CN106093392A - The integrated testing method secrete body separation outside a kind of urine, being enriched with and detecting and detection chip - Google Patents
The integrated testing method secrete body separation outside a kind of urine, being enriched with and detecting and detection chip Download PDFInfo
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- CN106093392A CN106093392A CN201610386872.5A CN201610386872A CN106093392A CN 106093392 A CN106093392 A CN 106093392A CN 201610386872 A CN201610386872 A CN 201610386872A CN 106093392 A CN106093392 A CN 106093392A
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Abstract
The present invention provides a kind of outer integrated testing method secreted body separation, be enriched with and detect, including: designing and assemble double membrane filtration chip, build chip ELISA examination criteria curve, sample collection, note sample and cleaning, chip ELISA detects, Data Management Analysis.The present invention also provides for secreting the integrated detection chip that body separates, is enriched with and detects outside a kind of urine.The reaction system of the present invention and detection method, can be used for detecting transitional cell bladder carcinoma sample or the content of body is secreted by bladder cancer cell lines culture fluid supernatant China and foreign countries, this method has the feature of high degree of specificity, and human body will not be had any impact or wound, detection method, also without expensive and accurate experimental apparatus (such as supercentrifuge, fluorescence microscope), has great application prospect.
Description
Technical field
The invention belongs to field of biological technology detection, be specifically related to develop outside a kind of integrated urine secrete body separate, enrichment and
The micro-fluid chip of ELISA detection.
Background technology
Bladder cancer refers to the malignant tumor in bladder mucosa.It is the modal malignant tumor of urinary system, is also
One of big kinds of tumor of whole body ten.Account for first of China's Genitourinary system sickness rate, and its sickness rate is the most secondary in west
In carcinoma of prostate, occupy the 2nd.Inspection method includes routine urianlysis, urine sediment, urine tumor marker, abdominal part and basin
Chamber B ultrasonic etc. check.Decide whether that row cystoscope, IVU, pelvic cavity CT are or/and pelvic MRI according to above-mentioned inspection result
Clarify a diagnosis Deng inspection.Wherein, cystoscopy is the main method of diagnosing bladder cancer.But cystoscope detects costly,
It is not particularly suited for the popularization and application of different medical unit or poverty-stricken area, the most urgently finds a kind of for bladder cancer detection
Biomarker detect.Secreting outward body (exosomes) is that a kind of diameter is about 40-120nm, has double-deck plasma membrane knot
The vesicle of structure, by cell by exocytosis release to extracellular gap or biological body fluid.In recent years, secrete outward body and become new
Study hotspot, Yin Qinei comprises the multi-signal such as mRNAs, microRNAs and albumen molecule specific to derived cell, at signal
Playing an important role in conduction and immune system, and reduced microenvironment interference by complete membranous structure parcel, this makes it in disease
Sick diagnosis aspect has the superiority of uniqueness, is research biomarker and the new biomaterial carrying out tumour immunity, simultaneously
Also there are treatment potentiality.
