CN106093392B - The integrated testing method and detection chip of a kind of urine excretion body separation, enrichment and detection - Google Patents
The integrated testing method and detection chip of a kind of urine excretion body separation, enrichment and detection Download PDFInfo
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- CN106093392B CN106093392B CN201610386872.5A CN201610386872A CN106093392B CN 106093392 B CN106093392 B CN 106093392B CN 201610386872 A CN201610386872 A CN 201610386872A CN 106093392 B CN106093392 B CN 106093392B
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Abstract
The present invention provides the integrated testing method of a kind of excretion body separation, enrichment and detection, including：Design and the double membrane filtration chips of assembling, build chip ELISA examination criteria curves, sample collection, note sample and cleaning, chip ELISA detections, Data Management Analysis.The present invention also provides the integrated detection chip of a kind of separation of urine excretion body, enrichment and detection.The reaction system and detection method of the present invention, content available for excretion body in detection transitional cell bladder carcinoma sample or bladder cancer cell lines nutrient solution supernatant, this method has the characteristics that high degree of specificity, and will not have any impact to human body or wound, detection method is also without expensive and accurate laboratory apparatus（Such as ultracentrifuge, fluorescence microscope）, have great application prospect.
Description
Technical field
The invention belongs to field of biological technology detection, and in particular to a kind of integrated urine excretion body separation of exploitation, enrichment and
The micro-fluid chip of ELISA detections.
Background technology
Carcinoma of urinary bladder refers to the malignant tumour in bladder mucosa occurs.It is the most common malignant tumour of urinary system, and
One of ten big kinds of tumor of whole body.First of China's Genitourinary system incidence is accounted for, and its incidence is only secondary in west
In prostate cancer, the 2nd is occupied.Inspection method includes routine urianlysis, urine sediment, urine tumor marker, belly and basin
Chamber B ultrasound etc. checks.Row cystoscope, intravenous urography, pelvic cavity CT or/and pelvic MRI are decided whether according to above-mentioned inspection result
Clarify a diagnosis Deng inspection.Wherein, cystoscopy is the main method of diagnosing bladder cancer.But cystoscope detection is costly,
The popularization and application of different medical unit or poverty-stricken area is not particularly suited for, is clinically urgently found a kind of for carcinoma of urinary bladder detection
Biomarker be detected.Excretion body（exosomes）It is that a kind of diameter is about 40-120nm, there is double-deck plasma membrane knot
The vesica of structure, is discharged into extracellular gap or biological body fluid by cell by exocytosis.In recent years, excretion body becomes newly
Research hotspot, Yin Qinei includes the multi-signal molecule such as mRNAs, microRNAs and albumen specific to derived cell, in signal
Play an important role in conduction and immune system, and wrapped up by complete membranous structure and reduce microenvironment interference, this makes it in disease
There is unique superiority in terms of sick diagnosis, be the new biomaterial studied biomarker and carry out tumour immunity, at the same time
Also there is treatment potentiality.
Research currently for excretion body is broadly divided into isolation technics and detection technique.Isolation technics includes ultracentrifugation
Method, filter centrifugation method, Density ultracentrifugation, immunomagnetic beads combination supercentrifugation and chromatography etc..Detection technique master
Including：Scanning electron microscope observes form（But pretreatments and preparation above more demanding, the standard of sample of the SEM to sample
The standby stage is more complicated, is not suitable for carrying out excretion body a large amount of quickly measurements, and due to excretion body have passed through pretreatment and
Preparation process, can not accurately carry out the measurement of excretion bulk concentration）, dynamic light scattering technique（But due to dynamic light scattering technique
It is the fluctuation data for measuring light intensity, so the light-intensity variation signal of bulky grain can cover the light-intensity variation signal of smaller particle, institute
Be not suitable for the measurement of complicated excretion body sample not of uniform size with dynamic light scattering, the size for being only suitable for preparing by chromatography is equal
The dimensional measurement of one excretion body, and the concentration of excretion body in sample can not be measured.）, flow cytomery technology（Streaming
Cell instrument can not only detect size, the quantity of vesica, but also the source of vesica can be detected by fluorescent marker, by vesica into
Row classification, therefore, flow cytometer are to carry out that vesica is quick, the optimal selection of high throughput, multi-parameter detection.However, conventional flow
The sample that formula cell instrument is directed to is mainly cell, and the detectable limit for scattering light is typically 300-500nm, and most cells external capsule
