CN109182444A - A kind of method and apparatus of detection Pathogen category distribution and drug resistance - Google Patents
A kind of method and apparatus of detection Pathogen category distribution and drug resistance Download PDFInfo
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- CN109182444A CN109182444A CN201811060507.0A CN201811060507A CN109182444A CN 109182444 A CN109182444 A CN 109182444A CN 201811060507 A CN201811060507 A CN 201811060507A CN 109182444 A CN109182444 A CN 109182444A
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Abstract
A method of detection Pathogen category distribution and drug resistance, from it is known include or may comprising pathogen cells body fluid in by centrifugation or filter obtain pathogen sample；Pathogen sample and antibiotic are respectively taken a certain amount of, simultaneously or substep instills at least one container, the bottom of container has plane quadrupole gold microelectrode array, applying voltage signal on microelectrode can produce inhomogeneous field, so that pathogen cells is formed dielectrophoresis directed movement, assembles to form several pathogen cells aggregations for arriving several hundred to container bottom center；Three-dimensional Raman spectrum imaging is carried out by pathogen cells aggregation described in laser scanning, can obtain and distinguish different pathogen cells distribution and Drug resistance status in pathogen cells aggregation.It is above-mentioned for detecting the device of Pathogen category distribution and drug resistance the present invention also provides realizing.It is analyzed by the real-time detection of these Raman spectrums, to carry out Pathogen category distribution and selection resistance to fungicide.
Description
Technical field
The present invention relates to physical field more particularly to a kind of optical detective technology, especially a kind of detection Pathogen category
The method and apparatus of distribution and drug resistance.
Background technique
At present in clinical infection medical consultations, because can not quickly determine the type and drug resistance of pathogen, the normal basis of doctor
Generally using broad-spectrum antibiotic and antibiotic is used in combination in experience, and often inaccurate therapeutic effects, causes abuse of antibiotics problem.And it is close
Year abuse of antibiotics problem is more shown in that seriously the intractable drug-resistant bacteria generated by abuse of antibiotics is through to wreak havoc the whole world.Newest system
Count it was noted that, drug-fast bacteria causes global 700,000 people dead every year, focuses mostly in developing country, infers by number, arrive
The year two thousand fifty, whole world death case may be up to 10,000,000 people.
Since the individual amount of each pathogen is few, be unfavorable for carrying out Physiology and biochemistry and immunologic identification, need by
After in vitro culture reaches a certain level its number, then identified.Traditional pathogen detection method is mostly just for specific
, educable bacterium, and take a long time.The detection time that 2 days are about needed for cultivable bacteria, it is slower for growing
Strain needs 7 days or so, and the bacterium that can not be cultivated for laboratory is then helpless.And conventional method be difficult to differentiate between it is a certain
Infection is as one or caused by multiple bacterium pathogen.
Present some new pathogen method for identifying molecules have gradually developed, these methods are independent of culture living
Object, but cause of disease is identified based on the analysis of high-throughput gene sequencing data, Raman spectrum detection and biomolecule mass spectral analysis
Body.
The analysis of gene sequencing data is to generate the gene mutation of drug resistance based on bacterium or obtain drug resistant gene, passes through needle
Gene mutation or related drug resistant gene are detected so that it is determined that bacterial drug resistance to property.
U.S. Patent No. 4,847,198 disclose a kind of method using UV excitation Raman spectroscopy identification microorganism.
According to 4,847,198 patents, bacterial suspension is contacted by the Single wavelength in ultraviolet spectral range.Luminous energy used in part is inhaled
Receive and part luminous energy is launched.The luminous energy of transmitting, resonant check Raman scattering, is measured as either backscatters energy.To this
Energy is handled to generate the characteristic spectral of bacterium.
U.S. Patent No. 6,177,266 disclose a kind of use through substance assistant laser desorpted ionized flight time matter
(MALDI-TOF-MQ analyzes the specific biomarker of genus and species and bacterial strain that cell protein extract or entire cell generate to spectrum
The method that chemotaxonomy classification is carried out to bacterium.But same bacterium is due to the difference of condition of culture or chemical extraction methods
Different mass spectrums can be generated, so as to cause the error of analysis result.Even if strict control conditions above, mass spectrography still cannot be complete
Accurately determine bacterial species.In addition, compared with traditional drug sensitive test, MALDI-TOF MS technology can only distinguish sensitive strain and
Persister, can not obtain accurate MIC value, and the technology judges that persister and sensitive strain are needed first with known sensitive strain and resistance to
Medicine strain modeling.