It is broadly divided into isolation technics and detection technique currently for the research secreting outward body.Isolation technics includes ultracentrifugation
Method, filter centrifugation method, Density ultracentrifugation, immunomagnetic beads combine supercentrifugation and chromatography etc..Detection technique master
Including: sem observation form (but pretreatment and the preparation of sample are required higher by SEM above, the standard of sample
The standby stage is more complicated, is not suitable for externally secreting body and carries out a large amount of quickly measuring, and due to secrete outward body have passed through pretreatment and
Preparation process, it is impossible to carry out the outer measurement secreting bulk concentration accurately), dynamic light scattering technique is (but due to dynamic light scattering technique
It is the fluctuation data measuring light intensity, so oarse-grained light-intensity variation signal can cover the light-intensity variation signal of smaller particle, institute
Being not suitable for the complicated outer measurement secreting body sample not of uniform size with dynamic light scattering, the size being only suitable for being prepared by chromatography is equal
The outer dimensional measurement secreting body of one, and the concentration of body cannot be secreted in measuring samples China and foreign countries.), flow cytomery technology (streaming
Cell instrument is possible not only to detect the size of vesicle, quantity, and can be detected the source of vesicle by fluorescent labeling, is entered by vesicle
Row classification, therefore, flow cytometer be by vesicle quickly, the optimal choice of high flux, multiparameter detection.But, conventional flow
Formula cell instrument for sample be mainly cell, the detectable limit of scattered light is typically 300-500nm, and most cells external capsule
The diameter of bubble, all at below 300nm, is therefore difficult to the most qualitatively and quantitatively analyze.), nanoparticle follow-up analysis technology
(it is a kind of method of relatively new research nano-particle, observation nano-particle that can be direct and real-time.Owing to secreting outward body surface
There is the existence of the transmembrane molecules such as mark CD9, CD63 in face, under complicated background environment (in serum), can resist with fluorescence
Secrete body outside body tag, then under complex background, externally secrete the measurement of body with the fluorescence measurement functional realiey of NTA, however it is necessary that precision
Instrument), flow cytometry analysis technology (but flow cytometry once can only detect, outward for a mark
Secrete body the least, by current flow cytometer device detection, it is necessary to the first binding coated magnetic bead of foreign antibodies, need precision
Instrument and equipment, and complex operation, sensitivity are different), immune-blotting method technology (operating process is complicated) and quantitative fluorescent PCR examine
It is complicated that cls analysis microRNA(extracts nucleic acid step, needs expensive experimental apparatus).Therefore, development one can reliably, soon
Speed, secreting body especially bladder cancer patients source outer outside Non-invasive detection economically, to secrete the method for body the most necessary.Micro-fluid chip
Micro-fluid chip laboratory, refer to the sample involved in the fields such as biological and chemical to prepare, biological with chemical reaction, separate
On the chip that the basic operation units such as detection are integrated or the most integrated a piece several square centimeters, in order to complete different biologies or change
Learn course of reaction, and a kind of technology that its product is analyzed.It is life sciences, chemical science and information science signal detection
Important technological platform with Study on processing method.Have integration, analyze that speed is fast, high flux, less energy consumption, pollution are little, cheap,
The features such as safety.Therefore, build a kind of integrated outer body of secreting and separate, be enriched with, be detected as micro-fluid chip integrally, have huge
Economic benefit.
Secreting outward body can be with stable existence in urine, and especially urine sample can be big on the premise of atraumatic damages
Amount obtains.If it is possible to the cancer information secreted outside detection urine in body, then be expected to use it for cancer non-invasive diagnosis,
The clinical practices such as monitoring.But, it practice, owing to the outer body burden of secreting in unit volume urine is the lowest, use conventional method
What single-trial extraction obtained outer secretes body, cannot meet the requirement of subsequent detection at all, thus main currently for the detection secreting outward body
It is first the outer body of secreting in sample to be carried out concentration, takes certain sign to measure the most again.Therefore, the present invention is directed to above-mentioned
Problem, have developed a kind of integrated outer body of secreting and separates, is enriched with and is detected as micro-fluid chip integrally, secretes body cross-film outside utilizing simultaneously
This distinctive mark of PROTEIN C D63, establishes a kind of chip ELISA fast diagnosis method.The method is outside bladder cancer patients
Secrete the aspects such as health check-up survey, monitoring and there is certain application prospect.
Summary of the invention
It is an object of the invention to provide a kind of outer integrated testing method secreted body separation, be enriched with and detect, the method is
Secrete outward body chip ELISA detection method, can effectively detect bladder cancer patients inside and outside and secrete the amount of body, testing result high specificity,
Practical.