The diameter of bubble is difficult accurately qualitatively and quantitatively to be analyzed all in below 300nm.）, nanoparticle follow-up analysis technology
（It is a kind of method of relatively new research nano particle, nano particle can be observed directly and in real time.Due to excretion body surface
There is the presence of the transmembrane molecules such as marker CD9, CD63 in face, (such as in serum), can be resisted under complicated background environment with fluorescence
Body marks excretion body, then realizes the measurement under complex background to excretion body with the fluorescence measurement function of NTA, but needs precision
Instrument）, flow cytometry analysis technology（But flow cytometry can only be once detected for a marker, outside
Secrete that body is too small, detected by current flow cytometer device, it is necessary to first bind the coated magnetic bead of foreign antibodies, it is necessary to accurate
Instrument and equipment, and it is cumbersome, susceptibility is different）, immune-blotting method technology（Operating process is complicated）Examined with quantitative fluorescent PCR
Survey analysis microRNA（It is complicated, it is necessary to the laboratory apparatus of costliness to extract nucleic acid step）.Therefore, developing one kind can reliably, soon
Speed, economically the method for the excretion body in Non-invasive detection excretion body especially bladder cancer patients source is very necessary.Micro-fluid chip
Micro-fluid chip laboratory, refers to sample preparation involved in the fields such as biological and chemical, biology with chemically reacting, separating
The basic operation units such as detection are integrated or integrated substantially on one piece several square centimeters of chip, to complete different biologies or change
Learn reaction process, and a kind of technology analyzed its product.It is life science, chemical science and information science signal detection
With the important technological platform of Study on processing method.With integration, analyze speed is fast, high throughput, less energy consumption, pollution are small, cheap,
The features such as safe.Therefore, a kind of integrated excretion body separation, enrichment are built, detects the micro-fluid chip being integrated, is possessed huge
Economic benefit.
Excretion body can be stabilized in urine, and especially urine sample can be big on the premise of atraumatic damages
Amount obtains.If it is possible to detect the cancer information in urine excretion body, then be expected to use it for the non-invasive diagnosis of cancer,
The clinical practices such as monitoring.However, in fact, since the excretion body content in unit volume urine is very low, using conventional method
The excretion body that single-trial extraction obtains, can not meet the requirement of subsequent detection at all, so the detection currently for excretion body is main
It is that concentration first is carried out to the excretion body in sample, takes certain characterization to measure again afterwards.Therefore, the present invention is for above-mentioned
Problem, have developed the micro-fluid chip that a kind of integrated separation of excretion body, enrichment and detection are integrated, while utilize excretion body cross-film
This distinctive marker of PROTEIN C D63, establishes a kind of chip ELISA fast diagnosis methods.This method is outside bladder cancer patients
Secreting physical examination survey, monitoring etc. has certain application prospect.
The content of the invention
It is an object of the invention to provide the integrated testing method of a kind of separation of excretion body, enrichment and detection, this method is
Excretion body chip ELISA detection method, can effectively detect the amount of excretion body in bladder cancer patients body, testing result high specificity,
It is highly practical.
In order to achieve the object of the present invention, the present invention uses following technical scheme：A kind of separation of urine excretion body, enrichment and
The integrated testing method of detection, it is characterised in that comprise the following specific steps that：
1）Design and the double membrane filtration chips of assembling：Double membrane filtration chips are four layers of PMMA plates, from top to bottom each
Layer thickness is respectively 2 mm, 1 mm, 1 mm, and 2 mm, and chip outer diameter is 20 í, 40 í, 6 mm, adjacent two layers
Double faced adhesive tape is used between PMMA plates（DSA）Connection, be respectively cut on middle two layers of PMMA plate the consistent inlet opening of upper-lower position and
Waste liquid tap, the PMMA plates of the superiors are the PMMA flaggies for two circular holes for being cut with 1.8 mm of diameter；The second layer is cutting
There are the PMMA flaggies of two circular holes of 10 mm of diameter；Third layer is to cut two circular holes of 10 mm of diameter, while two circular holes
Between use width 1.5mm, length is the PMMA flaggies that the channel of 11mm is connected；Last layer, is PMMA tablets.Wherein, it is middle
Two layers of DSA glue is equipped between two layers of PMMA plate, the poly- carbon that aperture is respectively 200nm and 30nm is stained between this two layers of DSA glue
Acid esters film（Such as Fig. 1）, for the filtering of liquid and the collection of excretion body.
