CN110376105A - It is a kind of for determining the method and device of clinical sample small particles characteristic and concentration - Google Patents
It is a kind of for determining the method and device of clinical sample small particles characteristic and concentration Download PDFInfo
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- G—PHYSICS
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N2015/0096—Investigating consistence of powders, dustability, dustiness
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Abstract
The present invention discloses a kind of for determining the method and device of clinical sample small particles characteristic and concentration, by introducing control particle and probe, clinical sample small particles and concentration are determined by the concentration of known control particle, the characteristics such as the source of clinical sample small particles can be obtained by probe label simultaneously, reach while obtaining specific short grained characteristic and information in clinical sample, which can be further used for clinical diagnosis and therapy monitoring.Method is simple, works well, and compared to the prior art, apparatus of the present invention are low in cost, is not limited by factors such as the concentration of particle, simple to operate, is adapted to use in clinical setting.
Description
Technical field
The present invention relates to a kind of for determining the method and device of clinical sample small particles characteristic and concentration.
Technical background
Little particle generally refers to particle of the diameter less than 1 micron, is thus generically referred to as nano particle.They are typically found in
In liquid and air.Usually by monitoring or characterizing these short grained sizes, quantity (or concentration), as monitoring product or system
Make a kind of means of procedure quality.However, since little particle has the general small size of nanometer, so being difficult reliably to measure it
Size and the concentration for determining sample small particles.The existing short grained technology of characterization mainly has:
(1) transmission electron microscope (TEM).Transmission electron microscope (TEM) is provided to short grained accurate and high-resolution
Measurement, but its have the shortcomings that it is many.Firstly, TEM imaging depends on the density of sample, therefore the little particle in biological sample
(being mainly made of lipid film, protein and nucleic acid) is difficult to the imaging that is taken in the case where no manipulation.In addition, this method
Expensive equipment is not only needed, but also needs flower very great strength and is lot more time to obtain result.Therefore, it is often unsuitable for
It is used in clinical setting.
(2) dynamic light scattering (DLS).Dynamic light scattering (DLS) is the technology based on light scattering, is commonly used in nanoparticle
Measurement.Dynamic light scattering (DLS) is the size that particle in solution is measured by the strength fluctuation of the light of analysis sample scattering.
Since the 1960s is by invention, DLS has become a kind of particle size determination method of prevalence.However, DLS is by many
Limitation.For example, scattered light intensity depends not only on the concentration of particle, refractive index, electrolyte and the particle ruler of solvent are additionally depended on
Very little uniformity, and the latter is especially problematic for biological sample, because particle size therein is usually non-uniform.
(3) gradient centrifugation.Gradient centrifugation is normally used for the particle in analytical solution because the rate of settling of particle with
Particle size is related.However, sinking speed depends not only on centrifugal force and particle size, the viscosity and density of solution are additionally depended on.
Although this method can be used for determining particle size, it is likely difficult to the concentration for determining particle.If using centrifugal method, also
Need to spend the time to complete required program and operation heavy equipment, to limit its effectiveness in clinical setting.
(4) nano-particle trace analysis (NTA).Nano-particle trace analysis (NTA) is for determining sample small particles
Size and concentration.With this method, the Blang of single particle is independently tracked using the light scattered from incident laser light source
Movement directly measures diffusion constant, and the diffusion constant is related to the hydrodynamic radius of single particle, therefore can use
In derivation particle size.But there are many disadvantages by NTA, including no size resolution, the folding for needing to know in advance tracked particle
Penetrate the interference of rate and mechanical oscillation.
(5) adjustable resistance pulse sensing (TRPS).The adjustable resistance pulse sensing (TRPS) of nanoparticle can be used to directly
It measures particle concentration and high-resolution analysis is carried out to granularity and surface charge.Apply electricity on the hole filled with electrolyte
Pressure, can produce ionic current.When particle passes through the hole, it can cause current blockade event, and this event can be used for measurement
Grain concentration and granular size.
Above-mentioned all characterizing methods all highlight the importance of determining particle size, and are mainly used for determining little particle
Size.However, Accurate Determining particle size may be difficult, it is highly dependent on analysis condition, so being unrepeatable.
Moreover, the demand of measurement particle size is in quality of production control or research environment.In clinical application, clinical sample
In particle even derive from the organ or cell of same type, size is also non-uniform, it is thus determined that partial size is not
It is required.On the contrary, the concentration and characteristic of particle can provide enough information for certain clinical applications.It is therefore desirable to little particle
Characteristic (for example, particle is come wherefrom or from which cell or organ) and concentration characterized, to be used for diagnostic monitoring
Disease or illness, or the safety and effect of monitoring treatment.
Summary of the invention
For above-mentioned problem, the present invention proposes a kind of method and apparatus for characterizing clinical sample small particles, really
The characteristic or source and concentration of clinical sample small particles are determined, to be used for diagnostic monitoring disease or illness, or the peace of monitoring treatment
Full property and effect.Specific technical solution is as follows:
A method of for determining clinical sample small particles characteristic and concentration, comprise the following steps that
1): the clinical sample containing candidate particles being contacted into incubation with detection reagent first；The detection reagent contains at least one
A probe for candidate particles in marker samples；Then mixture is mixed to form with the particle that compares for being combined with probe again；
2): the mixture in step 1) being loaded into the reaction vessel equipped with watch window, and observes and/or shoot photo note
Record；
3): according to the record that photo is observed and shot in step 2 as a result, determining the characteristic and concentration of candidate particles in sample, and
The characteristic and concentration information are applied to clinical diagnosis or therapy monitors.
