CN205861533U - A kind of flow cell - Google Patents
A kind of flow cell Download PDFInfo
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- CN205861533U CN205861533U CN201620587927.4U CN201620587927U CN205861533U CN 205861533 U CN205861533 U CN 205861533U CN 201620587927 U CN201620587927 U CN 201620587927U CN 205861533 U CN205861533 U CN 205861533U
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- flow cell
- capillary tube
- filter post
- column
- detection
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Abstract
This utility model provides a kind of flow cell, and this flow cell being used for detection includes capillary body, the adding mouth being positioned at capillary tube one end, is positioned at the flow export of the capillary tube other end and is positioned at the filter post within capillary tube and trapping column.Described flow cell can be greatly improved detection speed and contribute to improving and quickly detect the quality of data.
Description
Technical field
This utility model relates to technology for detection field, relates more specifically to a kind of flow cell that can be used for light detection.
Background technology
In detection field, often need all kinds of materials are carried out light detection.Derive based on " double-antibody method "
Panimmunity response analysis method: radioimmunology, euzymelinked immunosorbent assay (ELISA), chemoluminescence method, time-resolved fluorescence method and fluorescence are exempted from
Epidemic disease methods etc., can be used for determining pathogenic microorganism, and disease to the specific proteins detection by quantitative of human body thus carries out auxiliary diagnosis
Or monitoring etc., purposes is widely.
The usual practice of method is analyzed in this kind of immunoreation: capture antibody is fixed on solid phase carrier, then with antigen
(target protein) reacts, and reacts with traget antibody after washing again, washing, adds substrate detection absorbance or Direct-detection Optical letter
Number, whole detection process about needs 2 hours or longer, it is impossible to meet the needs of clinical emergency.
In sum, this area is still in the urgent need to developing work efficiency height and can quickly improve the inspection of the detection quality of data
Survey method.
Utility model content
The purpose of this utility model is to provide one and detection speed can be greatly improved and contribute to improving and quickly detect data
The flow cell of quality.
This utility model first aspect, it is provided that a kind of flow cell for detection, including capillary body, is positioned at capillary tube
The adding mouth of one end, it is positioned at the flow export of the capillary tube other end and is positioned at the filter post within capillary tube and trapping column.
In another preference, within described capillary tube filter post and trapping column to filter post-catching for Acquisition Detection thing
Obtain the sequence interval arrangement of post.
In another preference, the flow export end of described capillary tube is additionally provided with clamping, supports and fixing device.
In another preference, described trapping column number is n, and described filter post number is n+1, and wherein, n is more than or equal to 1
Positive integer.
In another preference, described microcapillary tube height L1For 50-200mm, and internal diameter d1For 0.5-2mm.
In another preference, described filter column length L3For 3-5mm.
In another preference, the aperture d of described filter post3For 5-20 μm.
In another preference, described capture column length L4For 1-3mm.
In should be understood that in the range of this utility model, above-mentioned each technical characteristic and the most specifically retouching
Can be combined with each other between each technical characteristic stated, thus constitute new or preferred technical scheme.As space is limited, at this not
Tire out the most one by one and state.
Accompanying drawing explanation
Fig. 1 shows the structure of this utility model flow cell.
Fig. 2 shows cTnI standard curve.
Fig. 3 shows CA199 standard curve.
Fig. 4 shows CA125 standard curve.
Fig. 5 shows SCCA standard curve.
Fig. 6 shows AFP standard curve.
Fig. 7 shows CEA standard curve.
Detailed description of the invention
The present inventor, by extensively in-depth study, is surprised to find that one can be greatly improved detection speed and have first
Help improve the flow cell quickly detecting the quality of data.Complete this utility model on this basis.
Term explanation
Unless otherwise defined, what whole technology the most used herein and scientific terminology were respectively provided with such as art is common
The identical meanings that technical staff is generally understood that.
Below in conjunction with specific embodiment, this utility model is expanded on further.Should be understood that these embodiments are merely to illustrate this
Utility model rather than limit scope of the present utility model.The experimental technique of unreceipted actual conditions in the following example is logical
Often according to normal condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are weight
Percentage ratio and parts by weight.
Experiment material used in following example and reagent the most all can obtain from commercially available channel.
Flow cell
Flow cell of the present utility model includes following structure: air pressure pump, capillary tube, filter post, trapping column etc..
1. the long 50-200mm of the pipe shaft of capillary tube, internal diameter 0.5-2mm；
2. the long 5-20mm of air pressure pump docking port, internal diameter 5-10mm；
3. filter column length 3-5mm；
4. capture column length 1-3mm；
5. the material of capillary tube is light transmissive material, such as: polystyrene, glass etc.；
6. the material filtering post is column glass fibre, aperture 5-20 μm, plays the effect filtering liquid sample；
7. trapping column is polymer aggregation, such as: polystyrene microsphere aggregation, many empty microsphere and agarose gel etc.,
Surface is fixed with the trapping agent for object, such as the antibody for target antigen or the oligonucleotide fragment for target nucleic acid
Deng；
8. filter post and trapping column are tightly packed in capillary tube；
9. the trapping column in Fig. 1 a is only for a kind of object, is used for detecting simple target thing, the trapping column of Fig. 1 b for
Plurality of target thing, for detection plurality of target thing simultaneously.
The making of flow cell
Selecting internal diameter is the capillary tube of 2mm, glass or polystyrene material；
Select the first filter post adaptive with capillary inner diameter size, the most tightly packed central position to capillary tube
Put；
The polymer securing trapping agent in appropriate amount adds capillary tube from top to bottom, is stopped by the first filter post；
Second filter post, the most tightly packed to capillary tube, with the first filter common squeeze polymer of post, form capture
Post.
Reagent and equipment:
CTnI (cTnI) and 1 pair of monoclonal antibody (Ab1 and Ab2), commercially available product；
Alpha-fetoprotein (AFP) and 1 pair of monoclonal antibody (Ab1 and Ab2), commercially available product；
Carcinoembryonic antigen (CEA) and 1 pair of monoclonal antibody (Ab1 and Ab2), commercially available product；
CA125 (CA125) and 1 pair of monoclonal antibody (Ab1 and Ab2), commercially available product；
CA199 (CA199) and 1 pair of monoclonal antibody (Ab1 and Ab2), commercially available product；
Squamous cell carcinoma antigen (SCCA) and 1 pair of monoclonal antibody (Ab1 and Ab2), commercially available product；
SA-PE complex (SA-PE), commercially available product；
BSA (BSA), commercially available product；
Biotin Biotin, commercially available product；
Surface is through the polystyrene microsphere that particle diameter is 20 μm of carboxyl modified, commercially available product；
Common agents, buffer are autogamy；
Detector: USB4000-FL (Ocean Optics of the U.S.)；
Light source: 532nm LASER Light Source.
Embodiment 1: the detection of cTnI in human serum
1. prepare
1.1Ab1 removes in advance containing the little molecule of amino and foreign protein (dialyse or cross chromatographic column), measures its concentration.
1.2 in 1.5ml polypropylene centrifuge tube, accurately weighs about 5mg N-hydroxysulfosuccini-mide
(NHSS), standby (moistureproof).
1.3 in 1.5ml polypropylene centrifuge tube, accurately weighs about 5mg N-ethyl-N ' (3-
Dimethylainopropyl)-carbodiimide (EDC), standby (moistureproof).
2. polystyrene microsphere (Beads) activation
2.1 microsphere stock solution vortex DL instrument suspendibles 20 seconds, pipette 200 μ L microspheres (being equivalent to 2.5 × 106 microspheres) in
In 1.5ml polypropylene centrifuge tube.
2.2 15000rpm are centrifuged 2 minutes (arranging 3 minutes), remove supernatant.
2.3 add 100 μ L distilled water, and vortex DL instrument suspendible 20 seconds, 15000rpm is centrifuged 2 minutes, removes supernatant.
2.4 repeat 2.3.
2.5 add 80 μ L 0.1mol/L phosphate buffer (PBS), pH 6.2, vortex DL instrument suspendible 20 seconds.
(NHSS) is diluted to 50mg/ml with distilled water by 2.6.(now with the current)
2.7 take 10 μ L 50mg/ml (NHSS) is added in Beads solution, vortex DL instrument suspendible 20 seconds.
EDC is diluted to 50mg/ml with distilled water by 2.8.(now with the current)
2.9 take 10 μ L 50mg/ml EDC is added in Beads solution, vortex DL instrument suspendible 20 seconds.
2.10 lucifuge, 37 DEG C hatch 20 minutes.
2.11 15000rpm are centrifuged 2 minutes, remove supernatant.
2.12 add 250 μ L 50mmol/L 2-(N-morpholino) ethanesulfonic acid (MES) (MES)
pH5.0。
2.13 15000rpm are centrifuged 2 minutes, remove supernatant.
2.14 repeat 2.12,2.13, carry out lower step experiment at once.
3. cross-link, close and store
3.1 add in 20 Ab1 processed for μ g to the Beads activated, vortex DL instrument suspendible 20 seconds.
3.2 lucifuges, 37 DEG C react 2 hours, every 15 minutes mixing once.
3.3 add 1ml PBS-TBN, and vortex DL instrument suspendible 20 seconds, 10000rpm is centrifuged 2 minutes.Remove supernatant
(PBS-TBN consist of the phosphate buffer of 10mmol/L pH7.4,0.02% polysorbas20,1mg/ml calf blood pure
Albumen and 0.05% sodium azide).
3.4 add 500 μ L PBS-TBN, and vortex DL instrument suspendible 20 seconds, 10000rpm is centrifuged 2 minutes.Remove supernatant.
3.5 add 500 μ L PBS-TBN, vortex DL instrument suspendible 20 seconds.
3.6 2-8 DEG C keep in Dark Place, obtain Ab1-Beads.
Making the most of the present utility model
Ab1-Beads, vortex DL instrument suspendible 20 seconds, takes 50 μ l, by " making of the present utility model " described this reality of making
With novel, repeat this process and can make a in multiple Fig. 1.
Fig. 1 illustrates:
1. the pipe shaft length 50～200mm of capillary tube, internal diameter 0.5～2mm；
2. air pressure pump docking port length 5～20mm, internal diameter 5～10mm；
3. filter column length 3～5mm；
4. capture column length 1～3mm；
5. the material of capillary tube is light transmissive material, such as: polystyrene, glass etc.；
6. the material filtering post is column glass fibre, aperture 5～20 μm, plays the effect filtering liquid sample；
7. trapping column is polymer aggregation, such as: polystyrene microsphere aggregation, many empty microsphere and agarose gel etc.,
Surface is fixed with the trapping agent for object, such as the antibody for target antigen or the oligonucleotide fragment for target nucleic acid
Deng；
8. filter post and trapping column are tightly packed in capillary tube；
9. a of Fig. 1 only has for the trapping column of a kind of object, is used for detecting simple target thing, the b of Fig. 1 contain for
The trapping column of plurality of target thing, for detection plurality of target thing simultaneously.
5.Ab2 labelling Biotin
5.1Ab2 pretreatment
5.2Biotin labelling reacts
Take the Ab1 10 μ L of above-mentioned pretreatment, add the 1mg/ml NHSS-Biotin DMSO solution of 25 μ L, mixing, 4 DEG C
Refrigerator lucifuge is reacted 2 hours.
6.Ab2 labelling PE
Label taking has remembered Biotin-Ab2, adds in pH7.4 phosphate buffer, and the final concentration of 5 μ g/ml of Ab2 are simultaneously introduced
SA-PE, PE total concentration is 60 μ g/ml, obtains Ab2-PE, and cumulative volume is 5ml, and 4 DEG C keep in Dark Place standby.
The preparation of 7.cTnI antigen standard
With PBS-TBN preparation cTnI standard substance and quality-control product solution, it is shown in Table 1.
The preparation of 8.cTnI standard curve
Take the STD0 mixing of the Ab2-PE and 50 μ l of 5 μ l, by a in Fig. 1 and air pressure pump closed butt joint, it is ensured that close
Envelope, then inserts the lower end of a in mixed liquor, makes mixed liquor move back and forth in capillary tube 3 times (1 time reciprocal about 40 seconds), often
Secondary trapping column of all passing through, finally gets mixed liquor capillary tube, then washs trapping column, the fluorescence intensity of detection trapping column with PBS.
Same operation obtains the fluorescence intensity of STD1～STD4, inserts table 1 relevant position, and with LgC as abscissa, LgF is as vertical coordinate
Make curve chart, as shown in Figure 2.