2）Build chip ELISA examination criteria curves：
1. transitional cell bladder carcinoma sample 200mL is collected, using Ultracentrifugation Method（2,000g room temperatures centrifuge 10-
15min, and cell and its fragment are removed, 100,000 g rotating speeds of supernatant are surpassed from 1-2h, the rear PBS with pH 7.4 is resuspended
Excretion body precipitates,
2. will 1. gained excretion body suspension, according to 1:3, 1:9, 1:27 and 1:81 different proportion is diluted, afterwards
Above-mentioned a certain amount of excretion body suspension stoste and each 300 μ L of dilution are taken respectively, with the flow velocity miniflow syringe pump of 40 μ L/min
It is injected into step 1）Double membrane filtration chips in,
3. after, each double membrane filtration chips continue to inject 300 μ L anti-CD63 with identical flow velocity（1:200-1:
500 dilutions, concentration is 2-5 μ g/mL）Afterwards, incubation 1-1.5h is carried out under the conditions of 25 DEG C,
4. cleaning double membrane filtration chips with 300-400 μ L PBS buffer, repeat three-four times, cleaning terminates backward each
500 a certain amount of air of μ L are injected to drain liquid in double membrane filtration chips with identical flow velocity in double membrane filtration chips,
5. the HRP of 300 μ L streptavidins mark is injected to each double membrane filtration chips（1:2000-1:5000 dilutions,
Concentration is 0.2-0.5 μ g/mL）, and be placed in wet box and be incubated 1-2h in 37 DEG C,
4. and 5. three-four times 6. repeat step.
7. 300 μ L TMB nitrite ions, and 37 DEG C of colour developing 5-30min in darkroom are injected to each double membrane filtration chips,
8. data acquisition is carried out with mobile phone imaging system,
9. finally processing analysis is carried out to gathered data, so as to build mark to laptop using wireless signal transmission
Directrix curve.
3）Sample collection, note sample and cleaning：Healthy volunteer/10 ml of transitional cell bladder carcinoma sample is collected, using 2,
000 g room temperatures centrifuge 10-15 minutes, and remove cell and its relic, by supernatant with miniflow syringe pump with the stream of 40 μ L/min
Speed is continuously injected into double membrane filtration chips, injected slurry volume 8mL, finally with the PBS of 400 μ LpH 7.4 with the flow velocity of 40 μ L/min
It is injected into double membrane filtration chips, repeats the step 3-4 times, completes the cleaning of capture sample.
4）Chip ELISA is detected：Using step 2）3. -9., to step 3）The excretion body captured is detected.
5）Data Management Analysis：Bring testing result into standard curve, carry out the comparison of excretion bulk concentration.
6）According to step 2）-5）Excretion body is substituted with PBS buffer, negative control group is set up and carries out experimental control.
The design and assembling of micro-fluid chip are carried out in the present invention first.Chip body PMMA is carried out using laser cutting machine
The cutting assembling of plate.The thickness range of PMMA plates is 1-2mm.Sample inlet and outlet hose outside diameter be 1.5-1.8mm, the hose with
Chip body is glued firmly.
Excretion body ELISA examination criteria curves are built, will be surpassed from from the excretion body liaison series doubling dilution in urine
（1;1:3; 1:9; 1:27; 1:81）Into different concentration, standard curve is built afterwards.Subsequently, sample collection measurement result with
Known standard curve is contrasted, to judge the content of patient's body excretion body.
While using above-mentioned technical proposal, the present invention can also be used or combined using technology further below
Scheme：
When injecting liquid into double membrane filtration chips, Liquid sample introduction velocity interval is 30-60 μ L/min, is injected with miniflow
Pump or autosampler carry out sample injection.
The cleaning solution used in the sample cleaning, is phosphate buffer PBS（pH 7.4 ± 0.2）, washing times
Typically 2-4 times.
When being loaded into double membrane filtration chips, each sample is 2-3 and repeats, per 300 μ L of hole.
