forward scattered light (FSC) and side scattered light (SSC) can be performed simultaneously, depending on the fact that the size of the fluorescently labeled microspheres and the uniformity of the internal particle properties.
The invention has the beneficial effects that:
in the invention, in the process of carrying out multi-index detection on multiple fluorescence-labeled microspheres by using a flow-type dot-matrix analyzer, each group of microspheres is subjected to further limited sorting of fluorescence intensity, so that the aim of reducing or avoiding cross reaction between adjacent microspheres caused by overlapping of fluorescence labeling levels between the adjacent microspheres is fulfilled, and the accuracy of multi-index detection is improved.
The method provided by the invention can be used for the multi-index simultaneous detection process of a flow type dot matrix instrument, a Luminex instrument, a liquid chip instrument and other similar detection platforms.
Drawings
FIG. 1 shows the concentration and dispersion trend of FSC/SSC and dual fluorescence intensity before and after sorting by flow cytometry for dual fluorescence labeled microspheres used in the flow cytometer of the present invention. The scattered spots in the enclosed area in the figure are the microspheres which drift across the area and fall on the signal positions of other groups of fluorescent microspheres, and are the main cause of cross reaction.
FIG. 2 shows the validation results of the dual fluorescence labeled microspheres used in the flow cytometer of the present invention after sorting by flow cytometry.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and the accompanying drawings. It is understood by those skilled in the art that the examples are for illustrative purposes only and should not be construed as limited to the methods described in this example, but rather construed to include any and all variations of the methods of practice provided herein and which become apparent therefrom.
The specific operation method for controlling the occurrence of the cross reaction in the detection of the flow-type dot matrix instrument comprises the following steps:
1.a group of microspheres with the same code is prepared into a single microsphere suspension by PBS or other buffer solution (the microsphere suspension is dispersed into single particles by vortex oscillation for 10s and then ultrasonic treatment for 20s by an ultrasonic instrument), and then the machine is used for carrying out pre-experiment. Adjusting the flow rate and the sample concentration according to the number of the microspheres; if the sample is read at a rate of more than 2000 samples per second, the sample is diluted by a factor of two, and the flow rate is reduced such that the concentration of target microspheres passing through the sample chamber is about 200 samples per second.
2. Analyzing the distribution data of the group of microspheres in forward scattering light FSC and side scattering light SSC, and determining whether the FSC and SSC are necessary to be selected restrictively according to the distribution of the target microsphere group in the two parameters. If the microspheres are not distributed and concentrated in the quadrant, and part of the microspheres are scattered around the main colony, a sorting gate can be established according to FSC data to sort out microsphere colonies with concentrated colony center areas, the diameters of the part of microspheres are relatively close, the difference between the microspheres is small, and therefore the particle size uniformity is good; microspheres with diameters smaller or larger than a set standard size, which are scattered around the colony, and two or more microsphere adhesive bodies are not selected. In addition, a microsphere community closer to the center in the main community can be sorted according to SSC data distribution, and the density of the microsphere particles in the main community is consistent with the internal complexity; or a combination of both.
3. The energy released by the fluorochrome of the labeled microsphere after being excited by excitation light with a certain wavelength can emit fluorescence with a specific wavelength, so that a detection channel suitable for the wavelength needs to be selected according to different types of fluorescein labeled by the microsphere (for example, the excitation light wavelength of the fluorochrome CY5.5 is about 680nm, the emission peak value of the fluorochrome is about 710nm, the excitation light wavelength of APC is 650nm, and the emission light wavelength is about 660 nm). And displaying the distribution of the fluorescence intensity marked by each microsphere detected on the two channels on the flow-type sorting instrument, and selecting a microsphere community closer to the center in the main community according to the distribution conditions of the concentration trend and the dispersion degree of the fluorescence intensity of all the microspheres, wherein the fluorescence intensity of part of the microspheres is relatively concentrated and the dispersion degree is small. According to the number of microspheres for the desired purpose, selecting a region with 80%, 60% or 20% of the center of the microsphere colony as a sorting target (for example, if the number of microspheres before sorting is 500 ten thousand, and the number of microspheres for the desired purpose is 100 ten thousand, then selecting the position of the microsphere with about 20% of the center of the colony and setting a gate), and setting the mode before sorting as "Purify". If the door is set to be larger, the microspheres scattered around the A community can be selected, in the process of analyzing the mixed microspheres, the part of the scattered microspheres can fall into the distribution area of the microsphere community of other groups (such as B), and the reaction signals of the A scattered microspheres can be calculated into the signal data of the B microspheres to cause misreading; if the door is set to be smaller, the collected target microspheres are insufficient, and the subsequent coating and analysis process is difficult to complete.
