CN107741417A - A kind of method of quick detection cell biological processes in situ - Google Patents

A kind of method of quick detection cell biological processes in situ Download PDF

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Publication number
CN107741417A
CN107741417A CN201710898955.7A CN201710898955A CN107741417A CN 107741417 A CN107741417 A CN 107741417A CN 201710898955 A CN201710898955 A CN 201710898955A CN 107741417 A CN107741417 A CN 107741417A
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cell
deuterium
deuterated
raman
raman spectrum
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CN107741417B (en
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宋之
宋一之
徐嘉宝
王允
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Shanghai deuterium peak Medical Technology Co.,Ltd.
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SHANGHAI HESEN BIOTECHNOLOGY Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

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Abstract

The present invention relates to a kind of method of quick detection cell biological processes in situ, based on competent cell to material absorbing containing deuterium, conversion, metabolism or storage after, can be in the different phase of cell biological processes, the labeled in situ of deuterium is realized in cell, cell is detected by Raman spectrum, whether there is the presence of deuterium in identification cell, and then obtain the Biochemical processes on cell, cell quality or function relevant information.The inventive method can be used for biology and the medical domains such as the drug susceptibility test, environmental microorganism metabolism, human body cell detection of bacterium infection.Compared with prior art, the inventive method has detection cell for the activity of most of important biomolecule molecules and the potentiality of metabolism, and it allows research to use more complicated deuterated material, including natural products, medicine and the deuterate mixture containing a variety of chemical substances.Functional study of the inventive method to cell under the natural conditions of individual cell level is very valuable.

Description

A kind of method of quick detection cell biological processes in situ
Technical field
The invention belongs to the detection technique of biotechnology, is related to a kind of method of quick detection cell biological processes in situ, It is specifically related to utilize the mark cell biological processes of substrate selective containing deuterium, and the state by unicellular Raman collection of illustrative plates to mark Analyzed, so as to identify the method for the characters such as cell metabolism, synthesis or function and its be led in biosynthesis and medical test etc. Application in domain.
Background technology
The research of Cytobiology and molecular biology has promoted our understanding to life entity on the earth.Pass through modified biological molecule Available for monitoring cellular change or function in natural environment.With the green fluorescent protein for obtaining Nobel chemistry Prize in 2008 (GFP) discovery for representative brings the revolution in chemical biology field.However, many small biomolecule and metabolites are such as Nucleic acid, glycan and lipid etc. can not be marked with the reporter of this genetic coding.Passed through selectivity although in the past several years Modify species in itself, let us generates new opinion to cell processes, but these technologies are generally relatively slow, anti-to biochemistry Poor selectivity is answered, it is with high costs and complicated.
The key of metabolic function detection is quick and special reaction on the premise of physiological condition is maintained.It is same using stabilization Position element marks specific objective microbe, and by 16S rRNA-SIP methods and NanoSIMS technologies, can be to specific The microorganism of function and character is identified and analyzed.It is right but the isotope that 16S rRNA-SIP can only combine 13- carbon uses 15- nitrogen etc. is helpless, limits its use, and its sensitivity is relatively low, cell can only be analyzed in population rank. NanoSIMS sensitivities are high, but cell is the same by irreversible destruction with 16S rRNA-SIP technologies in research process, And experiment and the data analysis operation of its high equipment price and complexity, it also limit its use.
Deuterium is the stable isotope of hydrogen, deuterated not influence atomic size, molecular shape, chemical bond lengths or rigidity (Franco,M.I.,et al.Molecular vibration-sensing component in Drosophila melanogaster olfaction.Proc.Natl.Acad.Sci.U.S.A.108,3797-3802(2011)).Deuterium-oxide Raman Technology can detect active microorganism (Berry, D., et with very high sensitivity and selectivity on individual cell level al.Tracking heavy water(D2O)incorporation for identifying and sorting active microbial cells.Proc.Natl.Acad.Sci.U.S.A.112,E194-E203(2015)).Cell is containing deuterium-oxide Culture medium in cultivate after, the Raman spectrum of active microorganism is in 2170-2300cm-1Between produce C-D letter Number peak, because, in the usually not any signal in this region, therefore this is unique not by the Raman spectrum of deuterium-labeled cell C-D signals can be used for the identification active microorganism of high accuracy and sensitivity.But up to this point, by adding deuterium-oxide Active cell is marked, there is following problem：1) just for microbial cell, without being adapted to eukaryotic cells Method；2) just for separated culture classical microorganism pure culture bacterial strain, not for unknown, not yet trained Foster or real mixed bacterial；3) training of its culture medium all artificially to prepare during microbial cell is marked by deuterium-oxide Support base, it is impossible to the real living environment of reacting cells.
Therefore, develop a set of deuterium-labeled single celled Raman detection method in original position that is based on and be applied to biology, chemistry and medical science Field, there are good prospect and value.
The content of the invention
The purpose of the present invention is just to provide for a kind of method of quick detection cell biological processes in situ, side of the invention Method is based on deuterium isotope marks, and the quick detection in situ of cell biological processes is realized using Raman detection technology.
The purpose of the present invention can be achieved through the following technical solutions：
The present invention provides a kind of method of quick detection cell biological processes in situ, competent cell to material absorbing containing deuterium, After conversion, metabolism or storage, the labeled in situ of deuterium can be realized in cell in the different phase of cell biological processes, led to Cross Raman spectrum to detect cell, identify in cell whether there is the presence of deuterium, and then obtain the biochemistry on cell Process, cell quality or function relevant information.
The method of the present invention can be used for Biochemical processes, cell quality or the function for understanding cell.
Heretofore described material containing deuterium, for the material containing deuterium element, include but is not limited to：Deuterium-oxide, deuterated nucleic acid, deuterium For amino acid, deuterated fatty, deuterated aliphatic acid, deuterated medicine, deuterated sugared, deuterated protein, or other deuterated organic metabolism bottoms Thing, or by one or more of deuterated mixtures formed etc. in above deuterated compound.
Further, the deuterated carbon source includes deuterated glucose or deuterated naphthalene.
The natural abundance of deuterium in the seawater is 0.0156%, when implementing of the invention, the abundance of deuterium in the material containing deuterium Between 5%-100%, for marking the cell of specific trait or function, and Raman can be passed through under the guidance of the present invention The Raman spectrum detection device such as spectrometer is identified and detected.
When implementing of the invention, the material containing deuterium can be by buying (such as the Reagent Company such as Sigma) or allowing not Substrate containing deuterium is in deuterium-oxide (D2O the displacement reaction of hydrogen/deuterium occurs in) and is readily available.
(1) present invention provides a kind of method of quick detection cell biological processes in situ.