Pathogen fining detection is not carried out in clinical diagnosis mostly at present, especially in some severe infections of diagnosis and treatment such as sepsis
When disease, can not fast accurate medication often result in life danger.
Summary of the invention
The purpose of the present invention is to provide the method for a kind of detection Pathogen category distribution and drug resistance, this inspections
The method of the distribution of survey Pathogen category and drug resistance will solve the type for surveying pathogen in the prior art and the method for drug resistance is multiple
Miscellaneous, ineffective technical problem.
The present invention provides the methods of a kind of detection Pathogen category distribution and drug resistance, include the following steps:
1) from it is known include or may comprising pathogen cells body fluid in by centrifugation or filtering obtain pathogen sample；
2) pathogen sample and antibiotic are respectively taken using micro syringe it is a certain amount of, while or substep instill at least one container
In, the bottom of the upper end opening of the container, the container has plane quadrupole gold microelectrode array, applies on microelectrode
Making alive signal makes pathogen cells form dielectrophoresis directed movement to generate inhomogeneous field, assembles to container bottom center
Form the pathogen cells aggregation between 2 to 1000；
3) pass through pathogen cells aggregation described in laser scanning and carry out three-dimensional Raman spectrum imaging, pass through Raman spectrum
Real-time detection analysis, carry out Pathogen category distribution and Resistance detection.
Further, the pathogen cells aggregation includes the pathogen cells of two or more numbers, passes through three Wellas
Graceful spectral scan imaging can distinguish different pathogen distribution and Drug resistance status；
Further, the resolution ratio of the three-dimensional Raman spectroscopy scans is less than 10 microns；
The present invention also provides the device of detection the Pathogen category distribution and drug resistance of realizing the above method, the device packets
At least one container, the upper end opening of the container are included, the bottom of the container is provided with plane quadrupole gold microelectrode battle array
Column, the plane quadrupole gold microelectrode array connect a voltage signal generator, the voltage signal generator and one
A iontophoretic electrode connection, the upper end of the container is provided with a lighting source；It further include having a CCD camera, in institute
The first beam splitter, an object lens are disposed on the CCD camera imaging direction stated, the lower end of first beam splitter is set
It is equipped with a CCD camera, first beam splitter, object lens, CCD camera, lighting source are on a central axes；It is described
The first beam splitter and the horizontal planes of the object lens be in 45 degree of angle；It further include having a Raman spectrum excitation light source,
In the optical path of the Raman spectrum excitation light source transmitting laser irradiation sample, it is disposed with a collimation lens, second point
Shu Jing, laser scanning device, the laser scanning device and a beam shaping system connection, the laser scanning device
It is arranged in 45 degree of optical path of first beam splitter with a beam shaping system；It further include having a spectrometer,
A coupled lens, optical filter, the second beam splitter, the light are disposed on the light absorbing direction of the spectrometer
Spectrometer, coupled lens, optical filter are arranged in 45 degree of optical path of second beam splitter.
The principle of the present invention is: applying voltage signal in plane quadrupole gold microelectrode array, produces under voltage signal driving
Raw inhomogeneous field forms dielectrophoretic motion, forms a cell aggregation to realize and collect pathogen cells using dielectrophoresis
Then body carries out three-dimensional Raman spectrum imaging to the pathogen cells aggregation using three-dimensional Raman spectroscopy scans, passes through
The real-time observation and detection of these Raman spectrums, can analyze different pharmaceutical to the Raman spectrum of the stimulation of pathogen cells
Variation and pathogen cells Species distributing situation, to carry out Pathogen category distribution and selection resistance to fungicide.This is because different
Pathogen cells molecular structure is different, the Raman spectrum that every kind of pathogen cells have its exclusive, by analyzing these Ramans
Spectrum can identify pathogen cells type.In addition, after biochemical action occurs for drug and pathogen cells, pathogen cells point
Minor structure can change, and react on the Raman spectrum of pathogen immediately, and have the pathogen of drug resistance thin drug
Born of the same parents do not have the variation of molecular structure, and pathogen cells spectrum remains unchanged.So as to quickly reflect pathogen pair
Whether there is or not drug resistances for the drug of application.Can also obtain pathogenic growth can be completely inhibited about how many drug concentration simultaneously, this
It is to significantly affect the successful important diagnostic parameter for the treatment of, provides one entirely for high-flux medicaments sifting and cell drug research
New technology platform and method.
The present invention is compared with prior art, its technical effect is that actively and apparent.The present invention is determined using Raman spectrum
The characteristic of cell interior, the cytogenetic variation of real-time dynamic monitoring biomolecule.The cell of study of disease, drug or toxin
Reaction；Pathogen cells are analyzed using microflow control technique and three-dimensional Raman spectroscopy scans imaging technique, can without
Pathogen cells are directly treated in the case where sample pretreatment and are collected manipulation and raman spectroscopy measurement, so as to maximum limit
Degree ground maintains the physiological activity of pathogen cells to be measured.The present invention has many advantages, such as extremely low sample consumption, Noninvasive, makes it
It is widely used in pathogen cells analysis and drug susceptibility screening etc..Present invention incorporates electrophoresis microflow control techniques, three-dimensional
Raman spectrum detection technique can use a drop body fluid and realize pathogen quick diagnosis.It is directly against pathogenic organisms point
Minor structure variation, which carries out Raman spectrum Testing and appraisal, can complete pathogen in one hour independent of the breeding of pathogen
Type publication, the measurement of drug resistance, minimum inhibitory concentration (MIC).Instruct doctor referring to Pathogen category, drug susceptibility and disease
Individual human Difference of Metabolism prescription realizes accuracy and the safety of clinical application, and also exactly precisely medical treatment is most crucial for this
Essential requirement.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram being distributed for detecting Pathogen category with the device of drug resistance of the invention.Portion
Part title is as follows:
1: pass container；2: sample；3: pathogen cells aggregation；4: lighting source；5: iontophoretic electrode；6: voltage signal hair
Raw device；7: object lens；8: the first beam splitters；9:CCD camera；10: beam shaping system；11: laser scanning device；12: Raman
Excitation of spectra light source；13: collimation lens；14: the second beam splitters；15: optical filter；16: coupled lens；17: spectrometer.
Fig. 2 is by the present invention using the Raman spectrogram after a kind of Escherichia coli and Ciprofloxacin effect.
Specific embodiment
Embodiment 1
As shown in Figure 1, the present invention provides a kind of for detecting the device of Pathogen category distribution and drug resistance, including at least one
A container 1, the upper end opening of the container 1, the bottom of the container 1 are provided with plane quadrupole gold microelectrode array, institute
The quadrupole gold microelectrode stated connects a voltage signal generator 6, the voltage signal generator 6 and an iontophoretic electrode 5
Connection, the upper end of the container 1 is provided with a lighting source 4；
Further include having a CCD camera 9, is disposed with first beam splitting on 9 imaging direction of CCD camera
Mirror 8, object lens 7, the lower end of first beam splitter 8 are provided with a CCD camera 9, first beam splitter 8, object lens
7, CCD camera 9, lighting source 4 are on a central axes；The horizontal plane of first beam splitter 8 and the object lens 7 is in
45 degree of angle；
Further include having a Raman spectrum excitation light source 12, emits laser irradiation sample in the Raman spectrum excitation light source 12
Optical path in, be disposed with a collimation lens 13, the second beam splitter 14, laser scanning device 11, the laser scanning
Device 11 and a beam shaping system 10 connect, and the laser scanning device 11 and a setting of beam shaping system 10 exist
In 45 degree of optical path of first beam splitter 8.
Further include having a spectrometer 17, a coupling is disposed on the light absorbing direction of the spectrometer 17
Lens 16 are closed, optical filter 15, the second beam splitter 14, the spectrometer 17, coupled lens 16, optical filter 15 are arranged in described
In 45 degree of optical path of the second beam splitter 14.
Further, first beam splitter 8 and second beam splitter 14 are arranged in parallel.