4. The multiple fluorescence labeled microspheres can adopt a mode of multiple fluorescein combined sorting, and are combined or not combined with the sorting of FSC/SSC, so that fluorescein, microsphere size and microsphere particle properties are finally selectively sorted out, wherein the fluorescein, microsphere size and microsphere particle properties are more uniform than those before sorting (namely the diameter and the complexity of the microspheres and the complexity of internal particles are almost the same, the quantity of the encoded fluorescein is also almost the same, and the properties are presented on a flow type sorter, namely, the properties are that the colony distribution of the microspheres in corresponding quadrants is extremely concentrated), and the microspheres are used for preparing a detection reagent source of the multiple microspheres.
5. The effectiveness detection method after the separation of the restrictive microspheres comprises the following steps:
a) and (3) according to the fluorescence intensity distribution condition of fluorescein marked by the microspheres, combining or not combining the microspheres sorted by the FSC/SSC sorting method, uniformly mixing, and detecting on a flow type dot matrix instrument again.
b) Observing the distribution of the fluorescence intensity of different microspheres on one or more fluorescein detection channels marked by the sorted microspheres, comparing the dispersion of the fluorescence intensity of the different microspheres in the different fluorescence channels before and after sorting, and specifically analyzing whether the microspheres positioned on the right side or above the center of the community (the fluorescence signal values of the part of microspheres exceed the theoretical signal intensity of the group of microspheres) and the microspheres positioned on the left side or below the center of the community (the fluorescence signal values of the part of microspheres are lower than the theoretical signal intensity of the group of microspheres) have cross-region drift to cause cross reaction with the adjacent microspheres in a fluorescence signal quadrant.
c) And determining whether further sorting is needed or not according to the checking result of the effectiveness after the primary sorting.
Examples
In this embodiment, a specific method for controlling the occurrence of cross reaction in the detection of the flow-type dot-matrix analyzer includes the following steps:
(1) an appropriate amount of microspheres labeled with double fluorescent labels (labeled dyes 1.APC and 2.APC-Cy7, respectively) to be sorted was prepared into a concentration of about 5X 10 with PBS5Single microsphere suspension per ml.
(2) The distribution of the protein on the FSC/SSC channel and on the fluorescein 1 and fluorescein 2 detection channels is observed by detection on a flow sorter.
As shown in fig. 1, before sorting: most microspheres have a relatively concentrated region on the FSC/SSC, but more scattered microspheres are still present outside the relatively concentrated region, and the part of microspheres are individuals with larger difference in size, internal particle properties and coded fluorescence compared with standard microspheres in the main population; images of fluorescein 1 and fluorescein 2 show that, although most of the microspheres are relatively concentrated in the intensity distribution of both fluorescein on their respective detection channels, some of the microspheres exhibit a phenomenon of either excessively strong or excessively weak fluorescence intensity.
(3) According to the distribution conditions of microspheres on an FSC/SSC channel and fluorescein 1 and fluorescein 2 detection channels, the size and granularity (FSC/SSC) of the microspheres and the tendency of relatively uniform and concentrated fluorescence intensity of two fluorescein marks are comprehensively considered, the microspheres with 10% of the microsphere distribution center area are selected as a sorting target, and the sorting mode is set to be 'Purify'; and (3) uniformly mixing the sorted microspheres, detecting the microspheres again in a flow cytometer, and observing the distribution conditions of the microspheres subjected to the restrictive sorting on the FSC/SSC and the fluorescein 1 and fluorescein 2 detection channels.
As shown in fig. 1, after sorting: according to the concentrated and discrete trend conditions of FSC/SSC and dual fluorescence intensity of microspheres before sorting, in an experiment, relatively concentrated restrictive sorting (the final sorting rate is 10-15%) is carried out on FSC/SSC and fluorescein marking intensity of the microspheres respectively, partial microspheres with large dispersion are removed, scattered sporadic microspheres around a microsphere main colony are removed, and the remaining microspheres in the center of the colony are microspheres with consistent size, internal particle properties and coded fluorescence.
(4) And taking unsorted fluorescence coding microspheres as negative control, respectively testing two groups of microspheres with the same codes before and after sorting on a flow-type dot matrix instrument, and analyzing and judging whether the microspheres subjected to restrictive sorting are obviously reduced in the dispersion level of the fluorescence intensity of fluorescein marked by the microspheres compared with the unsorted microspheres.