Under prokaryotes or Eukaryotic normal habitat, deuterium-oxide is mixed, deuterium constituent content is reached 5%- 60%, deuterium can form C-D keys during NADH or NADPH transmits electronics with the carbon in cell.It is in view of only active This reaction can just occur for cell, therefore, identification and screening to the cell with metabolic activity can be realized by the technology.
When material containing deuterium is deuterated carbohydrate or other organic metabolism substrates, it is possible to realize cell other Identification on bioprocess.For example, realize specific substrates metabolic marker with deuterated compound.
(2) present invention enters including a kind of can realize to unknown or uncultured microorganisms characters and/or function Row knows method for distinguishing.
After water sampling containing microorganism, in the case where not carrying out any preceding processing to sample, by water sample and deuterium-oxide Mixing, makes D2O/H2O ratios are 1:9 to 5:5, mixed sample shakes 6 to 72 hours.Afterwards by filtering, UF membrane, from The heart, magnetic material coating etc. mode the microorganism in sample is separated with impurity, the microbial cell after separation be stored in from In sub- water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, gathered by Raman spectrometer unicellular Raman collection of illustrative plates.In 2000-2300cm in its Raman spectrum of active microorganism in sample-1Between there is characteristic peak.Tool There is the cell at the peak, cell active under this condition can be identified as.
By calculating ratio of the cell with this feature peak in all detected cells, can calculate in sample has Ratio shared by the cell of activity.
(3) present invention, which includes one kind, microorganism to be resisted in natural environment is identified under conditions of not changing in situ environment The method of the resistance of raw element.
The a large amount of appearance and propagation of antibiotic resistant bacteria (ARB) cause huge risk to environmental and public health, Increasing bacterium and antibiotics resistance gene (ARG) with antibiotic resistance are found in medical care precess and environment. After human body is by resistant bacterium infection, if the resistance or sensitivity of the bacterial antibiotic can not be judged in a short time Spectrum, the treatment time of preciousness can be lost, can further promote bacterium to evolve using broader spectrum of antibiotic to be emergent, to more More antibiotic produce resistance.But at present, the bacterial resistance based on culture detects, it is necessary to which 48-72 hours could divide bacterium Class and its resistance make more accurate judgement.May although predicting bacterium from gene aspect based on the detection of resistant gene The resistant properties having, but do not expressed for resistant gene or resistance that new unknown resistant gene is brought then can not be accurate Really judge.
Present invention utilizes the Raman detection of deuterium stable isotope (Raman-DIP) technology, is not changing microorganism growth Under conditions of the chemical composition of environment, the quick detection in situ of bacterial resistance is realized.This method can be used for micro- in environmental sample Biotic resistance detects.
A kind of achievable embodiment is：After water sampling containing microorganism, any preceding place is not being carried out to sample In the case of reason, water sample is mixed with deuterium-oxide, makes D2O/H2O ratios are 1:9 to 5:5, artificially add in the sample certain density The mixture of single antibiotic or two kinds and two or more antibiotic, mixed sample shake 6 to 72 hours, passed through afterwards The modes such as filter, UF membrane, centrifugation, magnetic material coating separate the microorganism in sample with impurity, and the microorganism after separation is thin Born of the same parents preserve in deionized water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, pass through Raman spectrum Instrument gathers single celled Raman collection of illustrative plates.Have in sample for try in the microorganism of antibiotic resistance its Raman spectrum 2000-2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as with antibiotic resisting under this condition The cell of property.By calculating ratio of the cell with this feature peak in all detected cells, it can calculate in sample and have There is the ratio shared by the cell of antibiotic resistance.
(4) present invention, which includes one kind, (including to be not limited to blood, urine, excrement to human body fluid or other secretion Just, saliva, sputum, seminal fluid, vaginal fluid etc.) in microorganism carry out what is quickly judged and identify to the resistance of antibiotic Method.
Present invention utilizes the Raman detection of deuterium stable isotope (Raman-DIP) technology, realize blood of human body or its The quick detection in situ of bacterial resistance in his humoral sample.
After human body fluid containing microorganism or secretion collection, add in detection liquid.Detection liquid composition selects as needed Select, main to include human body fluid blood or secretion, 10-60% deuterium-oxide, nutrient, certain density single antibiotic or Two kinds and the mixture of two or more antibiotic, mixed sample shake 1 to 24 hours.Afterwards by filtering, UF membrane, from The modes such as the heart, magnetic material coating separate the microorganism in sample with other cells and impurity, the microbial cell after separation Preserve in deionized water.Take the microorganism after the separation of certain volume to be positioned on the chip for raman spectroscopy measurement, pass through Raman spectrometer gathers single celled Raman collection of illustrative plates.Have in sample for trying the microorganism of antibiotic resistance its Raman spectrum In in 2000-2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as that there is antibiosis under this condition The cell of plain resistance.Under the conditions of different antibiotic, the appearance situation of C-D characteristic peaks in sample, it can quickly select most to have Imitate the antibiotic of restraining and sterilizing bacteria.
(5) present invention can be quickly judged the cell in eukaryotic cells with specific trait or function including a kind of With knowledge method for distinguishing.
Deuterium-oxide is added in the nutrient solution of eukaryotic cells, the timing of culture one is carried out under condition of culture to be studied Between, the cell in sample is separated with impurity by modes such as filtering, UF membrane, centrifugation, magnetic material coatings, it is thin after separation Born of the same parents preserve in deionized water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, pass through Raman spectrum Instrument gathers single celled Raman collection of illustrative plates.Have in sample in its Raman spectrum of the eukaryotic of metabolic activity in 2000-2300cm-1 Between there is characteristic peak.Cell with the peak, the cell under this condition with metabolic activity can be identified as.
(6) present invention can identify method of the microorganism to the metabolic pathway of specific substrates including a kind of.(including it is deuterated mixed Close substrate)
Deuterated carbohydrate or other deuterated carbon sources are added to the culture of single microorganism or multiple-microorganism Base, by being metabolized or using these carbon sources and being integrated into cell, C-D keys are formed in cell Raman collection of illustrative plates.Pass through filtering, film point From, centrifugation, magnetic material coating etc. mode the microorganism in sample is separated with impurity, the microbial cell after separation is stored in In deionized water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, gathered by Raman spectrometer single The Raman collection of illustrative plates of cell.Have in sample in its Raman spectrum of the microbial single-cell of the metabolism carbon source function in 2000- 2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as that there is carbon metabolism function under this condition Cell.
(7) present invention includes a kind of method for the synthesis that can detect DNA/RNA.
Deuterated nucleic acid is added to the culture medium of microorganism, certain time is cultivated under the common condition of culture of the microorganism Afterwards, C-D keys will can be formed by the use of nucleic acid molecules as substrate and synthetic DNA/RNA cell.By filtering, UF membrane, from The heart, magnetic material coating etc. mode the microorganism in sample is separated with impurity, the microbial cell after separation be stored in from In sub- water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, gathered by Raman spectrometer unicellular Raman collection of illustrative plates.Its Raman spectrum is in 2000-2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as Nucleic acid synthetic DNA/RNA cell more can be efficiently utilized under this condition.