In order to realize the purpose of the present invention, the present invention by the following technical solutions: secrete outside a kind of urine body separate, enrichment and
The integrated testing method of detection, it is characterised in that comprise the following specific steps that:
1) design and assemble double membrane filtration chip: described pair of membrane filtration chip is four layers of PMMA plate, every thick layer from top to bottom
Degree is respectively 2 mm, 1 mm, 1 mm, and 2 mm, and chip outside dimension is 20 40 6 mm, adjacent two layers PMMA
Connect with double faced adhesive tape (DSA) between plate, middle two-layer PMMA plate is respectively cut the consistent inlet opening of upper-lower position and waste liquid
Tap, the PMMA plate of the superiors is the PMMA flaggy of two circular holes being cut with diameter 1.8 mm；The second layer is straight for being cut with
The PMMA flaggy of two circular holes of footpath 10 mm；Third layer is to cut two circular holes of diameter 10 mm, simultaneously between two circular holes
Use width 1.5mm, the PMMA flaggy that the channel of a length of 11mm is connected；Last layer, for PMMA flat board.Wherein, middle two-layer
It is provided with two-layer DSA glue between PMMA plate, is stained with aperture between this two-layer DSA glue and is respectively the Merlon of 200nm and 30nm
Film (such as Fig. 1), for the collection filtering and secreting outward body of liquid.
2) chip ELISA examination criteria curve is built:
1. collection transitional cell bladder carcinoma sample 200mL, employing Ultracentrifugation Method (2,000g room temperatures are centrifuged 10-15min, and
Removing cell and fragment thereof, by supernatant with 100,000 g rotating speed surpasses from 1-2h, after with the PBS of pH 7.4, resuspended outer to secrete body heavy
Form sediment,
2. by secreting body suspension outside 1. gained, it is diluted according to the different proportion of 1:3,1:9,1:27 and 1:81, distinguishes afterwards
Take and above-mentioned a certain amount of outer secrete body suspension stock solution and each 300 μ L of diluent, inject with miniflow syringe pump with the flow velocity of 40 μ L/min
In double membrane filtration chips of step 1),
3., after, it is dilute that each pair of membrane filtration chip continues to inject 300 μ L anti-CD63(1:200-1:500 with identical flow velocity
Releasing, concentration is 2-5 μ g/mL) after, carry out hatching 1-1.5h under the conditions of 25 DEG C,
4. clean double membrane filtration chip with 300-400 μ L PBS, repeat three-four times, clean and terminate backward each pair of film
Inject 500 a certain amount of air of μ L with identical flow velocity in filtrating chip and drain liquid in double membrane filtration chip,
5. the HRP(1:2000-1:5000 dilution of 300 μ L streptavidin labellings, concentration is injected to each pair of membrane filtration chip
For 0.2-0.5 μ g/mL), and be placed on and hatch 1-2h in 37 DEG C in wet box,
4. and 5. three-four times 6. step is repeated.
7. 300 μ L TMB nitrite ions, and 37 DEG C of colour developing 5-30min in darkroom are injected to each pair of membrane filtration chip,
8. data acquisition is carried out by mobile phone imaging system,
The most finally utilize transmission of wireless signals to arrive notebook computer, carry out Treatment Analysis to gathering data, thus it is bent to build standard
Line.
3) sample collection, note sample and cleaning: collect healthy volunteer/transitional cell bladder carcinoma sample 10 ml, use 2,
000 g room temperature is centrifuged 10-15 minute, and removes cell and relic thereof, by supernatant with miniflow syringe pump with the stream of 40 μ L/min
Speed is continuously injected in double membrane filtration chip, and injected slurry volume is 8mL, finally with the PBS of 400 μ LpH 7.4 with the flow velocity of 40 μ L/min
It is injected in double membrane filtration chip, repeats this step 3-4 time, complete to capture the cleaning of sample.
4) chip ELISA detection: use step 2) 3.-9., the outer body of secreting being captured step 3) detects.
5) Data Management Analysis: bring testing result into standard curve, carries out the outer comparison secreting bulk concentration.
6) according to step 2)-5) secrete body so that PBS replacement is outer, set up negative control group to carry out experimental control.