The method for being previously used for determining clinical sample small particles characteristic and concentration, including step are further are as follows:
A): the clinical sample containing candidate particles is contacted into incubation with detection reagent；The detection reagent contains for marking sample
The first probe of candidate particles, first probe can mutually be tied with first marker in its source is indicated on candidate particles in this
It closes；Then the first mixture is obtained with control particle mixing again, the control particle contains second wrapped up by the second marker
Probe；Second marker is similar to the first marker；
B): the first mixture being obtained in step a), further cultivation is contacted with the magnetic-particle for being coated with the first binding partners,
Obtain the second mixture；First binding partners can specifically with the first marker on candidate particles and compare particle
On the second marker combine；
C): the second mixture will be obtained in step b) and be loaded into the reaction vessel equipped with watch window, and by that can generate
The equipment in magnetic field attracts and is fixed to together by magnetic-particle and by the candidate particles and control particle that magnetic-particle is captured
The inner surface of reaction vessel watch window, to observe and/or shoot photo record；
D): according to the record that photo is observed and shot in step c) as a result, determining the characteristic and concentration of candidate particles in sample, and
The characteristic and concentration information are applied to clinical diagnosis or therapy monitors.
The method for being previously described for determining clinical sample small particles characteristic and concentration, is further described below:
S1: the clinical sample containing candidate particles is contacted into incubation with detection reagent；The detection reagent contains for marking sample
The first probe and third probe of candidate particles in this, first probe can be marked with indicating the first of its source on candidate particles
Note object combines；Then with compare particle and be mixed to form mixture, the control particle is combined with the second probe, and described second visits
Needle is wrapped up by the second marker of control particle；Second marker is similar to the first marker；
S2: the mixture in step S1 is loaded into the reaction vessel equipped with watch window, and observes and/or shoot photo note
Record；
S3: according to the record that photo is observed and shot in step S2 as a result, determining the characteristic and concentration of candidate particles in sample, and
The characteristic and concentration information are applied to clinical diagnosis or therapy monitors.
As the technical solution of one of them, be previously described for determine clinical sample small particles characteristic and concentration method
Described in clinical sample first pass through complete human body cell removal processing in advance, the candidate particles are the work in clinical sample
The cell fragment or aggregation of extracellular vesica, dead cell that cell is secreted.
As the technical solution of one of them, be previously described for determine clinical sample small particles characteristic and concentration method
It further include dilution step, which and compare before particle mixes after clinical sample contacts with detection reagent cultivation.
Be previously described for determine clinical sample small particles characteristic and concentration method, probe used be fluorescence probe or
Person can chemiluminescent enzyme, and the first, second, and third probe have differentiable emission wavelength.
The method for being previously described for determining clinical sample small particles characteristic and concentration as a preferred technical solution, institute
The control particle stated can be non-magnetic particle, further preferably granules of polystyrene, diameter dimension is preferably 50~
100nm, 50~250nm, 50~500nm, 50~1000nm or 500~2000nm.
The method for being previously described for determining clinical sample small particles characteristic and concentration as a preferred technical solution, institute
The first binding partners on the magnetic-particle stated are antibody, nucleic acid, peptide, aptamer, peptidomimetic, ligand, receptor are one of or it
Combination；The diameter dimension of the magnetic-particle is preferably 0.5~3.5 μm, 0.5~2 μm or 0.5~1.5 μm.
The method for being previously described for determining clinical sample small particles characteristic and concentration as a preferred technical solution, institute
Stating the first probe is the probe for having binding affinity to nucleic acid or lipid film, is used to indicate whether candidate particles are originated from dead cell
Or dead cell；The third probe is used to determine the characteristic of candidate particles, i.e. whether candidate particles have specifically
Marker.
The method for being previously described for determining clinical sample small particles characteristic and concentration, the clinical diagnosis includes in liver
Poison, acute kidney injury, heart attack and septic shock；The therapy monitoring includes chemotherapy, cell therapy, immune treatment
Method and radiotherapy.
For inventive method above-mentioned, the present invention also provides one kind for determining clinical sample small particles characteristic and concentration
Device, including reaction vessel, the scanner-recorder positioned at reaction vessel watch window side, and connect with scanner-recorder
Calculating and data storage device, the reaction vessel is for holding the mixture above-mentioned containing candidate particles.
As the technical solution of one of them, it is previously described for determining the dress of clinical sample small particles characteristic and concentration
It sets, further includes for causing candidate particles and compareing the excitation light source of the probe generation fluorescence in particle, the excitation light source hair
The exciting light of injection is irradiated to the watch window of reaction vessel at a certain angle.