(8) present invention includes a kind of method that can detect protein synthesis.
A certain or several specific deuterated amino acid are added in the nutrient solution of cell, the amino acid can be utilized to close Into the cell of protein, C-D keys will be formed.By modes such as filtering, UF membrane, centrifugation, magnetic material coatings by sample Microorganism separates with impurity, and the microbial cell after separation preserves in deionized water.The cell of certain volume is taken to be positioned over confession On the chip of raman spectroscopy measurement, single celled Raman collection of illustrative plates is gathered by Raman spectrometer.Its Raman spectrum is in 2000- 2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as more efficiently utilizing ammonia under this condition The cell of base acid synthetic proteins；It can make cell that there is the amino acid at the peak, can be identified as being utilized by such cell high-efficient Amino acid.
(9) present invention can detect fat absorption, conversion and the method for storage including a kind of.
By the hydrogen in fat with after deuterated, add in the nutrient solution of cell, can absorb, convert and store the fat Cell, C-D keys will be formed.By filtering, UF membrane, centrifugation, magnetic material be coated with etc. mode by the microorganism in sample with it is miscellaneous Matter separates, and the microbial cell after separation preserves in deionized water.Take the cell of certain volume to be positioned over to survey for Raman spectrum On the chip of amount, single celled Raman collection of illustrative plates is gathered by Raman spectrometer.Its Raman spectrum is in 2000-2300cm-1Between go out Existing characteristic peak.The appearance at the peak, the process available for absorption, conversion and the storage for judging fat.
(10) present invention includes a kind of in-situ monitoring medicine in the method that intracellular transports and is metabolized.
Expose cells to deuterated medicine, and by single cell Raman spectrum monitor the time of occurrence at intracellular C-D peaks with And distribution of the C-D peaks in cell spaces, available for the metabolism and transport for judging medicine.
The present invention includes the presence at C-D peaks and the sxemiquantitative of intracellular deuterium elemental abundance in a kind of judgement cell Raman collection of illustrative plates Analysis method.
With the wavelength X at C-D peak maximumsdCentered on, setting range Ad, scope AdThe wavelength of boundary be X1And X2, Wherein X1<Xd<X2, and X2-X1>7nm, XdIt is generally in X1With X2Center.Setting range Bd, scope BdBoundary wavelength For X0And X1, wherein X1-X0=X2-X1(because Raman spectrum is discontinuous, therefore takes and can make value identical X as far as possible0Value). Setting range Cd, scope CdBoundary wavelength be X2And X3, wherein X3-X2=X2-X1.(because Raman spectrum is discontinuous, therefore take Value identical X as far as possible can be made3Value).Calculate C-D peak areas：
Sd=Sad-(Sbd+Scd)/2, wherein Sad, SbdAnd ScdRespectively scope Ad, BdAnd CdArea under interior Raman spectrum.
With the wavelength Y at C-H peak maximumshCentered on, setting range Ah, scope AhBorder be wavelength Y1And Y2, wherein Y1<Yh<Y2, and Y2-Y1>9nm, YhIt is generally in Y1With Y2Center.Setting range Bh, scope BhBorder be wavelength Y0And Y1, Wherein Y1-Y0=Y2-Y1.(because Raman spectrum is discontinuous, therefore takes and can make value identical Y as far as possible0Value).Setting range Ch, scope ChBoundary wavelength be Y2And Y3, wherein Y3-Y2=Y2-Y1.(because Raman spectrum is discontinuous, therefore takes and can make this It is worth identical Y as far as possible3Value).Calculate C-H peak areas：
Sh=Sah-(Sbh+Sch)/2, wherein Sah, SbhAnd SchRespectively scope Ah, BhAnd ChArea under interior Raman spectrum.
Calculate D abundance：
D%=Sd/(Sd+Sh)。
Can be with the calculating of sxemiquantitative and more intracellular deuterium abundance of elements by the abundance of the numerical value.
It can also be judged by the numerical value, whether with the presence of C-D peaks in Raman spectrogram.
Cell is exposed in the medium without deuterium under similarity condition, gathers multiple unicellular collection of illustrative plates, calculates D in cell The average value D of abundance0With standard deviation δ D0。
When cell is exposed in the medium containing deuterium, multiple unicellular collection of illustrative plates are gathered, calculate the abundance D of D in cell1.If D1≥ D0+3δD0, then it is assumed that obvious C-D peaks be present in the unicellular Raman collection of illustrative plates of cell.
Raman-DIP is in non-intruding cell, isotope sensitiveness (about 5% deuterated test limit) and individual cell level detection side Face has the advantages of being better than other Stable Isotopic Analysis technologies.Its lossless property is that the unicellular sorting in downstream and group credit analysis carry Possibility is supplied.Due to13C flag or15The substrate derivant of N marks is typically expensive, and wherein many not commercially available, Which has limited their applications in Raman study.This problem can be overcome by using less expensive deuterated substrate. There is Raman-DIP the cell for detecting most of important biomolecule molecules (including protein, lipid, carbohydrate and nucleic acid) to live Property and cell metabolism potentiality, and it allows research to use more complicated deuterated material, including natural products, medicine and containing more The deuterate mixture of kind chemical substance.Therefore, using these deuterium-labeled substrates Raman-DIP technologies individual cell level from Functional study under the conditions of so to cell is very valuable.
Brief description of the drawings
Classification of substances containing deuterium in Fig. 1 present invention；
The mechanism that deuterium in Fig. 2 deuterium-oxides is absorbed by active microorganism；
The deuterated substrates of Fig. 3 characterize specific metabolic pathway mechanism；
Influence of Fig. 4 difference deuterium content carbon sources to growth curve of bacteria；
Microorganism Raman spectrogram in river under the conditions of Fig. 5 difference antibiotic；
Microorganism D abundance in river under the conditions of Fig. 6 difference antibiotic；
Sputum bacterium Raman spectrogram under the conditions of Fig. 7 difference antibiotic；
Bacterium Raman collection of illustrative plates deuterium content under the different deuterated substrates of Fig. 8；
Fig. 9 THP-1 cell lines Raman collection of illustrative plates in deuterium-oxide culture；
Raman collection of illustrative plates of Figure 10 stem cells in deuterium-oxide culture.
Embodiment
The present invention provides a kind of method of quick detection cell biological processes in situ, competent cell to material absorbing containing deuterium, After conversion, metabolism or storage, the labeled in situ of deuterium can be realized in cell in the different phase of cell biological processes, led to Cross Raman spectrum to detect cell, identify in cell whether there is the presence of deuterium, and then obtain the biochemistry on cell Process, cell quality or function relevant information.