First the present invention carries out design and the assembling of micro-fluid chip.Laser cutting machine is utilized to carry out chip body PMMA
The cutting of plate assembles.The thickness range of PMMA plate is 1-2mm.Sample import and export hose outside diameter be 1.5-1.8mm, described flexible pipe and
Chip body is all glued firmly.
Build and outer secretes body ELISA examination criteria curve, will surpass and secrete body liaison series doubling dilution from outer in urine
(1;1:3; 1:9; 1:27;1:81) become different concentration, build standard curve afterwards.Follow-up, sample collection measurement result with
Known standard curve contrasts, and judges the outer content secreting body in the patient.
While using technique scheme, the present invention can also use or combine and use technology further below
Scheme:
When injecting liquid in double membrane filtration chip, Liquid sample introduction velocity interval is 30-60 μ L/min, with miniflow syringe pump or
Person's automatic sampler carries out sample injection.
The cleaning mixture that described sample uses in cleaning, for phosphate buffer PBS(pH 7.4 ± 0.2), washing times
Typically 2-4 time.
When sample-adding in double membrane filtration chips, each sample is 2-3 repetition, every hole 300 μ L.
The standing and reacting of the described ELISA Plate adding sample is biotin and streptavidin combination, and its reaction temperature is generally
35-37 DEG C, the time is usually 1-2 hour.
Described nitrite ion is tetramethyl benzidine (TMB), and chromogenic reaction is HRP and TMB association reaction, during chromogenic reaction
Between generally 5-30 minute, reaction temperature is generally 35-37 DEG C.
The reaction system of the ELISA detection method of the present invention, mainly includes following components: secrete outward body, biotin labeled
Anti-CD63, HRP, TMB nitrite ion of streptavidin, termination reactant liquor, sample cleaning mixture PBS and urine specimen.
Another technical problem to be solved by this invention is to provide outside a kind of urine secretes the collection that body separates, is enriched with and detects
Become detection chip, it is possible to complete outer secrete body separation, be enriched with and be that basis is done in detection.
The present invention solves technical problem and be the technical scheme is that secreting body outside a kind of urine separates, is enriched with and detects
Integrated detection chip, including base plate, top board and the detection plate between described base plate and top board, described detection plate includes
Layer detection plate and lower floor's detection plate, described top board is provided with liquid injection hole and the outage that cutting is formed, described liquid injection hole connects
Having fluid injection flexible pipe, described outage connects a discharge opeing flexible pipe, described upper strata detection plate and lower floor's detection plate are respectively equipped with into
Sample hole is corresponding with the position of described sample holes with liquid injection hole described in waste liquid tap, described outage and described waste liquid tap
Position corresponding；
It is pasted together by the first layers of two-sided is fixing between described top board and described upper strata detection plate, described first double faced adhesive tape
Layer is provided with the first corresponding with described liquid injection hole and described outage respectively perforate, described upper strata detection plate and described lower floor
Detection plate between be provided with two-layer the second layers of two-sided, this two-layer DSA layers of two-sided is equipped with respectively with described sample holes and institute
State the second perforate that waste liquid tap is corresponding, be stained with between the second perforate below sample holes that aperture is 200nm
One Merlon diaphragm, is stained with the second Merlon that aperture is 30nm between the second perforate below waste liquid tap
Diaphragm；It is communicated with described sample holes and the first passage of described waste liquid tap on described lower floor detection plate；Lower floor's detection plate
And be provided with the 3rd layers of two-sided between described base plate, described 3rd layers of two-sided be provided with respectively with described sample holes and described useless
The 3rd perforate that liquid tap is corresponding, is provided with the second channel corresponding with described first passage between described 3rd perforate.
While using technique scheme, the present invention can also use or combine and use technology further below
Scheme:
Described base plate, top board are, with levels detection plate, the PMMA plate that shape size is consistent.