The standing reaction of the ELISA Plate for adding sample combines for biotin and streptavidin, its reaction temperature is generally
35-37 DEG C, when the time is usually 1-2 small.
The nitrite ion is tetramethyl benzidine（TMB）, chromogenic reaction is HRP and TMB association reactions, during chromogenic reaction
Between generally 5-30 minutes, reaction temperature is generally 35-37 DEG C.
The reaction system of the ELISA detection method of the present invention, mainly including following components：Excretion body, biotin labeling
Anti-CD63, HRP, TMB nitrite ion of streptavidin, termination reaction solution, sample cleaning solution PBS and urine specimen.
Another technical problem to be solved by this invention is to provide the collection of a kind of urine excretion body separation, enrichment and detection
Into detection chip, the separation of excretion body can be completed, is enriched with and does basis for detection.
The technical proposal for solving the technical problem of the invention is：A kind of urine excretion body separation, enrichment and detection
Integrated detection chip, including bottom plate, top plate and the detection plate between the bottom plate and top plate, the detection plate include upper
Layer detection plate and lower floor's detection plate, the top plate are equipped with the liquid injection hole and outage that cutting is formed, are connected on the liquid injection hole
Have fluid injection hose, discharge opeing hose be connected with the outage, be respectively equipped with the upper strata detection plate and lower floor's detection plate into
Liquid injection hole described in sample hole and waste liquid tap is corresponding with the position of the sample holes, the outage and the waste liquid tap
Position it is corresponding；
Fixed and be pasted together by the first layers of two-sided between the top plate and the upper strata detection plate, described first pair
Face glue-line be equipped with respectively with the liquid injection hole and corresponding first perforate of the outage, the upper strata detection plate and described
Two layers of second layers of two-sided are equipped between lower floor's detection plate, be equipped with this two layers of DSA layers of two-sided respectively with the sample holes
The second perforate corresponding with the waste liquid tap, it is 200nm that aperture is stained between the second perforate below sample holes
The first makrolon diaphragm, be stained between the second perforate below waste liquid tap aperture be 30nm the second poly- carbon
Acid esters diaphragm；Lower floor's detection plate is equipped with the first passage for connecting the sample holes and the waste liquid tap；Lower floor examines
Be equipped with the 3rd layers of two-sided between drafting board and the bottom plate, the 3rd layers of two-sided be equipped with respectively with the sample holes and institute
Corresponding 3rd perforate of waste liquid tap is stated, second channel corresponding with the first passage is equipped between the 3rd perforate.
While using above-mentioned technical proposal, the present invention can also be used or combined using technology further below
Scheme：
The bottom plate, top plate and levels detection plate are the consistent PMMA plates of shape size.
The aperture of the liquid injection hole and the outage is the hole of 1.8mm, the sample holes and the waste liquid tap
Footpath is 10mm, and the liquid injection hole is arranged concentrically with sample holes, and the outage is arranged concentrically with the waste liquid tap.
The length for forming the PMMA plates of the bottom plate, top plate and detection plate is 40mm, width 20mm, the integrated inspection
The thickness for surveying chip is 6mm.
Compared with prior art, beneficial effects of the present invention are as follows：
1）It is used to integrate the separation of excretion body, enrichment and detection micro-fluid chip the present invention provides one kind, constructs at the same time
Chip ELISA detection method.The reaction system and detection method, it is thin available for detection transitional cell bladder carcinoma sample or carcinoma of urinary bladder
The content of excretion body, this method have the characteristics that high degree of specificity in born of the same parents system nutrient solution supernatant.
2）The detection object of the present invention is the urine discharged outside human body, is readily available, and human body will not be caused any
Influence or wound, detection method is also without expensive and accurate laboratory apparatus（Such as ultracentrifuge, fluorescence microscope）, have
Very big application prospect.
Brief description of the drawings
Attached drawing is used for and is embodied case combination, and the invention will be further described.
Fig. 1 is double membrane filtration chip structure schematic diagrames.
Fig. 2 is double membrane filtration chip pictorial diagrams.
Fig. 3 is the testing result comparison diagram of double membrane filtration chips.
Fig. 4 is excretion body ELISA detection principle diagrams.
Fig. 5 is excretion body chip ELISA examination criteria curves.
Fig. 6 is the separation of excretion body, enrichment and overhaul flow chart.
Fig. 7 is ELISA detection method clinical detection box traction substation.
Fig. 8 is ROC curve analysis result.
Embodiment
With reference to instantiation, the present invention is further explained.It should be noted that these examples be merely to illustrate the present invention without
For limiting the scope of the invention.
Be not specified in case study on implementation specific experimental condition and test method according to normal condition and method or manufacturer
Proposed condition is practiced.
Not specified various instruments and reagent are commercial product well known in the art in the present invention, can pass through business
Approach buying obtains.
Embodiment 1：The separation of excretion body and the detection of chip ELISA method in transitional cell bladder carcinoma, referring to the drawings 1-8.
Please refer to Fig.1-7, the integrated testing method of a kind of urine excretion body separation, enrichment and ELISA detections, including it is as follows
Step：
1）On the premise of patient knows and Ethics Committee is agreed to, bladder cancer patients are collected（Clinical biopsy have proven to for
Carcinoma of urinary bladder）16 parts of urine specimen, healthy 8 parts, every part of 100ml of contributor, removes thin under room temperature 2, the centrifugal condition of 000g
Born of the same parents and big fragment, then obtain supernatant liquor.
2）By 1）Obtained supernatant, under the conditions of 4 DEG C, 100,000g ultracentrifugations 60 minutes, discard supernatant take it is heavy
Form sediment, and excretion body is resuspended with the PBS buffer of 1mL pH 7.4（Such as Fig. 1）.
3）By 1）Gained supernatant, is pumped using miniflow, is injected into 40 μ L/min of flow velocity in double membrane filtration chips, continuously
Filtering 8 times.
4）By 2）Gained excretion body, is serially diluted according to 3 times of multiple proportions, is preserved respectively, in case subsequent builds standard is bent
Line.
5）By 4）The standby excretion body of institute carries out chip ELISA detections（Principle such as Fig. 2）, first to 4）Chips inject antibody,
1 is added per hole：200-1:The anti-CD63 of 500 diluted 300 μ L（biotin-labeled）Antibody（I.e. concentration is 2-5 μ
g/mL）, 25 DEG C of incubation 1-1.5h.
6）To 5）Middle injection 300-400 μ L PBS buffer is cleaned, in triplicate.
7）To 6）500 μ L air are injected to drain liquid in chip with identical flow velocity in each chip.
8）To 7）Each chip injects the HRP of 300 μ L streptavidins mark（1:2000-1:5000 dilutions, concentration are
0.2-0.5 μg/mL）, and be placed in wet box and be incubated 1-2h in 37 DEG C.9）Repeat step 6）With 7）.
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）Use image capturing system（Such as from imaging system in mobile phone）Carry out data acquisition.
12）By 11）The data obtained, into laptop, RGB analyses is carried out using ImageJ by wireless signal transmission,
Build standard curve（Such as Fig. 4-5）, while use above-mentioned 1）-12）Step carries out bladder cancer patients excretion body content detection, goes forward side by side
Row ROC curve is analyzed and structure box traction substation.Testing result such as Fig. 6.
Embodiment 2, integrates detection chip, referring to the drawings 1-3.
The micro-fluid chip of the present invention includes bottom plate 1, top plate 2 and the detection plate between the bottom plate and top plate,
Liquid detecting chamber is equipped with the detection plate, the excretion body in urine can be collected in wherein by the liquid detecting chamber, and be led to
Cross the requirement that repeatedly note sample enrichment reaches detection.
The detection plate includes upper strata detection plate 3 and lower floor's detection plate 4, and the top plate 2 is equipped with the fluid injection that cutting is formed
Hole 5 and outage 12, are connected with liquid injection pipe 6 on the liquid injection hole 5, are connected with drain pipe 13 on the outage 12, it is described on
Sample holes 7 and waste liquid tap 8, the liquid injection hole 5 and the sample holes 7 are respectively equipped with layer detection plate 3 and lower floor's detection plate 4
Position it is corresponding, the position of the outage 12 and the waste liquid tap 8 is corresponding.
Fixed and be pasted together by the first layers of two-sided 9 between the top plate 2 and the upper strata detection plate 3, described the
One layers of two-sided 9 is equipped with to be examined with the liquid injection hole 5 and corresponding first perforate 14 of the outage 12, the upper strata respectively
Two layers of second layers of two-sided 15 are equipped between drafting board 3 and lower floor's detection plate 4, are equipped with this two layers of DSA layers of two-sided 15
Second perforate 16 corresponding with the sample holes 7 and the waste liquid tap 8 respectively, the second perforate positioned at the lower section of sample holes 7
The first makrolon diaphragm 10 that aperture is 200nm is stained between 16, positioned at the lower section of waste liquid tap 8 the second perforate 16 it
Between be stained with aperture be 30nm the second makrolon diaphragm 17.
Lower floor's detection plate 4 is equipped with the first passage 11 for connecting the sample holes 7 and the waste liquid tap 8.
The 3rd layers of two-sided 18 is equipped between lower floor's detection plate 4 and the bottom plate 1, the 3rd layers of two-sided 18 is equipped with
The 3rd perforate 19 corresponding with the sample holes 7 and the waste liquid tap 8 respectively, is equipped with and institute between the 3rd perforate 19
State the corresponding second channel 20 of first passage 11.
The bottom plate 1, top plate 2 and levels detection plate are the consistent PMMA plates of shape size.
The length for forming the PMMA plates of the bottom plate 1, top plate 2 and detection plate is 40mm, width 20mm, the miniflow
The integral thickness of body chip is 6mm.
First layers of two-sided 9, the second layers of two-sided 15 and the 3rd layers of two-sided 18 are DSA double faced adhesive tapes.
The liquid injection pipe 5 and the drain pipe 13 are sterile hose.
The aperture of the liquid injection hole 5 and the outage 12 is 1.8mm, the sample holes 7 and the waste liquid tap 8
Aperture be 10mm, the liquid injection hole 5 is arranged concentrically with sample holes 7, and the outage 12 and the waste liquid tap 8 are concentric
Set.
In the present embodiment, the cutting that chip body PMMA plates are carried out using laser cutting machine is assembled, the thickness model of PMMA plates
Enclose for 1-2mm, the fluid injection pipe outside diameter as sample inlet and outlet hose is 1.5-1.8mm, and the hose and chip body use glue
Bonding is firm.
The operation principle of micro-fluid chip of the present invention is：By urine to be detected by liquid injection pipe 5 according to certain flow rate
Be injected into liquid injection hole 5, and thus enter upper strata detection plate 3 sample holes 7, urine through being arranged on upper strata detection plate 3 into
The filtering for the first makrolon diaphragm 10 that the aperture of the lower section of sample hole 7 is 200nm, magazine and macromolecular cell is removed, filtrate
Enter in the sample holes 7 of lower floor's detection plate 4, and entered by passage 11 in the waste liquid tap 8 in lower floor's detection plate 4, warp
Filtering by aperture for the second makrolon diaphragm 17 of 30nm, excretion body are collected in the waste liquid tap of the version 14 of lower interlayer
In 8, filtrate is entered in the waste liquid tap 8 of upper strata detection plate 3 through the second makrolon diaphragm 17, and can be via outage
12 and drain pipe 13 be discharged.
The case of the chip of the present invention in actual use is as described in Example 1.