As shown in fig. 2, before sorting: when the double fluorescence labeling microspheres are detected on a flow-type dot matrix instrument, the discrete level of the fluorescence intensity of the microspheres is higher no matter on a detection channel of fluorescein 1 or fluorescein 2, especially in a region with lower fluorescein level; after sorting: the double fluorescence labeling microspheres used by the flow-type dot matrix analyzer are subjected to restrictive sorting on the flow-type cell sorter according to the FSC/SSC and the dispersion condition of the fluorescence intensities of fluorescein 1 and fluorescein 2, and then the detection result on the flow-type dot matrix analyzer shows that most microspheres with too low or too high fluorescence intensities are better eliminated, and compared with the unsorted microspheres, the double fluorescence labeling microspheres are obviously reduced.
In other words, the limited sorting of the fluorescence-encoded microspheres is verified to significantly reduce the fraction of unsorted fluorescence-encoded microspheres that have too high or too low a fluorescence intensity and tend to drift to the region adjacent to the encoded microspheres, thereby causing cross-reactivity.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications can be made on the basis of the above description, and all the implementation methods cannot be exhaustive, and the obvious variations or modifications introduced in the technical scheme of the present invention are within the protection scope of the present invention.
Claims (8)
1.A method for controlling cross-reactions in a flow-cytometer comprising: the flow cytometry sorting technology is utilized to perform restrictive sorting on the same group of microspheres with uneven fluorescence labeling level according to the difference of the fluorescein level carried by each microsphere, and partial microspheres with even fluorescence labeling level, concentrated fluorescence intensity distribution and small dispersion are sorted out and combined with other groups of fluorescence microspheres which are also sorted out to form new multiple fluorescence coding microspheres for detection.
2. The method of claim 1 for controlling cross-reactions in flow-cytometer detection, wherein the limiting sorting comprises sorting microspheres according to their distribution data in forward scattered light FSC and/or side scattered light SSC.
3. The method according to claim 2, wherein if the microspheres are not distributed in the quadrant and part of the microspheres are scattered around the main population, a sorting gate is established according to the FSC data to sort out the microsphere population concentrated in the central area of the population, and the microspheres scattered around the population and two or more microsphere adhesives having a diameter smaller than or larger than a predetermined standard size are not selected.
4. The method of claim 2, wherein if the distribution of the microspheres in the quadrant is not concentrated and part of the microspheres are scattered around the main colony, the microspheres with the same particle density and internal complexity concentrated in the center of the colony are sorted according to the SSC data distribution, and the microspheres with large internal property difference scattered around the colony are not selected.
5. The method of claim 2, wherein the restricted sorting further comprises selecting a detection channel appropriate for the wavelength of the microsphere-labeled fluorescein.
6. The method for controlling cross-reaction in flow-type dot-matrix instrument detection according to claim 3, 4 or 5, wherein the area near the center of microsphere colony is selected as the sorting target according to the number of microspheres for the desired purpose by taking into account the size and granularity of microspheres and the tendency of relatively uniform and concentrated fluorescence intensity of fluorescein labels according to the distribution of microspheres on FSC/SSC channels and detection channels corresponding to different fluorescein.
7. The method of any of claims 1-5 for controlling cross-reactivity in flow-format microarray assays, wherein the multiple fluorescently labeled microspheres are sorted with or without FSC/SSC in a multi-fluorescein combination, and a set of microspheres with fluorescein, microsphere size, and microsphere particle properties more uniform than those before sorting are selected for use as a source of assay reagents for preparing the multiple microspheres.