The method of the present invention can be used for Biochemical processes, cell quality or the function for understanding cell.
Heretofore described material containing deuterium, for the material containing deuterium element, include but is not limited to：Deuterium-oxide, deuterated nucleic acid, deuterium For amino acid, deuterated fatty, deuterated aliphatic acid, deuterated medicine, deuterated sugared, deuterated protein, or other deuterated organic metabolism bottoms Thing, or by one or more of deuterated mixture formed etc. in above deuterated compound, as shown in Figure 1.
Further, the deuterated carbon source includes deuterated glucose or deuterated naphthalene.
The natural abundance of deuterium in the seawater is 0.0156%, when implementing of the invention, the abundance of deuterium in the material containing deuterium Between 5%-100%, for marking the cell of specific trait or function, and Raman can be passed through under the guidance of the present invention The Raman spectrum detection device such as spectrometer is identified and detected.
When implementing of the invention, the material containing deuterium can be by buying (such as the Reagent Company such as Sigma) or allowing not Substrate containing deuterium is in deuterium-oxide (D2O the displacement reaction of hydrogen/deuterium occurs in) and is readily available.
(1) present invention provides a kind of method of quick detection cell biological processes in situ.
Under prokaryotes or Eukaryotic normal habitat, deuterium-oxide is mixed, deuterium constituent content is reached 5%- 60%, deuterium can form C-D keys during NADH or NADPH transmits electronics with the carbon in cell.It is in view of only active This reaction can just occur for cell, therefore, identification and screening to the cell with metabolic activity can be realized by the technology.
The mechanism that deuterium in deuterium-oxide is absorbed by active microorganism is as shown in Figure 2.
When material containing deuterium is deuterated carbohydrate or other organic metabolism substrates, it is possible to realize cell other Identification on bioprocess.For example, realize specific substrates metabolic marker with deuterated compound.
Fig. 3 is with deuterated glucose (glucose-d12) exemplified by, illustrate and realize specific substrates generation with deuterated compound Thank to the mechanism of mark.
(2) present invention enters including a kind of can realize to unknown or uncultured microorganisms characters and/or function Row knows method for distinguishing.
After water sampling containing microorganism, in the case where not carrying out any preceding processing to sample, by water sample and deuterium-oxide Mixing, makes D2O/H2O ratios are 1:9 to 5:5, mixed sample shakes 6 to 72 hours.Afterwards by filtering, UF membrane, from The heart, magnetic material coating etc. mode the microorganism in sample is separated with impurity, the microbial cell after separation be stored in from In sub- water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, gathered by Raman spectrometer unicellular Raman collection of illustrative plates.In 2000-2300cm in its Raman spectrum of active microorganism in sample-1Between there is characteristic peak.Tool There is the cell at the peak, cell active under this condition can be identified as.
By calculating ratio of the cell with this feature peak in all detected cells, can calculate in sample has Ratio shared by the cell of activity.
(3) present invention, which includes one kind, microorganism to be resisted in natural environment is identified under conditions of not changing in situ environment The method of the resistance of raw element.
The a large amount of appearance and propagation of antibiotic resistant bacteria (ARB) cause huge risk to environmental and public health, Increasing bacterium and antibiotics resistance gene (ARG) with antibiotic resistance are found in medical care precess and environment. After human body is by resistant bacterium infection, if the resistance or sensitivity of the bacterial antibiotic can not be judged in a short time Spectrum, the treatment time of preciousness can be lost, can further promote bacterium to evolve using broader spectrum of antibiotic to be emergent, to more More antibiotic produce resistance.But at present, the bacterial resistance based on culture detects, it is necessary to which 48-72 hours could divide bacterium Class and its resistance make more accurate judgement.May although predicting bacterium from gene aspect based on the detection of resistant gene The resistant properties having, but do not expressed for resistant gene or resistance that new unknown resistant gene is brought then can not be accurate Really judge.
Present invention utilizes the Raman detection of deuterium stable isotope (Raman-DIP) technology, is not changing microorganism growth Under conditions of the chemical composition of environment, the quick detection in situ of bacterial resistance is realized.This method can be used for micro- in environmental sample Biotic resistance detects.
A kind of achievable embodiment is：After water sampling containing microorganism, any preceding place is not being carried out to sample In the case of reason, water sample is mixed with deuterium-oxide, makes D2O/H2O ratios are 1:9 to 5:5, artificially add in the sample certain density The mixture of single antibiotic or two kinds and two or more antibiotic, mixed sample shake 6 to 72 hours, passed through afterwards The modes such as filter, UF membrane, centrifugation, magnetic material coating separate the microorganism in sample with impurity, and the microorganism after separation is thin Born of the same parents preserve in deionized water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, pass through Raman spectrum Instrument gathers single celled Raman collection of illustrative plates.Have in sample for try in the microorganism of antibiotic resistance its Raman spectrum 2000-2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as with antibiotic resisting under this condition The cell of property.By calculating ratio of the cell with this feature peak in all detected cells, it can calculate in sample and have There is the ratio shared by the cell of antibiotic resistance.
(4) present invention, which includes one kind, (including to be not limited to blood, urine, excrement to human body fluid or other secretion Just, saliva, sputum, seminal fluid, vaginal fluid etc.) in microorganism carry out what is quickly judged and identify to the resistance of antibiotic Method.
Present invention utilizes the Raman detection of deuterium stable isotope (Raman-DIP) technology, realize blood of human body or its The quick detection in situ of bacterial resistance in his humoral sample.
After human body fluid containing microorganism or secretion collection, add in detection liquid.Detection liquid composition selects as needed Select, main to include human body fluid blood or secretion, 10-60% deuterium-oxide, nutrient, certain density single antibiotic or Two kinds and the mixture of two or more antibiotic, mixed sample shake 1 to 24 hours.Afterwards by filtering, UF membrane, from The modes such as the heart, magnetic material coating separate the microorganism in sample with other cells and impurity, the microbial cell after separation Preserve in deionized water.Take the microorganism after the separation of certain volume to be positioned on the chip for raman spectroscopy measurement, pass through Raman spectrometer gathers single celled Raman collection of illustrative plates.Have in sample for trying the microorganism of antibiotic resistance its Raman spectrum In in 2000-2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as that there is antibiosis under this condition The cell of plain resistance.Under the conditions of different antibiotic, the appearance situation of C-D characteristic peaks in sample, it can quickly select most to have Imitate the antibiotic of restraining and sterilizing bacteria.
(5) present invention can be quickly judged the cell in eukaryotic cells with specific trait or function including a kind of With knowledge method for distinguishing.
Deuterium-oxide is added in the nutrient solution of eukaryotic cells, the timing of culture one is carried out under condition of culture to be studied Between, the cell in sample is separated with impurity by modes such as filtering, UF membrane, centrifugation, magnetic material coatings, it is thin after separation Born of the same parents preserve in deionized water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, pass through Raman spectrum Instrument gathers single celled Raman collection of illustrative plates.Have in sample in its Raman spectrum of the eukaryotic of metabolic activity in 2000-2300cm-1 Between there is characteristic peak.Cell with the peak, the cell under this condition with metabolic activity can be identified as.
(6) present invention can identify method of the microorganism to the metabolic pathway of specific substrates including a kind of.(including it is deuterated mixed Close substrate)
Deuterated carbohydrate or other deuterated carbon sources are added to the culture of single microorganism or multiple-microorganism Base, by being metabolized or using these carbon sources and being integrated into cell, C-D keys are formed in cell Raman collection of illustrative plates.Pass through filtering, film point From, centrifugation, magnetic material coating etc. mode the microorganism in sample is separated with impurity, the microbial cell after separation is stored in In deionized water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, gathered by Raman spectrometer single The Raman collection of illustrative plates of cell.Have in sample in its Raman spectrum of the microbial single-cell of the metabolism carbon source function in 2000- 2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as that there is carbon metabolism function under this condition Cell.
(7) present invention includes a kind of method for the synthesis that can detect DNA/RNA.
Deuterated nucleic acid is added to the culture medium of microorganism, certain time is cultivated under the common condition of culture of the microorganism Afterwards, C-D keys will can be formed by the use of nucleic acid molecules as substrate and synthetic DNA/RNA cell.By filtering, UF membrane, from The heart, magnetic material coating etc. mode the microorganism in sample is separated with impurity, the microbial cell after separation be stored in from In sub- water.Take the cell of certain volume to be positioned on the chip for raman spectroscopy measurement, gathered by Raman spectrometer unicellular Raman collection of illustrative plates.Its Raman spectrum is in 2000-2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as Nucleic acid synthetic DNA/RNA cell more can be efficiently utilized under this condition.
(8) present invention includes a kind of method that can detect protein synthesis.
A certain or several specific deuterated amino acid are added in the nutrient solution of cell, the amino acid can be utilized to close Into the cell of protein, C-D keys will be formed.By modes such as filtering, UF membrane, centrifugation, magnetic material coatings by sample Microorganism separates with impurity, and the microbial cell after separation preserves in deionized water.The cell of certain volume is taken to be positioned over confession On the chip of raman spectroscopy measurement, single celled Raman collection of illustrative plates is gathered by Raman spectrometer.Its Raman spectrum is in 2000- 2300cm-1Between there is characteristic peak.Cell with the peak, it can be identified as more efficiently utilizing ammonia under this condition The cell of base acid synthetic proteins；It can make cell that there is the amino acid at the peak, can be identified as being utilized by such cell high-efficient Amino acid.
(9) present invention can detect fat absorption, conversion and the method for storage including a kind of.
By the hydrogen in fat with after deuterated, add in the nutrient solution of cell, can absorb, convert and store the fat Cell, C-D keys will be formed.By filtering, UF membrane, centrifugation, magnetic material be coated with etc. mode by the microorganism in sample with it is miscellaneous Matter separates, and the microbial cell after separation preserves in deionized water.Take the cell of certain volume to be positioned over to survey for Raman spectrum On the chip of amount, single celled Raman collection of illustrative plates is gathered by Raman spectrometer.Its Raman spectrum is in 2000-2300cm-1Between go out Existing characteristic peak.The appearance at the peak, the process available for absorption, conversion and the storage for judging fat.
(10) present invention includes a kind of in-situ monitoring medicine in the method that intracellular transports and is metabolized.
Expose cells to deuterated medicine, and by single cell Raman spectrum monitor the time of occurrence at intracellular C-D peaks with And distribution of the C-D peaks in cell spaces, available for the metabolism and transport for judging medicine.
The present invention judges the presence at C-D peaks and the sxemiquantitative of intracellular deuterium elemental abundance in cell Raman collection of illustrative plates including two kinds Analysis method.
With the wavelength X at C-D peak maximumsdCentered on, setting range Ad, scope AdThe wavelength of boundary be X1And X2, Wherein X1<Xd<X2, and X2-X1>7nm, XdIt is generally in X1With X2Center.Setting range Bd, scope BdBoundary wavelength For X0And X1, wherein X1-X0=X2-X1(because Raman spectrum is discontinuous, therefore takes and can make value identical X as far as possible0Value). Setting range Cd, scope CdBoundary wavelength be X2And X3, wherein X3-X2=X2-X1.(because Raman spectrum is discontinuous, therefore take Value identical X as far as possible can be made3Value).Calculate C-D peak areas：
Sd=Sad-(Sbd+Scd)/2, wherein Sad, SbdAnd ScdRespectively scope Ad, BdAnd CdArea under interior Raman spectrum.
With the wavelength Y at C-H peak maximumshCentered on, setting range Ah, scope AhBorder be wavelength Y1And Y2, wherein Y1<Yh<Y2, and Y2-Y1>9nm, YhIt is generally in Y1With Y2Center.Setting range Bh, scope BhBorder be wavelength Y0And Y1, Wherein Y1-Y0=Y2-Y1.(because Raman spectrum is discontinuous, therefore takes and can make value identical Y as far as possible0Value).Setting range Ch, scope ChBoundary wavelength be Y2And Y3, wherein Y3-Y2=Y2-Y1.(because Raman spectrum is discontinuous, therefore takes and can make this It is worth identical Y as far as possible3Value).Calculate C-H peak areas：
Sh=Sah-(Sbh+Sch)/2, wherein Sah, SbhAnd SchRespectively scope Ah, BhAnd ChArea under interior Raman spectrum.
Calculate D abundance：
D%=Sd/(Sd+Sh)。
Can be with the calculating of sxemiquantitative and more intracellular deuterium abundance of elements by the abundance of the numerical value.
It can also be judged by the numerical value, whether with the presence of C-D peaks in Raman spectrogram.
Cell is exposed in the medium without deuterium under similarity condition, gathers multiple unicellular collection of illustrative plates, calculates D in cell The average value D of abundance0With standard deviation δ D0。
When cell is exposed in the medium containing deuterium, multiple unicellular collection of illustrative plates are gathered, calculate the abundance D of D in cell1.If D1≥ D0+3δD0, then it is assumed that obvious C-D peaks be present in the unicellular Raman collection of illustrative plates of cell.
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1：The influence that deuterated substrate grows to microorganism
The present embodiment checking cell cannot be distinguished by non-deuterate substrate and complete deuterated substrate in metabolic process.
Specific embodiment is as follows：
Choose escherichia coli DH5a, pseudomonas putida UWC1, (these strains are commercially available common to pseudomonas putida G7 Strain), the condition of culture of escherichia coli DH5a is 37 DEG C, and pseudomonas putida UWC1, pseudomonas putida G7 condition of culture are 30℃.The bacterium solution being incubated overnight in LB culture mediums, with 1:50 ratio is seeded to fresh MM culture mediums.Culture medium it is unique Carbon source is glucose or naphthalene.The ratio of deuterated glucose is respectively 0,5,10,25,50,75,100%, the ratio difference of deuterated naphthalene For 0,5,10,25,50,75,100%.200 μ L bacterium solution is pipetted to 96 orifice plates with liquid-transfering gun, the concussion and cultivate under the conditions of 30 DEG C. Bacterium solution using glucose as carbon source, detected per half an hour with multi-function microplate reader (Bio-Tek Synergy2) at its 600nm Absorbance, the bacterium solution using naphthalene as carbon source, measured its 600nm extinction at the 0th, 10,20,24,32,48,60 and 72 hour respectively Degree.
Fig. 4 is shown in influence of the different deuterium content carbon sources to growth curve of bacteria, and result of study confirms, pseudomonas putida UWC1 And G7 cannot be distinguished by deuterated glucose or naphthalene, and under conditions of the deuterated glucose of 0-100% and naphthalene growth curve without notable Sex differernce.There was no significant difference for growth curve under the deuterated glucose less than 75% for escherichia coli DH5a, in 100% deuterated Portugal Grow and be suppressed in the case of grape sugar.
The composition of MM culture mediums is shown in document Huang, W.E.et al.Chromosomally located gene with acquisition fusions constructed in Acinetobacter sp ADP1 for the detection of salicylate.Environ Microbiol 7:1339–1348(2015)。
Embodiment 2：The microorganism confrontation of non-pure culture unknown in river is being identified under conditions of not changing in situ environment The resistance of raw element
River was collected in Britain Times river in 2 months 2016, (sample collection point coordinates 51 ° of 44'47.0 " N, 1 ° of 15' 21.0 " W), take 200mL top layers river sample to be stored in glass sample bottle, preserved under the conditions of 4 DEG C.
2.4mL river samples are taken, add 1.6mL deuterium-oxides, and add various concentrations antibiotic (specific concentration is shown in Table 1), room Temperature culture 24 hours, rotating speed 500rpm.Negative control is that 2.4mL rivers sample adds 1.6mL deionized waters, similarity condition training Support.
Antibiotic concentration in the river sample of table 1
The mark river sample of 24 hours, with 5000g centrifuge 5 minutes, remove supernatant, add 4mL deionized waters 5000g from The heart 5 minutes, it is resuspended in after removing supernatant in the deionized water of same volume.After taking 1 μ L cells to be dried on calcirm-fluoride slide, use Raman spectrometer measures single celled Raman collection of illustrative plates.Raman spectroscopy measurement realizes (LabRAM HR by confocal Raman micro-platform Evolution,Horiba Scientific,UK).The measuring condition of Raman spectrum is：100 times of object lens (MPIanN, numerical apertures Footpath 0.9, Olympus), optical maser wavelength 532nm, power 4.2mW, 300l/mm grid, Spectral acquisition times 10s.
At least 50 unicellular collection of illustrative plates are gathered under the conditions of every kind of.
7 unicellular collection of illustrative plates under different condition are randomly selected, see Fig. 5, it can be seen that indivedual unicellular collection of illustrative plates are in 2000- 2300cm-1There are C-D peaks, show under conditions of the river chemical composition of cell is not changed, cell is successfully deuterium-labeled, And in not educable unknown microorganism, it was found that the microorganism resistant to carbenicillin and kanamycins.
Using the computational methods of D abundance in the content of the invention, the unicellular middle deuterium of each collected collection of illustrative plates can be calculated Abundance, it is drawn in figure, as shown in Figure 6.
Pass through unicellular ejection, DNA cloning, it is thus identified that the resistant microorganism identified by this method is potentially pathogenic Drug-resistant microorganism.
Embodiment 3：Bacterium infection resistance is identified from patient's sputum of the infection of the upper respiratory tract
Patient, male, 33 years old, the infection of the upper respiratory tract.Its sputum 2mL is collected, is mixed with 2mL deuterium-oxides.Mixed sample Five parts are bisected into, wherein four are separately added into different antibiotic, including kanamycins (50 μ g/mL), carbenicillin (100 μ G/mL), tetracycline (12 μ g/mL), chloramphenicol (6 μ g/mL), one does not add antibiotic as control.Sample is in 37 DEG C of conditions Lower culture 7 hours.
Sample is centrifuged 5 minutes with 5000g afterwards, removes supernatant, adds isometric deionized water mixing, and 5000g centrifuges 5 points Clock, it is resuspended in after removing supernatant in the deionized water of same volume.After taking 1 μ L cells to be dried on calcirm-fluoride slide, Raman is used The single celled Raman collection of illustrative plates of spectrometer measurement.Raman spectroscopy measurement realizes (LabRAM HR by confocal Raman micro-platform Evolution,Horiba Scientific,UK).Single celled Raman collection of illustrative plates is measured with Raman spectrometer.The survey of Raman spectrum Amount condition is：100 times of object lens (MPIanN, numerical aperture 0.9, Olympus), optical maser wavelength 532nm, power 4.2mW, 300l/ Mm grids, Spectral acquisition times 10s.At least 40 unicellular collection of illustrative plates are gathered under the conditions of every kind of.Raman figure under the conditions of calculating each The average value and standard deviation of spectrum, are drawn in figure, as described in Figure 7.
The result shows that the sputum of patient, can be in the original location by deuterium-oxide mark under conditions of without any sample pre-treatments Note.Resistant bacterial cell, there are C-D peaks on unicellular Raman collection of illustrative plates.
In summary result it is seen that, only exposed to carbenicillin whole bacteriums activity inhibited.
The patient has been taken with carbenicillin with being fully recovered behind the Amoxicillin of family.
Embodiment 4：Utilize the metabolic function of deuterated carbon substrate analysis microbial cell
Choose escherichia coli DH5a, pseudomonas putida UWC1, (these strains are commercially available common to pseudomonas putida G7 Strain), the condition of culture of escherichia coli DH5a is 37 DEG C, and pseudomonas putida UWC1, pseudomonas putida G7 condition of culture are 30℃.The bacterium solution being incubated overnight in LB culture mediums, with 1:50 ratio is seeded to fresh MM culture mediums.Culture medium it is unique Carbon source is glucose or naphthalene.The ratio of deuterated glucose is respectively 0,5,10,25,50,75,100%, the ratio difference of deuterated naphthalene For 0,5,10,25,50,75,100%.