Claims (10)
- A kind of 1. integrated testing method of the separation of urine excretion body, enrichment and the detection of non-diagnostic purpose, it is characterised in that including Following specific steps：1) design and assemble double membrane filtration chips：Double membrane filtration chips are four layers of PMMA plates, between adjacent two layers PMMA plates With double faced adhesive tape DSA connections, the consistent inlet opening of upper-lower position and waste liquid tap is respectively cut on middle two layers of PMMA plate, most Be cut with corresponding with inlet opening and waste liquid tap liquid injection hole and outage respectively on the PMMA plates on upper strata, the liquid injection hole and Connection flexible pipe is distinguished on outage, the bottom is PMMA tablets, is equipped with two layers of DSA glue between middle two layers of PMMA plate, this is two layers The polycarbonate membrane that aperture is respectively 200nm and 30nm is stained between DSA glue, for the filtering of liquid and the receipts of excretion body Collection；2) chip ELISA examination criteria curves are built：1. collecting and removing cell and its fragment, supernatant is surpassed and excretion body is resuspended with PBS precipitates from rear,2. will 1. gained excretion body suspension, be diluted according to different proportion, take a certain amount of excretion body suspension former respectively afterwards Liquid and dilution are injected into double membrane filtration chips of step 1) with miniflow syringe pump,3. after, each double membrane filtration chips continue to inject the anti-CD63 of biotin labeling, with 1:200-1:500 dilutions, its Concentration is 2-5 μ g/mL, after be incubated,4. being carried out cleaning double membrane filtration chips with PBS buffer, it is certain that cleaning terminates injection in backward each double membrane filtration chips The air of amount drains liquid in double membrane filtration chips,5. the HRP of 300 μ L streptavidins mark is injected to each double membrane filtration chips, with 1:2000-1:5000 dilutions, its is dense Spend for 0.2-0.5 μ g/mL, and be incubated in wet box,6. injecting TMB nitrite ions to each double membrane filtration chips, and develop the color in darkroom,7. data acquisition is carried out with imaging system,8. processing analysis is carried out to gathered data, so as to build standard curve；3) sample collection, note sample and cleaning：Urine specimen is collected, room temperature centrifuges and removes cell and its relic, supernatant is used Miniflow syringe pump is continuously injected into double membrane filtration chips, after with the PBS of pH7.4 double membrane filtration chips are injected into certain flow rate In, it is repeated several times, completes the cleaning of sample；4) chip ELISA is detected：Using step 2) 3. -8., the excretion body captured to step 3) is detected；5) Data Management Analysis：Testing result is substituted into standard curve, carries out the comparison of excretion bulk concentration.
- 2. the integrated detection side of the separation of urine excretion body, enrichment and the detection of a kind of non-diagnostic purpose as described in claim l Method, it is characterised in that when injecting liquid into double membrane filtration chips, the sample introduction velocity interval of liquid is 30-60 μ L/min, is used Miniflow syringe pump carries out sample injection.
- 3. the integrated detection side of the separation of urine excretion body, enrichment and the detection of a kind of non-diagnostic purpose as described in claim l Method, it is characterised in that the cleaning solution used in the sample cleaning is phosphate buffer PBS, its pH is 7.4, washing times For 2-4 times.
- 4. the integrated detection side of the separation of urine excretion body, enrichment and the detection of a kind of non-diagnostic purpose as described in claim l Method, it is characterised in that when being loaded into double membrane filtration chips, each sample repeats 2-3 times, per 300 μ L of hole.
- 5. the integrated detection side of the separation of urine excretion body, enrichment and the detection of a kind of non-diagnostic purpose as described in claim l Method, it is characterised in that it is biotin and strepto- parent to add standing reaction of the double membrane filtration chips of sample when carrying out wet box incubation Close element to combine, its reaction temperature is 35-37 DEG C, when the time is 1-2 small.
- 6. the integrated detection side of the separation of urine excretion body, enrichment and the detection of a kind of non-diagnostic purpose as described in claim l Method, it is characterised in that the nitrite ion is tetramethyl benzidine (TMB), and chromogenic reaction is reacted for HRP and TMB, chromogenic reaction Time is 5-30 minutes, and reaction temperature is 35-37 DEG C.
- A kind of 7. integrated detection chip of urine excretion body separation, enrichment and detection, it is characterised in that：Including bottom plate (1), top plate (2) and the detection plate between the bottom plate and top plate, the detection plate include upper strata detection plate (3) and lower floor's detection plate (4), the top plate (2) is equipped with the liquid injection hole (5) and outage (12) that cutting is formed, and note is connected with the liquid injection hole (5) Liquid hose (6), is connected with discharge opeing hose (13), the upper strata detection plate (3) and lower floor's detection plate (4) on the outage (12) On be respectively equipped with sample holes (7) and waste liquid tap (8), the liquid injection hole (5) is corresponding with the position of the sample holes (7), The outage (12) is corresponding with the position of the waste liquid tap (8)；The top plate (2) and the upper strata detection plate (3) Between by the first layers of two-sided (9) fixation be pasted together, first layers of two-sided (9) be equipped with respectively with the fluid injection Hole (5) and corresponding first perforate (14) of the outage (12), the upper strata detection plate (3) and lower floor's detection plate (4) two layers of second layers of two-sided (15) are equipped between, be equipped with this two layers of DSA layers of two-sided (15) respectively with the sample holes (7) and corresponding second perforate (16) of the waste liquid tap (8), between the second perforate (16) below sample holes (7) Be stained with the first makrolon diaphragm (10) that aperture is 200nm, the second perforate (16) below waste liquid tap (8) it Between be stained with aperture be 30nm the second makrolon diaphragm (17)；Lower floor's detection plate (4), which is equipped with, connects the sample introduction Hole (7) and the first passage (11) of the waste liquid tap (8)；The 3rd is equipped between lower floor's detection plate (4) and the bottom plate (1) Layers of two-sided (18), the 3rd layers of two-sided (18) be equipped with respectively with the sample holes (7) and the waste liquid tap (8) Corresponding 3rd perforate (19), is equipped with second channel corresponding with the first passage (11) between the 3rd perforate (19) (20)。
- 8. the integrated detection chip of a kind of urine excretion body separation as claimed in claim 7, enrichment and detection, its feature exist In：The bottom plate (1), top plate (2) and levels detection plate the PMMA plates that to be shape size consistent.