8. The method of claim 1, further comprising performing an effectiveness test on the microspheres obtained after the limited sorting, the method comprising:
1) uniformly mixing the sorted microspheres, and detecting on a flow type dot matrix instrument again;
2) observing the distribution of the fluorescence intensity of different microspheres on one or more fluorescein detection channels marked by the sorted microspheres, comparing the dispersion of the sorted microspheres in different fluorescence channels before and after sorting, and specifically analyzing whether the microspheres subjected to fluorescent signal restriction sorting have cross-region drift to a fluorescent signal region of an adjacent microsphere or not so as to cause cross reaction with the adjacent microsphere.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210104497.6A CN114544472A (en) | 2022-01-28 | 2022-01-28 | Method for controlling cross reaction in flow type dot matrix instrument detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210104497.6A CN114544472A (en) | 2022-01-28 | 2022-01-28 | Method for controlling cross reaction in flow type dot matrix instrument detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114544472A true CN114544472A (en) | 2022-05-27 |
Family
ID=81673826
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210104497.6A Pending CN114544472A (en) | 2022-01-28 | 2022-01-28 | Method for controlling cross reaction in flow type dot matrix instrument detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114544472A (en) |
Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030186465A1 (en) * | 2001-11-27 | 2003-10-02 | Kraus Robert H. | Apparatus used in identification, sorting and collection methods using magnetic microspheres and magnetic microsphere kits |
| US20050147961A1 (en) * | 2002-04-30 | 2005-07-07 | Esser Mark T. | Human papillomavirus multiplexed assay |
| CN101092487A (en) * | 2007-07-25 | 2007-12-26 | 天津大学 | Method for dyeing fluorescent microballons |
| CN103823065A (en) * | 2014-03-13 | 2014-05-28 | 福建卫生职业技术学院 | Mycotoxin detecting method based on cytometric bead array |
| CN103940798A (en) * | 2014-05-05 | 2014-07-23 | 武汉纽康度生物科技有限公司 | Solid fluorescent nanometer microsphere as well as preparation method and application thereof |
| CN104459135A (en) * | 2013-09-18 | 2015-03-25 | 深圳迈瑞生物医疗电子股份有限公司 | Device and method for detecting negative and positive property of sample and method for determining judgment standard |
| CN109115739A (en) * | 2018-08-06 | 2019-01-01 | 滴准生物科技（常州）有限公司 | A kind of hole internal calibration test method |
| WO2019140926A1 (en) * | 2018-01-22 | 2019-07-25 | 北京国科融智生物技术有限公司 | Functional bio-nano-magnetic bead fluorescence coding method and flow application thereof |
| CN110514575A (en) * | 2019-08-16 | 2019-11-29 | 迈克医疗电子有限公司 | The decoding apparatus and method of quantum point coding microball |
| CN110716053A (en) * | 2019-08-28 | 2020-01-21 | 苏州才博医学科技有限公司 | Novel method for detecting population reactive antibodies |
| CN111318238A (en) * | 2019-12-09 | 2020-06-23 | 彩科(苏州)生物科技有限公司 | Composite microsphere and preparation method and application thereof |
| CN112683873A (en) * | 2021-03-22 | 2021-04-20 | 泛肽生物科技(浙江)有限公司 | Liquid chip detection model construction method and device, and analysis method and device |
| CN113138270A (en) * | 2020-01-20 | 2021-07-20 | 上海纳衍生物科技有限公司 | Multi-target object detection method for blood sample |
| CN113234201A (en) * | 2021-04-23 | 2021-08-10 | 武汉珈源同创科技有限公司 | Multilayer coated quantum dot fluorescent coding microsphere and preparation method thereof |
| CN113552354A (en) * | 2021-07-21 | 2021-10-26 | 中国科学院苏州生物医学工程技术研究所 | Pepsinogen multiplex detection method and detection kit |
| CN113648942A (en) * | 2020-05-12 | 2021-11-16 | 国家纳米科学中心 | Carboxylated quantum dot coding fluorescent microsphere and preparation method and application thereof |
-
2022
- 2022-01-28 CN CN202210104497.6A patent/CN114544472A/en active Pending
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030186465A1 (en) * | 2001-11-27 | 2003-10-02 | Kraus Robert H. | Apparatus used in identification, sorting and collection methods using magnetic microspheres and magnetic microsphere kits |
| US20050147961A1 (en) * | 2002-04-30 | 2005-07-07 | Esser Mark T. | Human papillomavirus multiplexed assay |
| CN101092487A (en) * | 2007-07-25 | 2007-12-26 | 天津大学 | Method for dyeing fluorescent microballons |
| CN104459135A (en) * | 2013-09-18 | 2015-03-25 | 深圳迈瑞生物医疗电子股份有限公司 | Device and method for detecting negative and positive property of sample and method for determining judgment standard |