As the technical solution of one of them, it is previously described for determining the dress of clinical sample small particles characteristic and concentration
Set, the equipment that can generate magnetic field be equipped with below described image logger, so as to by magnetic-particle described in claim with
And the candidate particles captured by magnetic-particle and control particle attract together and are fixed to the interior table of reaction vessel watch window
Face, to observe and/or shoot photo.
As a preferred technical solution, it is previously described for determining the dress of clinical sample small particles characteristic and concentration
It sets, optical filter is equipped between described image logger and reaction vessel watch window, the optical filter only allows probe to generate
Fluorescence pass through.
As another technical solution, it is previously described for determining the device of clinical sample small particles characteristic and concentration,
The two sides of the reaction vessel watch window are equipped with prism, and the prism for being equipped with scanner-recorder side is located at reaction vessel sight
It examines between window and scanner-recorder.As a preferred technical solution, the reaction vessel watch window and image recording
Immersion oil layer is equipped between prism between device and reaction vessel watch window.
It is previously described for determining the device of clinical sample small particles characteristic and concentration, institute as a preferred technical solution,
Stating scanner-recorder is utilizing total internal reflection fluorescence microscope (TIRFM) or based on charge-coupled device (CCD), complementary metal oxide
The camera of semiconductor (CMOS) and other similar device.
The beneficial effects of the present invention are:
The present invention is same by the concentration that probe label can embody clinical sample small particles origin marking object and known control particle
When determine to short grained characteristic and concentration, and the characteristic and concentration information are used for clinical diagnosis and therapy and detected, method letter
It is single, and work well.Transmission electron microscope (TEM) apparatus of the present invention that compare are low in cost, and are not limited by the concentration of particle
System, it is simple to operate, it is adapted to use in clinical setting.
Detailed description of the invention
Fig. 1 is candidate particles in the present invention, control particle and magnetic-particle schematic diagram；
Fig. 2 is the reaction vessel observation window schematic diagram that the present invention fills the mixture containing candidate particles；
Fig. 3 is the present invention for observing equipped with magnetic-particle apparatus structure schematic diagram；
Fig. 4 is image schematic diagram of the present invention containing candidate particles, control particle；
Fig. 5 is the apparatus structure schematic diagram that the present invention uses utilizing total internal reflection fluorescence microscope；
In figure: 1, candidate particles；2, particle is compareed；3, magnetic-particle；4, reaction vessel；5, candidate particles and magnetic-particle knot
It is fit；6, particle and magnetic-particle combination are compareed；7, excitation light source；8, deflect light；9, particle to be observed；10, optical filter；
11, scanner-recorder；12, the equipment in magnetic field can be generated；13, calculating and data storage device；14, prism；15, immersion oil layer；
16, fluorescence/transmitting light.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, right below in conjunction with examples and drawings 1~5
Technical solution of the present invention is clearly and completely described.
The present embodiment is the source for determining the candidate particles with one or more specific marker objects, and estimates clinical sample
Candidate particles concentration in product.The specific marker object refer to the source (such as specific cells) that can reflect candidate particles and
The significant substance of derived cell situation (such as dead cell).These significant substances are also referred to as the characteristic of particle.These are special
Specific labels can reside in particle surface, can also exist on inside particle.The characteristic of measured particle and concentration
Information can be used for clinical application, such as the monitoring of medical diagnosis on disease and treatment method.
Candidate particles are the extracellular vesica of living cells secretion or the cell fragment of dead cell in the present embodiment.If to be measured
Particle has the label in indicator cells source, then this label can disclose the source of particle, and in sample candidate particles concentration
Then show its abundance.Therefore, the characteristic (or source) and concentration of candidate particles provide the snapshot of cell metabolism, and for more
The good safety for diagnosing the illness or monitoring treatment and/or effect provide strong supplementary means.
Determine that the specific implementation method of clinical sample small particles characteristic and concentration is as follows:
Human body cell complete in clinical sample is removed first.The clinical sample include human blood sample (serum or blood plasma),
Urine, saliva or the sample eluted in buffer etc..Human blood sample product are usually by blood platelet (2.65~2.9 μm of diameter), red thin
The leucocyte group that born of the same parents' (7~8 μm of diameter) and diameter range are 7~8 μm (small lymphocyte) to 15~30 μm (monocyte)
At.Urine sample may contain leucocyte, red blood cell and epithelial cell.Therefore candidate particles diameter be likely less than 2.5 μm or
1.0 μm of person.Human body cell and blood platelet in sample can be used centrifugal method and they removed from sample.For example, will face
Bed sample is centrifuged 15min at 2500G, it is sufficient to remove the blood platelet and human body cell in sample.Supernatant caused by being centrifuged
It can be used for the present embodiment small particles characteristic and concentration analysis.
Then a part of supernatant samples of above-mentioned centrifugation removal human body cell and blood platelet are contacted with detection reagent
It incubates.Incubation temperature is 15~37 DEG C, preferably 20~30 DEG C；Incubative time is 5~60min.Specific incubation temperature and time
It needs to optimize specific Testing index by experiment.The detection reagent contains the first probe, and first probe is appropriate
Buffer system in can be combined with indicating first marker in its source on candidate particles；100 μ l supernatants are taken in the present embodiment
The detection reagent of liquid sample and isometric (i.e. 100 μ l), which mix, to be incubated, and the Incubation mixtures of 2x are prepared into.
Since candidate particles concentration may be very high in above-mentioned Incubation mixtures, it is thus possible to need to dilute.The present embodiment
Selection is diluted 100 times to 10ml, since the probe not being combined in Incubation mixtures also is diluted by the dilution step,
Therefore dilution needs to be changed according to specific candidate particles and clinical application, and needs using from healthy individuals and patient
A large amount of samples optimize.
The Incubation mixtures for taking a part to dilute again are mixed with the particle that compares for being combined with the second probe, and it is mixed to form first
Close object.Then the first mixture is mixed to form the second mixture with the magnetic-particle for being enclosed with the first binding partners.Institute
The second probe is stated to be wrapped up by the second marker on control particle；And second marker is similar to the first marker；With
Convenient for the first binding partner binds on magnetic-particle.
Then the second mixture containing candidate particles 1, control particle 2, magnetic-particle 3 is loaded into equipped with watch window
Reaction vessel in observed, observation preferably carry out at room temperature, as shown in Figures 1 to 3, and shoot photo record.It is given in Fig. 4
An example of this image is gone out.The figure illustrates two kinds of particles, i.e. candidate particles 1 from clinical sample and right
According to particle 2.Due to control particle 2 concentration be it is known, can according in picture frame control particle 2 quantity survey
The concentration of candidate particles.In this example, there are 10 control particle 2 and 12 candidate particles respectively, therefore it is micro- to obtain a control
Grain is with the quantity of candidate particles than the ratio for 1.2.Assuming that the concentration of control particle is 1.5 × 10^6, then candidate particles is dense
Degree is 1.8 × 10^6.Multiple images frame can be used, and using mean concentration or the method for median concentration, more accurately to estimate
Count the concentration of particle to be measured.In this way, the concentration of candidate particles has been determined in clinical sample, and the characteristic of candidate particles (is come
Source etc.) also determined according to the marker contained by it, these information can be used in various medical applications.
Above-mentioned method can also some be flexible, for example, in certain embodiments, magnetic-particle may not be necessary.?
In the embodiment, image can be shot under the different depth of field, weaker signal will be shown by those of not focusing particle, such as
The signal of fruit granule be lower than focused particle signal (peak signal) 50%, then these particles be considered it is sightless
And in the picture without counting.The candidate particles rule being applied to simultaneously in control particle and clinical sample, so that it may right
Candidate particles in clinical sample count, and then diagnosis for disease or Treatment monitoring establish the critical concentration of candidate particles.
In the method for not magnetic-particle, two or more probes can be used.It is, for example, possible to use three probes,
In addition to the second probe for compareing particle, combinable two probes on candidate particles, one for determining coming for candidate particles
Source, i.e. the first probe；One characteristic (whether with specific marker) for determining candidate particles, as third probe.
In this case, which will provide three relevant informations: the concentration of candidate particles, which kind of cell class candidate particles come from
Whether type and particle to be measured are from dead or dead cell.
In mentioned-above two methods, used probe can be fluorescence probe, and the first, second probe, including
It is differentiable for not having the third used in the method for magnetic-particle to visit issued wavelength of fluorescence.In two methods, used
The first probe be the probe combined with nucleic acid or lipid film, can only penetrate into from dead or just in dead cell
In conjunction with candidate particles, therefore it can indicate the source of candidate particles, i.e., instruction candidate particles are from dead or dead
Die cell.The first marker combined on candidate particles with the first probe refer to be primarily present in an organ, a kind of cell,
Blood platelet, the epithelium of a kind of cancer cell or blood vessels or the marker (can be a kind of specific protein) in endothelial cell, with
Just test information is used to diagnose or therapy monitors.
In mentioned-above method, on particle to be measured used probe can be specific probe be also possible to it is non-specific
The probe of property.Specific probe be with a binding partner binds on candidate particles, and the binding partners are special
The opposite sex is combined with another marker (non-attribute tags object) on candidate particles, such as the antibody marked with FTIC.Therefore
Specific probe is indicated with the presence or absence of attribute tags object on the particle to be measured, and uses the attribute tags object of candidate particles as.And
It is because the first marker above particle to be measured has indicated the characteristic of particle to be measured when using nonspecific probe.
Nonspecific probe can be the fluorescent molecule in conjunction with lipid film, can also be with the dyestuff in conjunction with nucleic acid.With lipid knot
The example of the fluorescent molecule of conjunction includes BODIPY probe (by the ThermoFisher for being located at the city State of Massachusetts, US Waltham
Scientific company sale)；Probe in conjunction with nucleic acid includes various forms of SYBR Green molecules.
In clinical application, particle secrete from living cells (such as excretion body) or the generation of decomposing by dead cell are distinguished
Particle is being necessary.By dead cell film generate particle may be containing nucleic acid and the film of these particles is damaged and
Become to permeate, so certain can be with the dyestuff in conjunction with DNA or other nucleic acid, such as propidium iodide (PI) and SYTOX(are by position
ThermoFisher Scientific company in the Massachusetts city Waltham manufactures) or their derivative, these permeable films,
And these dyestuffs are these impermeable films under normal circumstances.Therefore these dyestuffs can be used for determining whether candidate particles come
From in dead cell, and fluorescence caused by them are determined for the concentration of these candidate particles, and then the information that will be measured
It can be used for monitoring the cytotoxicity or/and validity of therapy (such as chemotherapy use for cancer treatment).
Probe in addition to fluorescence probe can be used in certain examples, for example, chemiluminescence can also be used for be measured
Grain detection.There is the second binding partners of affinity, Ke Yiyong with the second upper marker on candidate particles (such as antibody)
Chemiluminescent molecule can generate chemiluminescent enzyme and be marked.Can chemiluminescent enzyme include alkaline phosphatase and peppery
Root peroxidase (HRP).Chemiluminescence may be implemented in the presence of suitable substrate in these enzymes, with meet the later period observation and
Shoot photo.
In mentioned-above two methods, used control particle is sample is introduced into when detection process starts outer
Carry out particle, it, can be with right and wrong for showing the correct progress of detection process, and the concentration for calculating the candidate particles in sample
Magnetic particle, diameter dimension specification include: 50~100nm, 50~250nm, 50~500nm, and 50~1000nm or 500~
2000nm.It is preferable to use granules of polystyrene for the control particle, the second probe thereon can be encapsulated in it is intragranular
Hydrophobic fluorescence molecule, marker thereon can pass through common chemical method (such as peptide bond) Lai Jinhang covalent coupling to particle
Surface.Control particle and its relevant product can obtain (such as real positioned at the Bangs of Indiana, USA from commercial source
Test room).
In the method using magnetic-particle, the magnetic-particle is marked with the first binding partners, the first knot
Closing gametophyte can be antibody, nucleic acid, peptide, aptamer, peptidomimetic, ligand, receptor one of which or combinations thereof.This first combine spouse
Physical efficiency is specifically combined with the first marker in clinical sample on candidate particles.In the method using magnetic-particle
Main purpose is to attract and fix candidate particles and control particle on the surface of the watch window of reaction vessel, so as to shooting figure
Picture.The suitable dimension of magnetic-particle is 0.5~3.5 μm, 0.5~2 μm or 0.5~1.5 μm.Magnetic-particle can also be easily from quotient
Industry source obtains (such as positioned at laboratory Bangs of Indiana, USA).As shown in Fig. 2, magnetic-particle 3 with compare particle
2 combine to form compound 5, combine to form compound 6 with candidate particles 1.From this figure, it can be seen that and to be measured not all
Grain 1 and control particle 2 are all captured by magnetic-particle 3.In some embodiments, there are one between the two sides of reaction vessel 4
Fixed space, so that the candidate particles 1 and control particle 2 that are not combined are combined and are attracted to magnetic-particle 3 far from those
The particle on watch window surface, therefore have no effect on measurement result.
According to the observation with the photo of shooting record, the characteristic and concentration of candidate particles, are used in the clinical sample calculated
The critical concentration of medical diagnosis on disease or Treatment monitoring, the particle with certain markers can be by testing from health with patient's
Sample is established.For example, when being used for therapy detection, when the particle from dead nephrocyte is more than certain critical concentration,
Indicate that kidney has been poisoned；For another example when being used to monitor the chemotherapy, immunotherapy or cell therapy of cancer, due to these therapies
It is all expected to kill cancer cell, while may also can kill healthy cell.In the clinical sample from patients undergoing chemotherapy, it is originated from dry
The concentration of the particle of cell (having certain stem cell-specific marker's objects) can be used for monitoring the safety of chemotherapy；And it is thin to be originated from cancer
The granule density of born of the same parents then can be used for the effect of monitoring chemotherapy.Similarly, the particle from liver and kidney can be during chemotherapy
It is respectively used to the damage of monitoring liver and kidney.
It is further for distinguishing bacterium and true in addition, method above-mentioned can be used for diagnosis bacterium or fungal infection
Bacterium infection.Since bacterial cell is significantly less than fungal cell in clinical sample, and the form of fungal cell and bacterial cell respectively has
Feature, therefore its cell size can be used or cellular morphology distinguishes, but can also be using using bacterial indicator or fungal cell
The specific marker object of characteristic is combined to determine, or using a variety of methods to distinguish bacterium and fungi.So can be with
Wen Pei and observation are carried out using the method for front, to determine the result distinguished into one.
Method above-mentioned can be also used for the aggregation in detection characterization blood sample, to indicate disease or health status.
For example, aggregation contains blood platelet or other ingredients in the blood sample of the patient with severe sepsis.Therefore assemble
Body concentration and characteristic (such as, if contain leucocyte or other ingredients) can be used for diagnosing or predicting whether patient suffers from seriously
Septicemia or septic shock.But, because the size of these aggregations is likely larger than normal blood cell, diagnosis whether
When with septicemia, the clinical blood sample used need not carry out separation removal normal blood cell, and the camera shooting used
Equipment can be lower with amplification factor.Concrete principle are as follows: because bigger than blood platelet with blood platelet or its segment in aggregation, this
A little aggregations can pass through blood platelet marker and size identification.Blood sample from patient suspected can prevent blood platelet
In the condition of solidification with fluorescent marker the special antibody of blood platelet (or the like) incubate, be then diluted to suitable dense
Degree is placed in observation device and carries out camera shooting record and analysis.Pass through size discrimination orthoplastocyte and aggregation when analysis.?
Same amplification factor similarly observes how much (or whether there is) and septicemia of aggregation in device, and same range of observation
Severity is related.Critical value can be established by testing the sample of a certain number of health and patient.
The device of observation and shooting photo noted earlier, primary element includes the incubation for holding candidate particles
The reaction vessel 4 of mixture, the scanner-recorder 11 positioned at 4 watch window side of reaction vessel, and with scanner-recorder 11
The calculating of connection and data storage device 13.In the embodiment using fluorescence probe, observation device used further includes being used for
Cause candidate particles 1 and compare particle 2 in probe generate fluorescence excitation light source 7, and the excitation light source 7 launch swash
It shines and is irradiated to the watch window of reaction vessel 4 at a certain angle, so that the residual light of light 8 deflects, reduce exciting light and penetrate
The result of interference observation and shooting.Excitation light source 7 appropriate includes that can emit with the wavelength light for being suitable for exciting probe
LED.For example, the maximum absorption wavelength (excitation wavelength) of propidium iodide dyestuff be 493nm, therefore can launch wavelength be 493nm
Or the LED light source of the light close to 493nm is suitable.In order to be further reduced the interference of exciting light, can remember in described image
Optical filter 10 is preferably provided between record device 11 and 4 watch window of reaction vessel, 10 permission probes of optical filter appropriate generate
Fluorescence pass through, and exciting light is stopped to pass through.For example, the emission maximum light of propidium iodide is 636nm, therefore can stop
The optical filter 10 that light source at or near 493nm allows the light at or near 636nm to pass through simultaneously is adapted as the iodine of probe
Change the third pyridine.When using multiple fluorescence probes, for example, the second probe for compareing particle and the first spy for candidate particles
When needle exists simultaneously, one or two excitation light source 7 can be used to excite two probes, while optical filter 10 should select to allow
The transmitting light of two probes passes through simultaneously.It the use of a variety of fluorescent probe techniques has been this field profession in fluoremetry
Personnel's common method.
In the embodiment using magnetic-particle 3, can be equipped under the scanner-recorder 11 of equipment therefor can generate magnetic
Equipment 12(such as magnet), so as to by magnetic-particle 3 and the candidate particles captured by magnetic-particle 31 and control
Grain 2 attracts and is fixed to together the inner surface of 4 watch window of reaction vessel, is observed and/or shot photo.
Scanner-recorder 11 used in the device of foregoing observations and color matching photo, is typically chosen based on charge-coupled device
(CCD), the camera of complementary metal oxide semiconductor (CMOS) and other similar device.It combines fluorescence microscope and nanometer
Grain trace analysis (NTA) technology is for monitoring and counting labeled particle.In certain embodiments, it is possible to use high-resolution
Digital camera can shoot image.The resolution limit of one standard picture recording device is that optical diffraction limit is about 200nm, more
Nian Lai has developed many technologies to realize that the resolution ratio for being lower than diffraction limit, such as Laser Scanning Confocal Microscope, photon tunnel are shown
Micro mirror (PTM), near field optic Random Maps microscope, 4Pi, structured illumination microscope and spatial modulation illumination.However, these are high
Resolution camera is too complicated and expensive for most of clinical applications.
Digital camera in some other embodiment with low resolution is equally applicable to certain clinical applications.This is
Because limiting factor here is granule density, and granule density can be adjusted by diluting.For example, can be incited somebody to action by dilution
Granule density is adjusted to less than 10^5/ml(or cm3).If there is the particle of 2x10^5/ml to be attracted to the watch window of 1x1cm
The picture field of surface or camera, if shooting them using 20,000,000 pixel digital cameras, each particle averagely has 100
Pixel.Theoretical limit is one particle of each pixel.Therefore, the digital camera of 20,000,000 pixels should have enough point
Resolution come detect every milliliter up to 20,000,000 particles particle concentration.In addition, being exactly image there is also another limiting factor
The area on surface.The image of one 1 × 1cm, surface area are 10^8 μm2, i.e., can at most accommodate 10^8 diameter is 1 μm micro-
Grain.If granule density be 2 × 10^5/ml, every 500 μm2There to be 1 particle, it, will when the use of camera pixel being 20,000,000
Every 5 μm2Picture area have 1 pixel, this is greater than about the optical diffraction limit of 200nm.Therefore, based on charge-coupled device
CCD digital camera can be to provide enough resolution ratio for certain clinical applications.In practical applications, each view can be shot
Multiple images, can also in image different zones and/or different images sampled and analyzed, to obtain to be measured
The mean concentration or median concentration of grain.If the signal strength of particle with granular size there are if correlativity, the letter of particle
Number intensity can also be used in the size for estimating particle.