- 9. the integrated detection chip of a kind of urine excretion body separation as claimed in claim 7, enrichment and detection, its feature exist In：The aperture of the liquid injection hole (5) and the outage (12) is 1.8mm, the sample holes (7) and the waste liquid tap (8) aperture is 10mm, and the liquid injection hole (5) is arranged concentrically with sample holes (7), and the outage (12) is arranged with the waste liquid Portal (8) be arranged concentrically.
- 10. the integrated detection chip of a kind of urine excretion body separation as claimed in claim 8, enrichment and detection, its feature exist In：The length for forming the PMMA plates of the bottom plate (1), top plate (2) and levels detection plate is 40mm, width 20mm, institute The thickness for stating integrated detection chip is 6mm.
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| CN107597216A (en) * | 2017-03-17 | 2018-01-19 | 李芳芳 | A kind of method that excretion body is separated using micro-fluid chip |
| CN108085245A (en) * | 2018-02-11 | 2018-05-29 | 中南大学 | A kind of room temperature isolates and purifies the casing filtering device of excretion body |
| CN111172009A (en) * | 2018-11-13 | 2020-05-19 | 中国科学院大连化学物理研究所 | Integrated exosome separation chip and preparation method and application thereof |
| CN109499631A (en) * | 2018-11-19 | 2019-03-22 | 深圳拓扑精膜科技有限公司 | A kind of micro-fluid chip of integrated anodised aluminium perforated membrane |
| CN109628277B (en) * | 2019-01-23 | 2022-02-01 | 东南大学 | System and method for separating and detecting tumor marker miRNA in exosome |
| CN109884315A (en) * | 2019-02-27 | 2019-06-14 | 华中科技大学 | A kind of external quick detection platform and detection method of PD-L1 excretion body |
| CN110186881A (en) * | 2019-05-17 | 2019-08-30 | 华南农业大学 | A kind of biosensor and method detecting Microcystin |
| CN110339874B (en) * | 2019-06-21 | 2021-05-11 | 中国科学院上海微系统与信息技术研究所 | Microfluidic device for exosome separation and surface protein detection and use method |
| CN112305233A (en) * | 2019-07-23 | 2021-02-02 | 南方科技大学 | Automatic urine collection and detection method and urine detection controller |
| CN110747111A (en) * | 2019-09-29 | 2020-02-04 | 山东大学 | Exosome filter equipment |
| CN112903403B (en) * | 2019-12-04 | 2022-09-27 | 中国科学院大连化学物理研究所 | Device for realizing exosome enrichment by using filter membrane and exosome enrichment method |
| CN111505318A (en) * | 2020-05-15 | 2020-08-07 | 刘大基 | Blood transfusion compatibility detection package analyzer |
| CN111961584B (en) * | 2020-08-24 | 2022-05-03 | 山东大学齐鲁医院 | Cerebrospinal fluid exosome RNA detection device, system and method based on microfluidic technology |
| CN113005033B (en) * | 2021-02-24 | 2022-08-26 | 清华大学 | Device and method for capturing and separating exosomes and cells in biological sample |
| CN114231523A (en) * | 2022-01-12 | 2022-03-25 | 西安交通大学 | Exosome separation method based on CD63 antibody coupling magnetic beads |
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| US9719129B2 (en) * | 2011-06-10 | 2017-08-01 | Hitachi Chemical Co., Ltd. | Methods for isolating vesicles from biological fluids |
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