| CN103823065A (en) * | 2014-03-13 | 2014-05-28 | 福建卫生职业技术学院 | Mycotoxin detecting method based on cytometric bead array |
| CN103940798A (en) * | 2014-05-05 | 2014-07-23 | 武汉纽康度生物科技有限公司 | Solid fluorescent nanometer microsphere as well as preparation method and application thereof |
| WO2019140926A1 (en) * | 2018-01-22 | 2019-07-25 | 北京国科融智生物技术有限公司 | Functional bio-nano-magnetic bead fluorescence coding method and flow application thereof |
| CN109115739A (en) * | 2018-08-06 | 2019-01-01 | 滴准生物科技（常州）有限公司 | A kind of hole internal calibration test method |
| CN110514575A (en) * | 2019-08-16 | 2019-11-29 | 迈克医疗电子有限公司 | The decoding apparatus and method of quantum point coding microball |
| CN110716053A (en) * | 2019-08-28 | 2020-01-21 | 苏州才博医学科技有限公司 | Novel method for detecting population reactive antibodies |
| CN111318238A (en) * | 2019-12-09 | 2020-06-23 | 彩科(苏州)生物科技有限公司 | Composite microsphere and preparation method and application thereof |
| CN113138270A (en) * | 2020-01-20 | 2021-07-20 | 上海纳衍生物科技有限公司 | Multi-target object detection method for blood sample |
| CN113648942A (en) * | 2020-05-12 | 2021-11-16 | 国家纳米科学中心 | Carboxylated quantum dot coding fluorescent microsphere and preparation method and application thereof |
| CN112683873A (en) * | 2021-03-22 | 2021-04-20 | 泛肽生物科技(浙江)有限公司 | Liquid chip detection model construction method and device, and analysis method and device |
| CN113234201A (en) * | 2021-04-23 | 2021-08-10 | 武汉珈源同创科技有限公司 | Multilayer coated quantum dot fluorescent coding microsphere and preparation method thereof |
| CN113552354A (en) * | 2021-07-21 | 2021-10-26 | 中国科学院苏州生物医学工程技术研究所 | Pepsinogen multiplex detection method and detection kit |
Non-Patent Citations (3)
| Title |
|---|
| ZHANG, C., FENG, J., DU, J., ZHUO, Z., YANG, S., ZHANG, W., WANG, W., ZHANG, S., IWAKURA, Y., MENG, G., FU, Y. X., HOU, B. AND TAN: "Macrophage-derived IL-1α promotes sterile inflammation in a mouse model of acetaminophen hepatotoxicity", 《CELL MOL IMMUNOL》, vol. 15, no. 11, 31 December 2018 (2018-12-31), pages 973 - 982, XP036624325, DOI: 10.1038/cmi.2017.22 * |
| 周丽, 周振英: "流式细胞仪研制的技术进展", 《现代医学仪器与应用》, no. 01, 30 March 2003 (2003-03-30), pages 13 - 19 * |
| 陈晓光: "《药理学研究的新技术与新方法》", 31 March 2014, 中国协和医科大学出版社, pages: 232 - 234 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11754499B2 (en) | Enzyme quantification | |
| US4717655A (en) | Method and apparatus for distinguishing multiple subpopulations of cells | |
| US4499052A (en) | Apparatus for distinguishing multiple subpopulations of cells | |
| JP4318859B2 (en) | Combined assay method | |
| Kettman et al. | Classification and properties of 64 multiplexed microsphere sets | |
| US7232691B2 (en) | Bioassay and biomolecular identification, sorting, and collection methods using magnetic microspheres | |
| Sklar et al. | Flow cytometry for drug discovery, receptor pharmacology and high-throughput screening | |
| US20090105963A1 (en) | Methods for Flow Cytometry Analyses of Un-Lysed Cells from Biological Fluids | |
| Givan | Flow cytometry: an introduction | |
| CA2089423A1 (en) | Dna fragment sizing and sorting by laser-induced fluorescence | |
| CN107677803B (en) | coding and decoding system and method for liquid phase chip analyzer | |
| CN114544472A (en) | Method for controlling cross reaction in flow type dot matrix instrument detection | |
| Yingyongnarongkul et al. | Parallel and multiplexed bead-based assays and encoding strategies | |
| AU618736B2 (en) | Automated analyzer and method for screening cells or formed bodies for enumeration of populations expressing selected characteristics | |
| EP4354144A1 (en) | Microfluidic chip for testing protein antigen, and method, kit and system | |
| CN110554178A (en) | Suspension type liquid biochip detection method | |
| US11592367B2 (en) | Method for analyzing and selecting a specific droplet among a plurality of droplets and associated apparatus | |
| WO2004063716A2 (en) | Assay products and procedures | |
| Lipoglavšek et al. | Application of flow cytometry for ecological monitoring of the rumen microbial ecosystem | |
| CN116046644A (en) | Method for flow sorting single nucleated cells | |
| Jett | Molecular cytometry | |
| WO2004027389A2 (en) | Assay products and procedures | |
| CN112760383A (en) | Group of qRT-PCR (quantitative reverse transcription-polymerase chain reaction) internal reference genes applied to lung adenocarcinoma cell subsets and application thereof | |
| FI91920B (en) | Process for improving the accuracy of a biospecific multi- parametric determination method | |
| CN115508317A (en) | Human inflammatory factor detection chip, composition and human inflammatory factor detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |