CN105158324A - Method for rapidly identifying cells - Google Patents
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- CN105158324A CN105158324A CN201510497548.6A CN201510497548A CN105158324A CN 105158324 A CN105158324 A CN 105158324A CN 201510497548 A CN201510497548 A CN 201510497548A CN 105158324 A CN105158324 A CN 105158324A
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Abstract
The invention discloses a method for rapidly identifying cells. The method comprises steps as follows: an inorganic and organic fingerprint spectrum library of cells is established firstly; inorganic and organic fingerprint spectra of to-be-detected cells are collected and then matched with fingerprint mass spectrometric data of known cells in the inorganic and organic fingerprint spectrum library of cells, and whether the to-be-detected cells and the known cells are the same kind is judged, wherein the fingerprint mass spectrometric data refer to the molecular weight and the number of specific protein in an organic fingerprint spectrum and the content of various heavy metal elements in an inorganic fingerprint spectrum; the cells after simple preprocessing can be directly subjected to automatic detection by instruments, the detection time of two instruments for each cell is shorter than 5 min, the detection cost is lower, the detection speed is high, and the accuracy of cell identification is higher through cross validation of organic and inorganic mass spectra and is up to 90%.
Description
Technical field
The present invention relates to a kind of method of identification of cell, specifically, relate to and a kind ofly set up inorganic, the organic fingerprint spectrum library of cell and utilize the method for the quick identification of cell of inorganic, organic fingerprint spectrum library, belong to inspection technology field.
Background technology
The method of current identification of cell mainly contains morphology discriminating, cell surface marker discriminating, cell factor discriminating, gene discriminating, albumen discriminating etc.Wherein Morphological Identification cell is a kind of the most basic method, this method is by the difference in the tool observes cellular morphologies such as microscope, fast more convenient, but, the factor such as cell growth characteristics and growing environment affects the change of cellular morphology, therefore, only lean on Morphological Identification target cell not to be a kind of authentication method reliably, need to identify Cell differentials accurately and efficiently in conjunction with other multiple methods.
The discriminating of cell surface marker, cell factor, gene and albumen discriminating etc. need to carry out Immunological Identification, and these detection methods waste time and energy; And only rely on and be difficult to accurately identify cell by means of a kind of method, accuracy is low; Need comprehensive many-sided testing result just can confirm, cause testing cost higher.
The authentication method of above cell all belongs to biological means, does not also utilize physical means to carry out the report of Rapid identification cell at present.
Summary of the invention
The technical problem to be solved in the present invention is for the above deficiency that exists and defect, provides a kind of method of quick identification of cell, adopts the method, have the advantage that testing cost is low, detection time is short, accuracy is high.
For solving above technical matters, the present invention by the following technical solutions: a kind of method of quick identification of cell, is characterized in that, described method comprises first sets up inorganic, the organic fingerprint spectrum library of cell; Gather inorganic, organic dactylogram of cell to be measured, then in inorganic with cell, organic fingerprint spectrum library, the fingerprint mass spectrometric data of known cell is mated, and determines whether one species cell.
A kind of prioritization scheme, described fingerprint mass spectrometric data refers to the content of each Heavy Metallic Elements in the molecular weight of characteristic protein in organic dactylogram, the quantity of characteristic protein and inorganic dactylogram.
Further, when the fingerprint mass spectrometric data similarity of known cell reaches more than 90% in inorganic, organic dactylogram and inorganic, the organic fingerprint spectrum library of cell of cell to be measured, one species cell can be judged as.
Further, set up inorganic, the organic fingerprint spectrum library of cell to comprise:
Collecting cell step, pre-treatment step, collection ICP-MS data set up inorganic dactylogram step, gather MALDI-TOF-MS data set up organic dactylogram step.
Further, collecting cell sample step comprises:
Cell cultivates 24h with 37 DEG C in incubator, collects 2 × 10
6the cell of the order of magnitude, 1600rpm is centrifugal 5min at 5 DEG C, abandoning supernatant, often kind of cell all parallelly cultivates 6 times.
Further, pre-treatment step comprises:
A) ICP-MS sample pretreatment: 50% cell pure water of collected by centrifugation is washed three times, shift in teflon counteracting tank, the 0.1mL hydrogen peroxide of the 0.5mL nitric acid and mass percent concentration 30% that add mass percent concentration 65% is placed and is spent the night;
Counteracting tank is put into microwave dissolver and is cleared up, and clears up complete uncapping, and shakes up for subsequent use.
B) MALDI-TOF-MS sample pretreatment: another 50% cell of collected by centrifugation is directly added 100 μ L cinnamic acids (CHCA), sinapic acid (SA) or 2 that concentration is 10mg/mL, in one in 5-dihydroxy-benzoic acid (DHB) matrix solution, cell Ultrasonic Cell Disruptor pulverizes 1-2 minute, centrifugal at 1600rpm, abandoning supernatant, above step is repeated once, then drips on target plate after adding 100 μ L matrix solutions dissolvings.
Further, gather ICP-MS data to comprise:
The condition of work of ICP-MS: power: 1550W; Sampling depth: 8mm; Flow rate of carrier gas: 1.07L/min; Quantitative test pattern, it is 3 that the collection of unit mass number is counted, and data acquisition heavy burden number of times is 3 times, and integral time, As was 1sec; Se, Cd are 2sec; Other elements are 0.3sec; Mark in online: mark in Li6, Sc, Ge, Y, In, Tb, Bi=1.0mg/L mixture of multi-elements; Wherein Ge (72) marks as in each element of mass number 23-82; In (115) marks as in each element of mass number 114-137; Bi (209) marks as in each element of mass number 202-238;
Set up inorganic dactylogram to comprise: processed data by ICP-MS working software, often kind of cell continuous sample introduction investigates its precision 6 times, the parallel preparation of same cell is also gone up machine for 6 times and is investigated its reappearance, and at different cell chulture time 0h, 1h, 3h, 5h, 19h carries out detecting the stability investigating result, finally collects each Heavy Metallic Elements concentration data in cell to be measured and draw fingerprint chromatogram after normalized; Using statistics software carries out significant difference analysis to different cell.
Further, gather MALDI-TOF-MS data to comprise:
The condition of work of MALDI-TOF-MS: nitrogen lasers light source, optical maser wavelength 377nm, linear positive ion operator scheme, accelerating potential 20kv, setting mass range 4800-30000m/z, Laser emission voltage 2500mv, laser frequency 50Hz;
Set up organic dactylogram to comprise: with MALDI-TOF-MS cell carried out to the full scan within the scope of molecular weight 0-50000Da, often kind of cell continuous sample introduction investigates its precision 6 times, the parallel preparation of same cell is also gone up machine for 6 times and is investigated its reappearance, and at different cell chulture time 0h, 1h, 3h, 5h, 19h carry out detecting the stability investigating result;
Application ProteinChip and BiomarkerWizard software is analyzed the organic finger-print of cell, marks the total protein peak of modeling cell;
Often kind of each total protein peak molecular weight of bacterium is averaged, standard deviation and the coefficient of variation, the total protein peak molecular weight coefficient of variation≤0.5%, take similarity as index, adopt average link-chain method between class, utilize Euclidean distance square measuring technique, the data obtained is inputted spss software and carry out cluster analysis.
Further, unknown cell is through collecting cell step, pre-treatment step, collection ICP-MS data set up inorganic dactylogram step, gather MALDI-TOF-MS data and obtain its inorganic dactylogram and organic dactylogram after setting up organic dactylogram step, import inorganic and organic fingerprint spectrum library accordingly respectively, the cluster analysis result in organic spectrum storehouse and the difference analysis result in inorganic spectrum storehouse can be obtained.
Further, the method for any one of claim 5-8 is comprised.
After the present invention adopts above technical scheme, compared with prior art, have the following advantages: just can directly go up machine after cell is carried out simple pre-service and realize Aulomatizeted Detect, two kinds of instruments are less than 5 minutes for the detection time of each cell, testing cost is lower, and detection speed is fast.By organic and inorganic mass spectrographic cross validation, there is higher accuracy to Identification cell lines, reach 90%; To same part sample preparation liquid continuous sample introduction 6 times; 5 parts are prepared to same sample and carries out detection contrast; Prepared by liquid successively at 0h to same sample, 4h, 8h, 12h, 24h, 48h sample introduction 6 times, the data importing software for calculation then obtained take similarity as index, adopt average link-chain method between class, utilize Euclidean distance square measuring technique, carry out cluster analysis by spss statistical software, there is good precision, reappearance and stability.
Below in conjunction with drawings and Examples, the present invention is described in detail.
Accompanying drawing explanation
Accompanying drawing 1 is the inorganic elements Fingerprint profiles of several cell in the embodiment of the present invention;
Accompanying drawing 2 be in the embodiment of the present invention a kind of cell at organic fingerprint chromatogram of different time;
Accompanying drawing 3 is the cluster analysis results in organic spectrum storehouse in the embodiment of the present invention;
Accompanying drawing 4 is the difference analysis results in inorganic spectrum storehouse in the embodiment of the present invention.
Embodiment
Be described the method setting up inorganic, the organic fingerprint spectrum library of cell below for human breast cancer cell Hela, the method is a kind of universal method, is not limited to Hela cell; For the ease of understanding the present invention, and the claim do not limited the present invention in any way and core content.
Embodiment, a kind of method of quick identification of cell, comprises and first sets up inorganic, the organic fingerprint spectrum library of cell; Gather inorganic, organic dactylogram of cell to be measured, then in inorganic with cell, organic fingerprint spectrum library, the fingerprint mass spectrometric data of known cell is mated, and determines whether one species cell.
Wherein, fingerprint mass spectrometric data refers to the content of each Heavy Metallic Elements in the molecular weight of characteristic protein in organic dactylogram, the quantity of characteristic protein and inorganic dactylogram.
When the similarity of both fingerprint mass spectrometric datas and cell fingerprint mass spectrometric data reaches more than 85%, one species cell can be judged as.
Specifically, set up inorganic, the organic fingerprint spectrum library of cell to comprise: collecting cell step, pre-treatment step, collection ICP-MS data set up inorganic dactylogram step, gather MALDI-TOF-MS data set up organic dactylogram step.
Collecting cell sample step:
Human breast cancer cell Hela cultivates 24h with 37 DEG C in incubator, collects 2 × 10
6the cell of the order of magnitude, 1600rpm is centrifugal 5min at 5 DEG C, abandoning supernatant, often kind of cell all parallelly cultivates 6 times.
Pre-treatment step:
A) ICP-MS sample pretreatment: 50% cell pure water of collected by centrifugation is washed three times, shift in teflon counteracting tank, the 0.1mL hydrogen peroxide of the 0.5mL nitric acid and mass percent concentration 30% that add mass percent concentration 65% is placed and is spent the night;
Counteracting tank is put into microwave dissolver and is cleared up, and clears up complete uncapping, and shakes up for subsequent use.
B) MALDI-TOF-MS sample pretreatment: another 50% cell of collected by centrifugation is directly added 100 μ L cinnamic acids (CHCA), sinapic acid (SA) or 2 that concentration is 10mg/mL, in one in 5-dihydroxy-benzoic acid (DHB) matrix solution, cell Ultrasonic Cell Disruptor pulverizes 1-2 minute, centrifugal at 1600rpm, abandoning supernatant, above step is repeated once, then drips on target plate after adding 100 μ L matrix solutions dissolvings.
Gather ICP-MS data:
The condition of work of ICP-MS: power: 1550W; Sampling depth: 8mm; Flow rate of carrier gas: 1.07L/min; Quantitative test pattern, it is 3 that the collection of unit mass number is counted, and data acquisition heavy burden number of times is 3 times, and integral time, As was 1sec; Se, Cd are 2sec; Other elements are 0.3sec; Mark in online: mark in Li6, Sc, Ge, Y, In, Tb, Bi=1.0mg/L mixture of multi-elements; Wherein Ge (72) marks as in each element of mass number 23-82; In (115) marks as in each element of mass number 114-137; Bi (209) marks as in each element of mass number 202-238.
As shown in Figure 1, set up inorganic dactylogram: by ICP-MS working software, data are processed, often kind of cell continuous sample introduction investigates its precision 6 times, the parallel preparation of same cell is also gone up machine for 6 times and is investigated its reappearance, and at different cell chulture time 0h, 1h, 3h, 5h, 19h carry out detecting the stability investigating result, finally collect each Heavy Metallic Elements concentration data in cell to be measured and draw fingerprint chromatogram after normalized; Using statistics software carries out significant difference analysis to different cell.
Gather MALDI-TOF-MS data:
The condition of work of MALDI-TOF-MS: nitrogen lasers light source, optical maser wavelength 377nm, linear positive ion operator scheme, accelerating potential 20kv, setting mass range 4800-30000m/z, Laser emission voltage 2500mv, laser frequency 50Hz.
As shown in Figure 2, set up organic dactylogram: with MALDI-TOF-MS cell carried out to the full scan within the scope of molecular weight 0-50000Da, often kind of cell continuous sample introduction investigates its precision 6 times, the parallel preparation of same cell is also gone up machine for 6 times and is investigated its reappearance, and at different cell chulture time 0h, 1h, 3h, 5h, 19h carry out detecting the stability investigating result.Application ProteinChip and BiomarkerWizard software is analyzed the organic finger-print of cell, marks the total protein peak of modeling cell.Often kind of each total protein peak molecular weight of bacterium is averaged, standard deviation and the coefficient of variation, the total protein peak molecular weight coefficient of variation≤0.5%, take similarity as index, adopt average link-chain method between class, utilize Euclidean distance square measuring technique, the data obtained is inputted spss software and carry out cluster analysis.
Inorganic, the organic fingerprint spectrum library of cell of complete set is set up according to the inorganic dactylogram of gained and organic dactylogram.
Adopt inorganic fingerprint base and organic fingerprint base intersection identification of cell kind:
As shown in Figure 3, Figure 4, unknown cell is through collecting cell step, pre-treatment step, collection ICP-MS data set up inorganic dactylogram step, gather MALDI-TOF-MS data and obtain its inorganic dactylogram and organic dactylogram after setting up organic dactylogram step, import inorganic and organic fingerprint spectrum library accordingly respectively, the cluster analysis result in organic spectrum storehouse and the difference analysis result in inorganic spectrum storehouse can be obtained, by the cross validation of two kinds of analysis results, qualification coincidence rate is greater than 90%.
Above method obtains qualification result, through the checking of gene discrimination method, in the present invention when the similarity of both fingerprint mass spectrometric datas and cell fingerprint mass spectrometric data reaches more than 90%, can be judged as one species cell; Conclusion is accurately believable.
Authentication method of the present invention be by cell after carrying out simple pre-service just can directly on machine realize Aulomatizeted Detect, two kinds of instruments are less than 5 minutes for the detection time of each cell, and testing cost is lower, and detection speed is fast.By organic and inorganic mass spectrographic cross validation, there is higher accuracy to Identification cell lines, reach 90%; To same part sample preparation liquid continuous sample introduction 6 times; 5 parts are prepared to same sample and carries out detection contrast; Prepared by liquid successively at 0h to same sample, 4h, 8h, 12h, 24h, 48h sample introduction 6 times, the data importing software for calculation then obtained take similarity as index, adopt average link-chain method between class, utilize Euclidean distance square measuring technique, carry out cluster analysis by spss statistical software, there is good precision, reappearance and stability.
Above-mentioned embodiment is exemplary; to enable those skilled in the art better understand content of the present invention; should not be understood as limiting the scope of the invention, as long as the improvement done according to technical solution of the present invention, all fall into protection scope of the present invention.
Claims (10)
1. a method for quick identification of cell, is characterized in that, described method comprises first sets up inorganic, the organic fingerprint spectrum library of cell; Gather inorganic, organic dactylogram of cell to be measured, then in inorganic with cell, organic fingerprint spectrum library, the fingerprint mass spectrometric data of known cell is mated, and determines whether one species cell.
2. the method for a kind of quick identification of cell as claimed in claim 1, is characterized in that, described fingerprint mass spectrometric data refers to the content of each Heavy Metallic Elements in the molecular weight of characteristic protein in organic dactylogram, the quantity of characteristic protein and inorganic dactylogram.
3. the method for a kind of quick identification of cell as claimed in claim 1, it is characterized in that, when the fingerprint mass spectrometric data similarity of known cell reaches more than 85% in inorganic, organic dactylogram and inorganic, the organic fingerprint spectrum library of cell of cell to be measured, one species cell can be judged as.
4. the method for a kind of quick identification of cell as claimed in claim 1, is characterized in that, sets up inorganic, the organic fingerprint spectrum library of cell and comprises:
Collecting cell step, pre-treatment step, collection ICP-MS data set up inorganic dactylogram step, gather MALDI-TOF-MS data set up organic dactylogram step.
5. the method for a kind of quick identification of cell as claimed in claim 1, is characterized in that, collecting cell sample step comprises:
Cell cultivates 24h with 37 DEG C in incubator, collects 2 × 10
6the cell of the order of magnitude, 1600rpm is centrifugal 5min at 5 DEG C, abandoning supernatant, often kind of cell all parallelly cultivates 6 times.
6. the method for a kind of quick identification of cell as claimed in claim 1, it is characterized in that, pre-treatment step comprises:
A) ICP-MS sample pretreatment: 50% cell pure water of collected by centrifugation is washed three times, shift in teflon counteracting tank, the 0.1mL hydrogen peroxide of the 0.5mL nitric acid and mass percent concentration 30% that add mass percent concentration 65% is placed and is spent the night;
Counteracting tank is put into microwave dissolver and is cleared up, and clears up complete uncapping, and shakes up for subsequent use;
B) MALDI-TOF-MS sample pretreatment: another 50% cell of collected by centrifugation is directly added 100 μ L cinnamic acids (CHCA), sinapic acid (SA) or 2 that concentration is 10mg/mL, in one in 5-dihydroxy-benzoic acid (DHB) matrix solution, cell Ultrasonic Cell Disruptor pulverizes 1-2 minute, centrifugal at 1600rpm, abandoning supernatant, above step is repeated once, then drips on target plate after adding 100 μ L matrix solutions dissolvings.
7. the method for a kind of quick identification of cell as claimed in claim 1, is characterized in that, gathers ICP-MS data and comprises:
The condition of work of ICP-MS: power: 1550W; Sampling depth: 8mm; Flow rate of carrier gas: 1.07L/min; Quantitative test pattern, it is 3 that the collection of unit mass number is counted, and data acquisition heavy burden number of times is 3 times, and integral time, As was 1sec; Se, Cd are 2sec; Other elements are 0.3sec; Mark in online: mark in Li6, Sc, Ge, Y, In, Tb, Bi=1.0mg/L mixture of multi-elements; Wherein Ge (72) marks as in each element of mass number 23-82; In (115) marks as in each element of mass number 114-137; Bi (209) marks as in each element of mass number 202-238;
Set up inorganic dactylogram to comprise: processed data by ICP-MS working software, often kind of cell continuous sample introduction investigates its precision 6 times, the parallel preparation of same cell is also gone up machine for 6 times and is investigated its reappearance, and at different cell chulture time 0h, 1h, 3h, 5h, 19h carries out detecting the stability investigating result, finally collects each Heavy Metallic Elements concentration data in cell to be measured and draw fingerprint chromatogram after normalized; Using statistics software carries out significant difference analysis to different cell.
8. the method for a kind of quick identification of cell as claimed in claim 1, is characterized in that, gathers MALDI-TOF-MS data and comprises:
The condition of work of MALDI-TOF-MS: nitrogen lasers light source, optical maser wavelength 377nm, linear positive ion operator scheme, accelerating potential 20kv, setting mass range 4800-30000m/z, Laser emission voltage 2500mv, laser frequency 50Hz;
Set up organic dactylogram to comprise: with MALDI-TOF-MS cell carried out to the full scan within the scope of molecular weight 0-50000Da, often kind of cell continuous sample introduction investigates its precision 6 times, the parallel preparation of same cell is also gone up machine for 6 times and is investigated its reappearance, and at different cell chulture time 0h, 1h, 3h, 5h, 19h carry out detecting the stability investigating result;
Application ProteinChip and BiomarkerWizard software is analyzed the organic finger-print of cell, marks the total protein peak of modeling cell;
Often kind of each total protein peak molecular weight of bacterium is averaged, standard deviation and the coefficient of variation, the total protein peak molecular weight coefficient of variation≤0.5%, take similarity as index, adopt average link-chain method between class, utilize Euclidean distance square measuring technique, the data obtained is inputted spss software and carry out cluster analysis.
9. the method for a kind of quick identification of cell as claimed in claim 1, it is characterized in that, unknown cell is through collecting cell step, pre-treatment step, collection ICP-MS data set up inorganic dactylogram step, gather MALDI-TOF-MS data and obtain its inorganic dactylogram and organic dactylogram after setting up organic dactylogram step, import inorganic and organic fingerprint spectrum library accordingly respectively, the cluster analysis result in organic spectrum storehouse and the difference analysis result in inorganic spectrum storehouse can be obtained.
10. the method for a kind of quick identification of cell as claimed in claim 9, is characterized in that, comprise the method for any one of claim 5-8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105628782A (en) * | 2015-12-30 | 2016-06-01 | 聚光科技(杭州)股份有限公司 | ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) analysis method |
| CN110501414A (en) * | 2019-08-24 | 2019-11-26 | 中国人民解放军陆军特色医学中心 | A kind of identification model, construction method and the application of VIM type and SPM type metalloenzyme pseudomonas aeruginosa |
-
2015
- 2015-08-14 CN CN201510497548.6A patent/CN105158324A/en active Pending
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105628782A (en) * | 2015-12-30 | 2016-06-01 | 聚光科技(杭州)股份有限公司 | ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) analysis method |
| CN110501414A (en) * | 2019-08-24 | 2019-11-26 | 中国人民解放军陆军特色医学中心 | A kind of identification model, construction method and the application of VIM type and SPM type metalloenzyme pseudomonas aeruginosa |
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