The aperture of described liquid injection hole and described outage is 1.8mm, described sample holes and the hole of described waste liquid tap
Footpath is 10mm, and described liquid injection hole is arranged concentrically with sample holes, and described outage is arranged concentrically with described waste liquid tap.
Constituting a length of 40mm of the PMMA plate of described base plate, top board and detection plate, width is 20mm, described integrated inspection
The thickness surveying chip is 6mm.
Compared with prior art, beneficial effects of the present invention is as follows:
1) the invention provides a kind of body of secreting outside integrated to separate, be enriched with and detect micro-fluid chip, construct chip simultaneously
ELISA detection method.This reaction system and detection method, can be used for detecting transitional cell bladder carcinoma sample or bladder cancer cell lines
The content of body is secreted by culture fluid supernatant China and foreign countries, and this method has the feature of high degree of specificity.
2) detection of the present invention to as if discharge the urine outside human body, be readily available, and human body will not be caused any
Impact or wound, detection method, also without expensive and accurate experimental apparatus (such as supercentrifuge, fluorescence microscope), has
The biggest application prospect.
Accompanying drawing explanation
Accompanying drawing for and be embodied as case combine the invention will be further described.
Fig. 1 is double membrane filtration chip structure schematic diagrams.
Fig. 2 is double membrane filtration chip pictorial diagram.
Fig. 3 is the testing result comparison diagram of double membrane filtration chip.
Fig. 4 secretes body ELISA Cleaning Principle figure outside being.
Fig. 5 secretes body chip ELISA examination criteria curve outside being.
Fig. 6 is that outer body of secreting separates, is enriched with and overhaul flow chart.
Fig. 7 is ELISA detection method Clinical detection box traction substation.
Fig. 8 is ROC curve analysis result.
Detailed description of the invention
Below in conjunction with instantiation, the present invention is expanded on further.It should be noted that these examples be merely to illustrate the present invention and not
For limiting the scope of the present invention.
Unreceipted concrete experimental condition and test method according to normal condition and method or manufacturer in case study on implementation
Proposed condition is practiced.
Various instruments not specified in the present invention and reagent are commercially available prod well known in the art, can pass through business
Approach buying obtains.
Embodiment 1: separation and the detection of chip ELISA method of body, 1-8 referring to the drawings secrete in transitional cell bladder carcinoma China and foreign countries.
Refer to Fig. 1-7, secrete body outside a kind of urine and separate, be enriched with and the integrated testing method of ELISA detection, including as follows
Step:
1) on the premise of patient knows the inside story and Ethics Committee is agreed to, (clinical biopsy has proven to as bladder to collect bladder cancer patients
Cancer) urine specimen 16 parts, healthy donor 8 parts, every part of 100ml, remove under room temperature 2, the centrifugal condition of 000g cell and
Big fragment, then obtains the supernatant.
2) by 1) supernatant that obtains, under the conditions of 4 DEG C, 100,000g ultracentrifugations 60 minutes, discard supernatant take heavy
Form sediment, and secrete body (such as Fig. 1) outward with the PBS of 1mL pH 7.4 is resuspended.
3) by 1) gained supernatant, utilize miniflow pump, be injected in double membrane filtration chip with flow velocity 40 μ L/min, continuously
Filter 8 times.
4) by 2) secrete body outside gained, carry out serial dilution according to 3 times of multiple proportions, preserve respectively, in case subsequent builds standard is bent
Line.
5) by 4) standby secreting body carries out chip ELISA detection (principle such as Fig. 2) outward, first to 4 in institute) chips injection antibody,
Every hole adds the anti-CD63(biotin-labeled of 300 μ L of 1:200-1:500 dilution) (i.e. concentration is 2-5 μ to antibody
G/mL), 25 DEG C of incubation 1-1.5h.
6) to 5) in inject 300-400 μ L PBS be carried out, in triplicate.
7) to 6) inject 500 μ L air to drain liquid in chip with identical flow velocity in each chip.