The composition of MM culture mediums is shown in document Huang, W.E.et al.Chromosomally located gene with acquisition fusions constructed in Acinetobacter sp ADP1 for the detection of salicylate.Environ Microbiol 7:1339–1348(2015)。
After being incubated overnight, 5000g is centrifuged 5 minutes, removes supernatant, adds isometric deionized water mixing, 5000g centrifugations 5 Minute, it is resuspended in after removing supernatant in the deionized water of same volume.After taking 1 μ L cells to be dried on calcirm-fluoride slide, with drawing The graceful single celled Raman collection of illustrative plates of spectrometer measurement.Raman spectroscopy measurement realizes (LabRAM HR by confocal Raman micro-platform Evolution,Horiba Scientific,UK).Single celled Raman collection of illustrative plates is measured with Raman spectrometer.The survey of Raman spectrum Amount condition is：100 times of object lens (MPIanN, numerical aperture 0.9, Olympus), optical maser wavelength 532nm, power 4.2mW, 300l/ Mm grids, Spectral acquisition times 10s.At least 30 unicellular collection of illustrative plates are gathered under the conditions of every kind of.According to method of the present invention, Intracellular D abundance is calculated, as a result as shown in Figure 8.
This result shows, by Raman-DIP technologies, can quick and easy identification there is a certain substrate utilization function Microorganism.In Fig. 8, as little as 5% glucose (p<Or naphthalene (p 0.001)<0.01) can be detected.It is visible in figure, cell The abundance of interior deuterium is the same as the linear positive correlation of concentration of deuterium in cell culture fluid, but the slope of fit correlation, at different bottoms There is significant difference in thing and microorganism.This difference can be used for determining whether metabolism of the different microorganisms to different substrates The difference of approach.
Embodiment 5：Eukaryotic cells of the Raman-DIP identifications with metabolic activity
Cultivated after the THP-1 cell lines for being commercialized purchase are thawed in RPMI culture mediums.Two kinds of RPMI are used to test, its Middle deuterium-oxide ratio is respectively 33% and 0%, and 37 DEG C are cultivated.Respectively at the 0th, 6,24,48 and 72h, take 2mL cells, containing 15min is fixed in 4%PFA PBS solution.Subsequent cell is separated by centrifuging with nutrient solution, and is washed with deionized (1000rpm, 5min, room temperature).In each sample, 10-20 unicellular collection Raman collection of illustrative plates are taken, in each is unicellular, After 25 sample spot collections of selection are average, preserved as the single celled Raman spectrogram.
As a result as shown in figure 9, the result shows, eucaryon zooblast can be marked using deuterium-oxide.
Embodiment 6：Stem cell of the Raman-DIP identifications with metabolic activity
Extraction and separating mesenchymal stem cell, are transferred in 96 orifice plates from ox bone marrow, and density is per the cell of hole 500.Use Containing low concentration glucose, 10% heat inactivated foetal calf serum (FBS), 1% penicillin/streptomysin, 2.5mg/L amphotericin Bs and The DMEM culture mediums of 2ng/mL bovine brain hypophysis fibroblast growth factors (FGF), at 37 DEG C, 95% humidity and 5%CO2Condition Lower culture.96 orifice plate outer perimeter holes add 200 μ L PBS solutions with vaporization prevention.Fresh DMEM medium twice is changed weekly.Remittance piece During up to 80%, cell is cleaned three times using 200 μ L PBS, and add the DMEM culture mediums containing 20-30% heavy water and continue point Pei Yang not 0,6,12,24,48 and 72h.Culture medium is removed at each time point, adds 200 μ L 4% (v/v) poly first Aldehyde, 20min is cultivated at room temperature, then with deionized water rinsing 3s to remove paraformaldehyde solution.Cell is placed in PBS：Ethanol (1: 1) -20 DEG C are positioned in solution, it is stand-by.Cell is separated with background solution by centrifuging, and is washed with deionized (1000rpm, 5min, room temperature).Single celled Raman collection of illustrative plates is measured with Raman spectrometer.The measuring condition of Raman spectrum is：100 Times object lens (MPIanN, numerical aperture 0.9, Olympus), optical maser wavelength 532nm, power 4.2mW, 300l/mm grid, spectrum are adopted Collect time 10s.In each sample, 10-20 unicellular collection Raman collection of illustrative plates are chosen, in each is unicellular, select 25 After sample spot collection is average, preserved as the single celled Raman spectrogram.
As a result as shown in Figure 10, the result shows, its metabolic activity can be identified with labeled stem cells using deuterium-oxide.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention. Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general Principle is applied in other embodiment without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability Field technique personnel do not depart from improvement that scope made and modification all should be the present invention's according to the announcement of the present invention Within protection domain.

Claims (20)

  1. A kind of 1. method of quick detection cell biological processes in situ, it is characterised in that competent cell to material absorbing containing deuterium, turn Change, after metabolism or storage, the labeled in situ of deuterium can be realized in cell, is passed through in the different phase of cell biological processes Raman spectrum detects to cell, identifies in cell whether there is the presence of deuterium, and then obtain the biochemistry mistake on cell Journey, cell quality or function relevant information.
  2. 2. according to the method for claim 1, it is characterised in that the material containing deuterium, for the material containing deuterium element, including But it is not limited to：Deuterium-oxide, deuterated nucleic acid, deuterated amino acid, deuterated fatty, deuterated aliphatic acid, deuterated medicine, deuterated sugared, deuterated egg White matter, or by one or more of deuterated mixtures formed in above deuterated compound.
  3. 3. according to the method for claim 1, it is characterised in that in the material containing deuterium the abundance of deuterium be 5%-100% it Between.
  4. 4. according to the method for claim 1, it is characterised in that the method detected by Raman spectrum to cell is： The Raman collection of illustrative plates of cell is gathered by Raman spectrometer, in 2000-2300cm in Raman spectrum-1Between there is characteristic peak, i.e. C-D The cell at peak, show there is the presence of deuterium into the cell.