In addition utilizing total internal reflection fluorescence microscope (TIRFM) also can be used to be imaged.Aobvious using total internal reflection fluorescent
When micro mirror (TIRFM) is imaged, device is as shown in figure 5, the two sides of 4 watch window of reaction vessel are equipped with prism 14 to be used for
Total internal reflection.And be equipped between 4 watch window of prism 14 and reaction vessel of 11 side of scanner-recorder and be equipped with immersion oil layer 15, with
Guarantee that two contact surface any surface finish are conducive to observation shooting.But since TIRFM only illuminates the object near glass surface (at a distance of about
200nm), therefore it is only capable of shooting image along glass surface, using with certain limitation.
Therefore, it is previously described for determining the principle of device of clinical sample small particles characteristic and concentration i.e. are as follows: (1) will contain
After candidate particles 1, the mixed solution for compareing particle 2 are loaded into reaction vessel 4, particle 9 to be observed is formed, reaction vessel is passed through
4 watch windows are (mountable to have microscope) to be observed and/or shoots lower 1 or multiple pictures by scanner-recorder 11, and is transmitted
It is stored and is calculated to the calculating and data storage device 13 being connect with scanner-recorder 11, while obtaining phase in clinical sample
The characteristic and concentration information for closing particle are further used for medical diagnosis and therapy monitoring.(2) when magnetic-particle, will contain
After thering is the mixed solution of candidate particles 1, control particle 2 and magnetic-particle 3 to be loaded into reaction vessel 4, to be observed is formed
Grain 9；Magnetic-particle 3 captures candidate particles 1, control particle 2 respectively, forms complex 5 and 6, and can produce in bottom of device
Under the action of the equipment 12 of magnetisation field, it is absorbed and fixed at the watch window of reaction vessel 4, is then shot and remembers by scanner-recorder 11
Then record is stored and is calculated with data storage device 13 by calculating.(3) when using fluorescence probe, excitation light source 7 is issued
Exciting light, exciting light are irradiated at an angle on the particle 9 to be observed with fluorescence probe, and it is glimmering to cause fluorescence probe generation
Light 16, while remaining particle 8 to be observed is reflected back, fluorescence 16 penetrates optical filter 10, is shot by scanner-recorder 11
Then record is stored and is calculated with data storage device 13 by calculating.(5) or excitation beam 7 at a certain angle according to
It is mapped on prism 14, on the surface for the watch window for then directing light to reaction vessel 4 by prism 14；Only close to observation
Those of window surface particle 9 to be observed is illuminated and issues fluorescence 16, and fluorescence 16 passes through immersion oil layer 15 by scanner-recorder
11 records, scanner-recorder 11 are connected to computing device 13.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive.Although in addition, it should be understood that originally
Specification is described in terms of embodiments, but only includes not one technical solution, and this narrating mode of specification is only
It is only for clarity that the skilled in the art should refer to the specification as a whole, the technical solution in embodiment can also
To be properly combined, form other embodiments that can be understood by those skilled in the art.
Claims (20)
1. a kind of method for determining clinical sample small particles characteristic and concentration, it is characterised in that: comprise the following steps that
1): the clinical sample containing candidate particles being contacted into incubation with detection reagent first；The detection reagent contains at least one
A probe for candidate particles in marker samples；Then mixture is mixed to form with the particle that compares for being combined with probe again；
2): the mixture in step 1) being loaded into the reaction vessel equipped with watch window, observation and/or shooting photo note
Record；
3): according to observation in step 2 and shooting recording as a result, determining the characteristic and concentration of candidate particles in sample, and should
Characteristic and concentration information are applied to clinical diagnosis or therapy monitors.
2. a kind of method for determining clinical sample small particles characteristic and concentration, it is characterised in that: comprise the following steps that
A): the clinical sample containing candidate particles is contacted into incubation with detection reagent；The detection reagent contains for marking sample
The first probe of candidate particles, first probe can mutually be tied with first marker in its source is indicated on candidate particles in this
It closes；Then again with compare particle mixing obtain the first mixture, it is described control particle contain by the second marker wrap up second
Probe；Second marker is similar to the first marker；
B): the first mixture obtained in step a) is contacted to further training with the magnetic-particle for being coated with the first binding partners
It educates, obtains the second mixture；First binding partners can specifically with the first marker on candidate particles and compare
The second marker on particle combines；
C): the second mixture will be obtained in step b) and be loaded into the reaction vessel equipped with watch window, and by that can generate
The equipment in magnetic field attracts and is fixed to together by magnetic-particle and by the candidate particles and control particle that magnetic-particle is captured
The inner surface of reaction vessel watch window, to observe and/or shoot photo record；
D): according to the record observed and shot in step c) as a result, determining the characteristic and concentration of candidate particles in sample, and should
Characteristic and concentration information are applied to clinical diagnosis or therapy monitors.