8) to 7) each chip inject 300 μ L streptavidin labellings HRP(1:2000-1:5000 dilution, concentration is
0.2-0.5 μ g/mL), and be placed in wet box and hatch 1-2h in 37 DEG C.9) step 6) and 7 is repeated).
10) to 9) in each filtrating chip inject 300 μ L TMB nitrite ions, and 37 DEG C of colour developing 5-30min in darkroom.
11) data acquisition is carried out with image capturing system (from imaging system in such as mobile phone).
12) by 11) the data obtained in notebook computer, utilizes ImageJ to carry out RGB analysis by transmission of wireless signals,
Build standard curve (such as Fig. 4-5), use above-mentioned 1 simultaneously)-12) step carries out secreting outside bladder cancer patients body burden detection, goes forward side by side
Row ROC curve is analyzed and builds box traction substation.Testing result such as Fig. 6.
Embodiment 2, integrated detection chip, referring to the drawings 1-3.
The micro-fluid chip of the present invention includes base plate 1, top board 2 and the detection plate between described base plate and top board,
Being provided with liquid detecting chamber in described detection plate, the outer body of secreting in urine can be collected in wherein by described liquid detecting chamber, and leads to
Cross repeatedly note sample enrichment and reach the requirement of detection.
Described detection plate includes upper strata detection plate 3 and lower floor's detection plate 4, and described top board 2 is provided with the fluid injection that cutting is formed
Hole 5 and outage 12, described liquid injection hole 5 connects and has liquid injection pipe 6, and described outage 12 connects a discharging tube 13, described on
It is respectively equipped with sample holes 7 and waste liquid tap 8, described liquid injection hole 5 and described sample holes 7 on layer detection plate 3 and lower floor's detection plate 4
Position corresponding, described outage 12 is corresponding with the position of described waste liquid tap 8.
It is pasted together by the first layers of two-sided 9 is fixing between described top board 2 and described upper strata detection plate 3, described the
One layers of two-sided 9 is provided with the first corresponding with described liquid injection hole 5 and described outage 12 respectively perforate 14, and described upper strata is examined
It is provided with two-layer the second layers of two-sided 15 between drafting board 3 and described lower floor detection plate 4, this two-layer DSA layers of two-sided 15 is equipped with
The second corresponding with described sample holes 7 and described waste liquid tap 8 respectively perforate 16, is positioned at the second perforate below sample holes 7
Be stained with the first Merlon diaphragm 10 that aperture is 200nm between 16, be positioned at the second perforate 16 below waste liquid tap 8 it
Between be stained with the second Merlon diaphragm 17 that aperture is 30nm.
It is communicated with described sample holes 7 and the first passage 11 of described waste liquid tap 8 on described lower floor detection plate 4.
Being provided with the 3rd layers of two-sided 18 between lower floor's detection plate 4 and described base plate 1, described 3rd layers of two-sided 18 is provided with
The 3rd corresponding with described sample holes 7 and described waste liquid tap 8 respectively perforate 19, is provided with and institute between described 3rd perforate 19
State the second channel 20 of first passage 11 correspondence.
Described base plate 1, top board 2 are, with levels detection plate, the PMMA plate that shape size is consistent.
Constituting a length of 40mm of the PMMA plate of described base plate 1, top board 2 and detection plate, width is 20mm, described miniflow
The integral thickness of body chip is 6mm.
Described first layers of two-sided the 9, second layers of two-sided 15 and the 3rd layers of two-sided 18 are DSA double faced adhesive tape.
Described liquid injection pipe 5 and described discharging tube 13 are sterile hose.
The aperture of described liquid injection hole 5 and described outage 12 is 1.8mm, described sample holes 7 and described waste liquid tap 8
Aperture be 10mm, described liquid injection hole 5 is arranged concentrically with sample holes 7, and described outage 12 is concentric with described waste liquid tap 8
Arrange.