  5. 5. according to the method for claim 4, it is characterised in that judge the presence at C-D peaks and born of the same parents in cell Raman collection of illustrative plates The method of interior deuterium elemental abundance is：
    With the wavelength X at C-D peak maximumsdCentered on, setting range Ad, scope AdThe wavelength of boundary be X1And X2, wherein X1<Xd<X2, and X2-X1>7nm, setting range Bd, scope BdThe wavelength of boundary be X0And X1, wherein X1-X0=X2-X1If Determine scope Cd, scope CdBoundary wavelength be X2And X3, wherein X3-X2=X2-X1, calculate C-D peak areas：
    Sd=Sad-(Sbd+Scd)/2, wherein Sad, SbdAnd ScdRespectively scope Ad, BdAnd CdArea under interior Raman spectrum；
    With the wavelength Y at C-H peak maximumshCentered on, setting range Ah, scope AhBorder be wavelength Y1And Y2, wherein Y1<Yh< Y2, and Y2-Y1>9nm, setting range Bh, scope BhBorder be wavelength Y0And Y1, wherein Y1-Y0=Y2-Y1, setting range Ch, model Enclose ChBoundary wavelength be Y2And Y3, wherein Y3-Y2=Y2-Y1, calculate C-H peak areas：
    Sh=Sah-(Sbh+Sch)/2, wherein Sah, SbhAnd SchRespectively scope Ah, BhAnd ChArea under interior Raman spectrum；
    Deuterium abundance of elements calculation formula is into the cell：D abundance：D%=Sd/(Sd+Sh)；
    Cell is exposed in the medium without deuterium under similarity condition, gathers multiple unicellular collection of illustrative plates, calculates the abundance of D in cell Average value D0With standard deviation δ D0,
    When cell is exposed in the medium containing deuterium, multiple unicellular collection of illustrative plates are gathered, calculate the abundance D of D in cell1If D1≥D0+3 δD0, then it is assumed that obvious C-D peaks be present in the unicellular Raman collection of illustrative plates of cell.
  6. 6. according to the method for claim 1, it is characterised in that the cell includes eukaryotic cells and prokaryotes are thin Born of the same parents.
  7. 7. according to the method for claim 1, it is characterised in that in prokaryotes or Eukaryotic normal habitat Under, material containing deuterium is mixed, during the particular organisms of cell, the carbon in active cell can form C-D keys with deuterium, pass through Raman spectrum detects to cell, if being realized in identification cell with the presence of deuterium to the cell with particular organisms process Identification and screening.
  8. 8. according to the method for claim 1, it is characterised in that competent cell to material absorbing containing deuterium, conversion, be metabolized or deposit Chu Hou, the labeled in situ of deuterium can be realized in cell, by Raman spectrum to thin in the different phase of cell biological processes Born of the same parents are detected, and identify whether there is the presence of deuterium in cell, and then realize to unknown or uncultured microorganisms characters It is identified with function.
  9. 9. according to the method for claim 8, it is characterised in that after the sample collection containing microorganism, sample is not being entered In the case of any pre-treatment of row, sample is mixed with deuterium-oxide, the microorganism in mixed sample is separated with impurity, separated Microbial cell afterwards preserves in deionized water, gathers single celled Raman collection of illustrative plates by Raman spectrometer, has in sample In 2000-2300cm in its Raman spectrum of the microorganism of activity-1Between there is characteristic peak, have the peak cell, be identified as Active cell under this condition, by calculating ratio of the cell with this feature peak in all detected cells, Calculate the ratio shared by cell active in sample.
  10. 10. according to the method for claim 1, it is characterised in that cell is to material absorbing containing deuterium, conversion, metabolism or storage Afterwards, the labeled in situ of deuterium can be realized in cell in the different phase of cell biological processes, in the mistake of cell in-situ mark Cheng Zhong, antimicrobial or antibiotic are added, cell is detected by Raman spectrum, whether identify has the presence of deuterium in cell, If there is the presence of deuterium in cell, the cell is the cell with antimicrobial or antibiotic resistance, by calculating there is deuterium to exist Ratios of the cell in all detected cells, calculate the ratio having in sample shared by the cell of antimicrobial or antibiotic resistance Example.
  11. 11. according to the method for claim 1, it is characterised in that in specific environment competent cell to material absorbing containing deuterium, turn Change, after metabolism or storage, the labeled in situ of deuterium can be realized in cell, is passed through in the different phase of cell biological processes Raman spectrum detects to cell, identifies whether there is the presence of deuterium in cell, and then it is thin to judge to survive in specific environment Born of the same parents.
  12. 12. according to the method for claim 11, it is characterised in that the cell in specific environment includes not yet being separately cultured Mixed microorganism flora cell.
  13. 13. according to the method for claim 11, it is characterised in that specific environment includes human body fluid or secretion.
  14. 14. according to the method for claim 11, it is characterised in that in specific environment competent cell to material absorbing containing deuterium, After conversion, metabolism or storage, the labeled in situ of deuterium can be realized in cell in the different phase of cell biological processes, During cell in-situ marks, antimicrobial or antibiotic are added, cell is detected by Raman spectrum, identified in cell Whether the presence of deuterium is had, if there is the presence of deuterium in cell, the cell is to have antimicrobial or antibiotic in the specific environment The cell of resistance, by calculating the ratio with cell existing for deuterium in all detected cells, calculate in sample in the spy Determine the ratio for having shared by the cell of antimicrobial or antibiotic resistance in environment.
  15. 15. according to the method for claim 1, it is characterised in that active microorganism is unicellular to thing containing deuterium in specific substrates After matter absorbs, converts, is metabolized or stored, the original position of deuterium can be realized in cell in the different phase of cell biological processes Mark, is detected by Raman spectrum to cell, identifies in cell whether there is the presence of deuterium, if there is the presence of deuterium in cell, Then the microbial single-cell is the cell with the substance metabolism functions containing deuterium under determination of the environment.
  16. 16. according to the method for claim 15, it is characterised in that the material containing deuterium includes deuterated carbon source.
  17. 17. according to the method for claim 1, it is characterised in that active microorganism is unicellular to be used as bottom by the use of deuterated nucleic acid Thing and synthetic DNA or RNA, and C-D keys are formed in intracellular, detected by Raman spectrum to unicellular, identifying in cell is The no presence for having deuterium, if there is the presence of deuterium in cell, the microbial single-cell is that nucleic acid can be utilized to synthesize under condition determination DNA or RNA cell.
  18. 18. according to the method for claim 1, it is characterised in that active microorganism is unicellular to utilize deuterated Amino acid synthesis Protein, and C-D keys are formed in intracellular, detected by Raman spectrum to unicellular, identify in cell whether there is depositing for deuterium , if there is the presence of deuterium in cell, the microbial single-cell for can be using Amino acid synthesis albumen under condition determination it is thin Born of the same parents, this deuterated amino acid are identified as the amino acid utilized by such cell high-efficient.
  19. 19. according to the method for claim 1, it is characterised in that active microorganism is unicellular can be absorbed, convert and store Deuterated fat, and C-D keys are formed in intracellular, detected by Raman spectrum to unicellular, identify in cell whether there is deuterium In the presence of if there is the presence of deuterium in cell, the microbial single-cell is deuterated fatty for that can be absorbed under condition determination, convert this Cell, and can determine whether the process of absorption, conversion and the storage of fat.
  20. 20. according to the method for claim 1, it is characterised in that competent cell can absorb, convert or be metabolized deuterated medicine Thing, and form C-D keys in intracellular, is detected by Raman spectrum to unicellular, monitor the time of occurrence at intracellular C-D peaks with And distribution of the C-D peaks in cell spaces, for judging the metabolism and transport of medicine.
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