Pathogen cells accumulation process of the invention is: on iontophoretic electrode 5, applying electricity by voltage signal generator 6
Signal is pressed to collect the control voltage of pathogen cells as dielectrophoresis, dielectrophoresis is the lower object of dielectric constant non-even
In strong electrical field, the directed movement of stress generation due to polarization.It is unrelated whether dielectric power size is charged with object, with object
Size, electrical properties, field strength, field strength change rate, the frequency of the electrical properties of surrounding medium and extra electric field it is related.In electricity
The voltage signal applied on swimming electrode 5 can produce inhomogeneous field, and pathogen cells is made to form dielectrophoresis directed movement, Xiang Rong
Device bottom centre assembles to form several pathogen cells aggregations 3 for arriving several hundred, pathogen cells aggregation almost spherical shape
Shape, diameter is up to more than ten microns；
Three-dimensional Raman spectroscopy scans imaging process of the invention are as follows: Raman spectrum excitation light source 12 emits laser first, passes through standard
Straight lens 13 collimate, and then by the second beam splitter 14, laser scanning device 11, beam shaping system 10, then pass through first
Beam splitter 8 emits, and focuses in pathogen cells aggregation 3 finally by object lens 7 and generates Raman diffused light, and a part of Raman dissipates
It penetrates light and collimates original optical path return further through object lens 7, and reflected by the first beam splitter 8, swept through beam shaping system 10, laser
Imaging apparatus 11, and reflected by the second beam splitter 14, it is focused finally by optical filter 15 and coupled lens 16 and enters spectrometer 17,
For pathogen cells raman spectroscopy measurement.Then the laser that Raman spectrum excitation light source 12 emits passes through laser scanning device 11
Scanning is to collect three-dimensional Raman spectral image.Raman spectrum excitation light source 12 use 1064nm laser-excitation source, by 50 times/
0.75 object lens 7 of NA focus on total laser intensity of about 50mW on sample.Using 0.8 μm of step-length, entire pathogen cells aggregation
The body time of integration is 20 seconds.
In the present embodiment, the process of detection is: the urine of patient is obtained first, by being centrifuged and filtering to take and obtaining pathogen sample
Pathogen sample 2 is taken 200 microlitres individually to instill in a container 1 by product 2 using micro syringe.In addition micro note is reused
Pathogen sample 2 is taken 200 microlitres to instill in another container 1 by emitter, and it is a certain amount of that antibiotic ciprofloxacin is added.Then,
Applying voltage letter on iontophoretic electrode 5 by voltage signal generator 6 can produce inhomogeneous field, make pathogen cells shape
At dielectrophoresis directed movement, assemble to form several pathogen cells aggregations 3 for arriving several hundred to container bottom center.Pass through electricity
The voltage of swimming electrode 5 is about 4 volts and 1MHz.Resulting electrophoretic force is directed toward four electrode centers.After twenty minutes, it is taken the photograph by CCD
As first 9 it can be seen that the pathogen cells being collected into form a compact three-dimensional globoid.
After sixty minutes, start using raman spectroscopy measurement, Raman spectrum excitation light source 12 first emits laser, passes through collimation
Lens 13 collimate, and then by the second beam splitter 14, laser scanning device 11, beam shaping system 10, then pass through first point
Beam mirror 8 emits, and focuses in pathogen cells aggregation 3 finally by object lens 7 and generates Raman diffused light, a part of Raman scattering
Light collimates original optical path further through object lens 7 and returns, and is reflected by the first beam splitter 8, through beam shaping system 10, laser scanning
Device 11, and reflected by the second beam splitter 14, it is focused finally by optical filter 15 and coupled lens 16 and enters spectrometer 17, used
In pathogen cells aggregation raman spectroscopy measurement.Then the laser that Raman spectrum excitation light source 12 emits is filled by laser scanning
11 scannings are set to collect three-dimensional Raman spectral image.
Ciprofloxacin belongs to Development of Fluoroquinolone Antibacterials, and the A for mainly acting on the DNA helicase of bacterial cell is sub- single
Position, can destroy original DNA molecular structure, inhibit the synthesis and duplication of DNA of bacteria, and then lead to bacterium Large Scale Death.It sees
It examines Raman line and obtains the Characteristic Raman spectral line of Escherichia coli.Ciprofloxacin and Escherichia coli effect after sixty minutes, obtain large intestine
The Raman line of bacillus integrally dies down, and two 782 cm-1 of phosphoric acid backbone peak, 1084 cm- of DNA are belonged in Raman spectrum
1 and 1155 cm-1 of deoxyribose-phosphate vibration peak and 1262 cm-1 spectral lines significantly reduce;Characterize the feature of A type (DNA) conformation
Peak 812 cm-1 and 979 cm-1,668 cm-1 disappear, and have 1075 cm-1 of new peak appearance, this illustrates the phosphoric acid bone of DNA
Frame has certain fracture, ineffective so as to cause the schizogamy of Escherichia coli.Measurement result is as shown in Figure 2.