In the present embodiment, the cutting utilizing laser cutting machine to carry out chip body PMMA plate assembles, the thickness model of PMMA plate
Enclosing for 1-2mm, the liquid injection pipe external diameter importing and exporting flexible pipe as sample is 1.5-1.8mm, and described flexible pipe and chip body all use glue
Bonding is firmly.
The operation principle of the micro-fluid chip of the present invention is: by urine to be detected by liquid injection pipe 5 according to certain flow rate
It is injected in liquid injection hole 5, and hence into the sample holes 7 of upper strata detection plate 3, urine is through being arranged on entering of upper strata detection plate 3
Aperture below sample hole 7 is the filtration of the first Merlon diaphragm 10 of 200nm, magazine and macromole cell is removed, filtrate
Enter in the sample holes 7 of lower floor's detection plate 4, and enter in the waste liquid tap 8 on lower floor's detection plate 4 by passage 11, warp
By the filtration of the second Merlon diaphragm 17 that aperture is 30nm, secrete outward the waste liquid tap that body is collected in the version 14 of lower interlayer
In 8, filtrate enters in the waste liquid tap 8 of upper strata detection plate 3 through the second Merlon diaphragm 17, and can be via outage
12 and discharging tube 13 be discharged.
The chip of present invention case in actual use is as described in Example 1.
Claims (10)
1. secrete the integrated testing method that body separates, is enriched with and detects outside a urine, it is characterised in that include following concrete step
Rapid:
1) design and assemble double membrane filtration chip: described pair of membrane filtration chip is four layers of PMMA plate, between adjacent two layers PMMA plate
Connect with double faced adhesive tape (DSA), middle two-layer PMMA plate be respectively cut the consistent inlet opening of upper-lower position and waste liquid tap,
Liquid injection hole corresponding with inlet opening and waste liquid tap respectively and outage, described liquid injection hole it is cut with on the PMMA plate of the superiors
With connect flexible pipe on outage respectively, the bottom is PMMA flat board, is provided with two-layer DSA glue between middle two-layer PMMA plate, and these are two years old
It is stained with aperture between layer DSA glue and is respectively the polycarbonate membrane of 200nm and 30nm, for the receipts filtering and secreting outward body of liquid
Collection；
2) chip ELISA examination criteria curve is built:
1. collect, and remove cell and fragment thereof, supernatant is surpassed and precipitates from the resuspended outer body of secreting of rear PBS,
2. 1. will secrete body suspension outside gained, be diluted according to different proportion, take that a certain amount of outer to secrete body suspension former the most respectively
Liquid and diluent miniflow syringe pump are injected in double membrane filtration chips of step 1),
3. after, each pair of membrane filtration chip continues to inject anti-CD63(1:200-1:500 dilution, and concentration is 2-5 μ g/mL)
After hatch,
4. it is carried out double membrane filtration chip with PBS, cleans injection in terminating backward each pair of membrane filtration chip certain
The air of amount drains liquid in double membrane filtration chip,
5. the HRP(1:2000-1:5000 dilution of 300 μ L streptavidin labellings, concentration is injected to each pair of membrane filtration chip
For 0.2-0.5 μ g/mL), and hatch in wet box,
6. inject TMB nitrite ion to each pair of membrane filtration chip, and develop the color in darkroom,
7. data acquisition is carried out by imaging system,
8. collection data are carried out Treatment Analysis, thus build standard curve；
3) sample collection, note sample and cleaning: collecting urine specimen, room temperature is centrifuged and removes cell and relic thereof, is used by supernatant
Miniflow syringe pump is continuously injected in double membrane filtration chip, after be injected into double membrane filtration chip with the PBS of pH 7.4 with certain flow rate
In, it is repeated several times, completes the cleaning of sample；
4) chip ELISA detection: use step 2) 3.-8., the outer body of secreting being captured step 3) detects；
5) Data Management Analysis: bring testing result into standard curve, carries out the outer comparison secreting bulk concentration.