3. a kind of method for determining clinical sample small particles characteristic and concentration, it is characterised in that: comprise the following steps that
S1: the clinical sample containing candidate particles is contacted into incubation with detection reagent；The detection reagent contains for marking sample
The first probe and third probe of candidate particles in this, first probe can be marked with indicating the first of its source on candidate particles
Note object combines；Then with compare particle and be mixed to form mixture, the control particle is combined with the second probe, and described second visits
Needle is wrapped up by the second marker of control particle；Second marker is similar to the first marker；
S2: the mixture in step S1 is loaded into the reaction vessel equipped with watch window, and observes and/or shoot photo note
Record；
S3: according to observation in step S2 and shoot recording as a result, determining the characteristic and concentration of candidate particles in sample, and should
Characteristic and concentration information are applied to clinical diagnosis or therapy monitors.
4. the method according to any one of claims 1 to 3 for determining clinical sample small particles characteristic and concentration,
It is characterized by: the clinical sample first passes through complete human body cell removal processing in advance, the candidate particles include living
The cell fragment or aggregation of extracellular vesica, dead cell that cell is secreted.
5. the method according to any one of claims 1 to 3 for determining clinical sample small particles characteristic and concentration,
It is characterized by also including dilution step, the dilution step be clinical sample contacted with detection reagent cultivate after dilute, then with
Compare particle mixing.
6. the method according to any one of claims 1 to 3 for determining clinical sample small particles characteristic and concentration,
It is characterized by: the probe be fluorescence probe or can chemiluminescent enzyme, and the first, second, and third probe has
Differentiable emission wavelength.
7. the method according to any one of claims 1 to 3 for determining clinical sample small particles characteristic and concentration,
It is characterized by: the control particle be non-magnetic particle, diameter dimension include 50~100nm, 50~250nm, 50~
500nm, 50~1000nm and 500~2000nm.
8. the method according to claim 2 for determining clinical sample small particles characteristic and concentration, it is characterised in that:
The first binding partners on the magnetic-particle include antibody, nucleic acid, peptide, aptamer, peptidomimetic, ligand, receptor or combinations thereof.
9. the method according to claim 2 or 8 for determining clinical sample small particles characteristic and concentration, feature exist
In: the diameter dimension of the magnetic-particle includes 0.5~3.5 μm, 0.5~2 μm and 0.5~1.5 μm.
10. the method according to claim 2 or 3 for determining clinical sample small particles characteristic and concentration, feature
Be: first probe includes the probe for having binding affinity to nucleic acid or lipid film, whether is used to indicate candidate particles
From dead cell or dead cell.
11. the method according to claim 3 for determining clinical sample small particles characteristic and concentration, feature exist
In: the third probe is used to determine the characteristic of candidate particles, that is, determines whether candidate particles have specific marker.
12. according to any one of claims 1 to 3 for determining the side of clinical sample small particles characteristic and concentration
Method, it is characterised in that: the clinical diagnosis includes hepatotoxicity diagnosis, acute kidney injury diagnosis, heart attack diagnosis and loses
Hypophosphatemic shock diagnosis；The therapy monitoring includes monitoring chemotherapy, cell therapy monitoring, immunotherapy monitoring and radiotherapy
Monitoring.
13. a kind of for determining the device of clinical sample small particles characteristic and concentration, it is characterised in that: including reaction vessel
(4), it is located at the scanner-recorder (11) of reaction vessel (4) watch window side, and the meter connecting with scanner-recorder (11)
It calculates and data storage device (13), the reaction vessel (4) needs for holding to contain described in claims 1 to 3 any one
Survey the mixture of particle.
14. according to claim 13 for determining the device of clinical sample small particles characteristic and concentration, feature exists
In: it further include for causing candidate particles (1) and compareing the excitation light source (7) of the probe generation fluorescence in particle (2).
15. special according to claim 1 for determining the device of clinical sample small particles characteristic and concentration described in 3 or 14
Sign is: being equipped with optical filter (10) between described image logger (11) and reaction vessel (4) watch window, the optical filter
(10) fluorescence that permission probe generates passes through.
16. according to claim 15 for determining the device of clinical sample small particles characteristic and concentration, feature exists
It is equipped with the equipment (12) that can generate magnetic field, below: described image logger (11) so as to by magnetic as stated in claim 2
Property particle (3) and the candidate particles (1) that are captured by magnetic-particle (3) and control particle (2) attract together and be fixed to anti-
The inner surface of container (4) watch window is answered, is conducive to observe and/or shoot photo.
17. according to claim 13 for determining the device of clinical sample small particles characteristic and concentration, feature exists
In: the two sides of reaction vessel (4) watch window are equipped with prism (14), and are equipped with the prism of scanner-recorder (11) side
(14) between reaction vessel (4) watch window and scanner-recorder (11).
18. according to claim 17 for determining the device of clinical sample small particles characteristic and concentration, feature exists
In: prism (14) and reaction vessel (4) watch window between reaction vessel (4) watch window and scanner-recorder (11)
Between be equipped with immersion oil layer (15).
19. according to claim 18 for determining the device of clinical sample small particles characteristic and concentration, feature exists
In: described image logger (11) is utilizing total internal reflection fluorescence microscope.
20. according to claim 16 for determining the device of clinical sample small particles characteristic and concentration, feature exists
In: described image logger (11) is the camera based on charge-coupled device and complementary metal oxide semiconductor.
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