Prove that pathogen is only Escherichia coli, without other bacterium infections.In addition, not using the cause of disease of Ciprofloxacin
Body Raman spectrum does not change, and obtains Raman spectrum using Ciprofloxacin and show apparent variation, shows Ciprofloxacin to disease
Escherichia coli in human urine are effective.
Notwithstanding the preferred embodiment of the present invention, it should be apparent to those skilled in the art that
Without departing from the spirit or scope of the present invention, it can carry out various modifications and change in the present invention.Therefore, this hair
It is bright to be intended to cover the modifications and variations of the invention come within the scope of the appended claims and their.
Claims (4)
1. a kind of method of detection Pathogen category distribution and drug resistance, characterized by the following steps:
1) from it is known include or may comprising pathogen cells body fluid in by centrifugation or filtering obtain pathogen sample；
2) pathogen sample and antibiotic are respectively taken using micro syringe it is a certain amount of, while or substep instill at least one container
In, the bottom of the upper end opening of the container, the container has plane quadrupole gold microelectrode array, applies on microelectrode
Making alive signal makes pathogen cells form dielectrophoresis directed movement to generate inhomogeneous field, assembles to container bottom center
Form the pathogen cells aggregation between 2 to 1000；
3) pass through pathogen cells aggregation described in laser scanning and carry out three-dimensional Raman spectrum imaging, pass through Raman spectrum
Real-time detection analysis, carry out Pathogen category distribution and Resistance detection.
2. the method for a kind of detection Pathogen category distribution and drug resistance as described in claim 1, which is characterized in that described
Pathogen cells aggregation includes the pathogen cells of two or more numbers.
3. the method for a kind of detection Pathogen category distribution and drug resistance as described in claim 1, which is characterized in that described
The resolution ratio of three-dimensional Raman spectroscopy scans is less than 10 microns.
4. a kind of device of detection Pathogen category distribution and drug resistance, including at least one container, which is characterized in that described
The upper end opening of container, the bottom of the container are provided with plane quadrupole gold microelectrode array, and the plane quadrupole gold is micro-
Electrod-array connects a voltage signal generator, the voltage signal generator and an iontophoretic electrode connection, described
The upper end of container is provided with a lighting source；It further include having a CCD camera, in the CCD camera imaging direction
On be disposed with the first beam splitter, an object lens, the lower end of first beam splitter is provided with a CCD camera, institute
The first beam splitter for stating, object lens, CCD camera, lighting source are on a central axes, first beam splitter and described
The horizontal plane of object lens is in 45 degree of angle；It further include having a Raman spectrum excitation light source, in the Raman spectrum exciting light
Source emits in the optical path of laser irradiation sample, is disposed with a collimation lens, the second beam splitter, laser scanning device, institute
The laser scanning device stated and a beam shaping system connection, the laser scanning device and a beam shaping system are set
It sets in 45 degree of first beam splitter of optical path, further includes having a spectrometer, in the absorption light of the spectrometer
Direction on be disposed with a coupled lens, optical filter, the second beam splitter, the spectrometer, coupled lens, optical filter
It is arranged in 45 degree of optical path of second beam splitter.
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| CN114196725A (en) * | 2021-12-03 | 2022-03-18 | 清华大学 | Method for identifying microorganism and system thereof |
| CN114544593A (en) * | 2022-03-08 | 2022-05-27 | 北京大学 | Embedded dielectrophoresis in-situ Raman spectrum system |
| US11698304B2 (en) | 2019-02-15 | 2023-07-11 | Wayne State University | Apparatuses, systems, and methods for detecting materials based on Raman spectroscopy |
| US11927520B2 (en) | 2019-05-30 | 2024-03-12 | Hewlett-Packard Development Company, L.P. | Rotating levitated particle imaging |
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| CN109182444B (en) | 2022-06-24 |
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