Secreting the integrated testing method that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 1, its feature exists
In, when injecting liquid in double membrane filtration chip, the sample introduction velocity interval of liquid is 30-60 μ L/min, uses miniflow syringe pump
Or automatic sampler carries out sample injection.
Secreting the integrated testing method that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 1, its feature exists
In, the cleaning mixture that described sample uses in cleaning is phosphate buffer PBS(pH 7.4 ± 0.2), washing times is 2-4
Secondary.
Secreting the integrated testing method that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 1, its feature exists
In, when sample-adding in double membrane filtration chips, each sample repeats 2-3 time, every hole 300 μ L.
Secreting the integrated testing method that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 1, its feature exists
In, adding the ELISA Plate of the sample standing and reacting when carrying out wet box and hatching and be biotin and streptavidin combines, it react warm
Degree is for 35-37 DEG C, and the time is 1-2 hour.
Secreting the integrated testing method that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 1, its feature exists
In, described nitrite ion is tetramethyl benzidine (TMB), and chromogenic reaction is HRP and TMB association reaction, and the chromogenic reaction time is
5-30 minute, reaction temperature was 35-37 DEG C.
7. outside a urine, secrete the integrated detection chip that body separates, is enriched with and detects, it is characterised in that: include base plate (1), top board
(2) the detection plate and between described base plate and top board, described detection plate includes upper strata detection plate (3) and lower floor's detection plate
(4), described top board (2) is provided with liquid injection hole (5) and the outage (12) that cutting is formed, and the upper connection of described liquid injection hole (5) has note
Liquid flexible pipe (6), the upper connection of described outage (12) has discharge opeing flexible pipe (13), described upper strata detection plate (3) and lower floor's detection plate (4)
On be respectively equipped with sample holes (7) and waste liquid tap (8), described liquid injection hole (5) is corresponding with the position of described sample holes (7),
Described outage (12) is corresponding with the position of described waste liquid tap (8)；
It is pasted together by the first layers of two-sided (9) is fixing between described top board (2) and described upper strata detection plate (3), described
First layers of two-sided (9) is provided with the first perforate (14) corresponding with described liquid injection hole (5) and described outage (12) respectively,
Being provided with two-layer the second layers of two-sided (15) between described upper strata detection plate (3) and described lower floor detection plate (4), this two-layer DSA is double
The second perforate (16) corresponding with described sample holes (7) and described waste liquid tap (8) respectively, position it is equipped with on face glue-line (15)
It is stained with, between second perforate (16) of sample holes (7) lower section, the first Merlon diaphragm (10) that aperture is 200nm, is positioned at
It is stained with, between second perforate (16) of waste liquid tap (8) lower section, the second Merlon diaphragm (17) that aperture is 30nm；Institute
State the first passage (11) being communicated with described sample holes (7) and described waste liquid tap (8) in lower floor's detection plate (4)；Lower floor
Being provided with the 3rd layers of two-sided (18) between detection plate (4) and described base plate (1), described 3rd layers of two-sided (18) is provided with respectively
Threeth perforate (19) corresponding with described sample holes (7) and described waste liquid tap (8), is provided with between described 3rd perforate (19)
The second channel (20) corresponding with described first passage (11).
Secreting the integrated detection chip that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 7, its feature exists
In: described base plate (1), top board (2) are, with levels detection plate, the PMMA plate that shape size is consistent.
Secreting the integrated detection chip that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 7, its feature exists
In: the aperture of described liquid injection hole (5) and described outage (12) is 1.8mm, described sample holes (7) and described waste liquid tap
(8) aperture is 10mm, and described liquid injection hole (5) is arranged concentrically with sample holes (7), and described outage (12) is arranged with described waste liquid
Portal (8) be arranged concentrically.
Secreting the integrated detection chip that body separates, is enriched with and detects outside a kind of urine the most as claimed in claim 1, its feature exists
In: constituting a length of 40mm of the PMMA plate of described base plate (1), top board (2) and levels detection plate, width is 20mm, institute
The thickness stating integrated detection chip is 6mm.
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