Table 1:cTnI standard substance and fluorescence intensity synopsis
| cTnI | STD0 | STD1 | STD2 | STD 3 | STD 4 |
| C(ng/ml) | 0 | 0.5 | 2 | 5 | 10 |
| Fluorescence intensity F | 33.7 | 702.6 | 1480.6 | 2910.7 | 5646.6 |
9. quality-control product detection
Preparing 2 cTnI quality-control product solution with PBS-TBN, concentration is respectively 1ng/ml and 8ng/ml, uses this utility model
Detection, fluorescence signal is brought standard curve into, must be measured concentration and be respectively 0.91ng/ml and 8.23ng/ml, meet good with theoretical value
Good.
The quick detection of embodiment 2:5 kind tumor markers
1, repeat the institute of " 1～6 " of embodiment 1 in steps, respectively obtain Ab1-Beads and Ab2-PE of AFP, CEA
Ab1-Beads and Ab2-PE, Ab1-Beads and Ab2-PE of CA125, Ab1-Beads and Ab2-PE of CA199, SCCA
Ab1-Beads and Ab2-PE, and prepare multiple as shown in Figure 1 b new for detecting this practicality of 5 kinds of tumor markerses simultaneously
Type.
2,5 kinds of tumor markers hybrid antigen standard substance and the preparation of quality-control product
Standard substance and the quality-control product of 5 kinds of tumor markerses as shown in table 2 is prepared, it may be assumed that without appointing in STD1 with PBS-TBN
What tumor markers, contains 5 kinds of tumor markerses simultaneously in STD2, other the like.
3. the preparation of standard curve
Respectively take 5 kinds of Ab2-PE of 3 μ l and add mix homogeneously in the STD0 of 50 μ l, the b in Fig. 1 is tight with air pressure pump
Docking, it is ensured that seal, then inserts the lower end of b in mixed liquor, and (1 time reciprocal to make mixed liquor move back and forth 3 times in capillary tube
About 40 seconds), every time by all trapping columns, finally mixed liquor is got capillary tube, then washs all trapping columns with PBS, point
Do not detect the fluorescence intensity of all trapping columns.Same operation obtains 5 kinds of antigens in STD1, STD2, STD3, STD4 standard solution
Corresponding fluorescence intensity, is shown in Table 3～7, and with LgC as abscissa, LgF make curve chart, corresponding standard curve for vertical coordinate
See Fig. 3-Fig. 7.
Table 2:5 kind tumor markers hybrid antigen standard substance and the concentration of quality-control product
| Tumor markers | STD0 | STD1 | STD2 | STD 3 | STD 4 | Quality Control 1 | Quality Control 2 |
| CA199 | 0U/ml | 5U/ml | 20U/ml | 100U/ml | 300U/ml | 40U/ml | 200U/ml |
| CA125 | 0U/ml | 10U/ml | 30U/ml | 100U/ml | 300U/ml | 30U/ml | 200U/ml |
| SCCA | 0U/ml | 1U/ml | 5U/ml | 30U/ml | 150U/ml | 5U/ml | 100U/ml |
| AFP | 0ng/ml | 5ng/ml | 20ng/ml | 100ng/ml | 300ng/ml | 20ng/ml | 200ng/ml |
| CEA | 0ng/ml | 2ng/ml | 10ng/ml | 50ng/ml | 200ng/ml | 5ng/ml | 150ng/ml |
Table 3:CA199 standard substance and fluorescence intensity synopsis
| CA199 | STD0 | STD1 | STD2 | STD 3 | STD 4 |
| C(U/ml) | 0 | 5 | 20 | 100 | 300 |
| Fluorescence intensity F | 13.7 | 713.7 | 1470.9 | 2839.2.7 | 5728.7 |
Table 4:CA125 standard substance and fluorescence intensity synopsis
| CA125 | STD0 | STD1 | STD2 | STD 3 | STD 4 |
| C(U/ml) | 0 | 10 | 30 | 100 | 300 |
| Fluorescence intensity F | 61.8 | 750.2 | 1493 | 2650 | 5127.2 |
Table 5:SCCA standard substance and fluorescence intensity synopsis
| SCCA | STD0 | STD1 | STD2 | STD 3 | STD 4 |
| C(U/ml) | 0 | 1 | 5 | 30 | 150 |
| Fluorescence intensity F | 10.7 | 740.2 | 1421 | 2550 | 5227.2 |
Table 6:AFP standard substance and fluorescence intensity synopsis
| AFP | STD0 | STD1 | STD2 | STD 3 | STD 4 |
| C(U/ml) | 0 | 5 | 20 | 100 | 300 |
| Fluorescence intensity F | 3.7 | 666.5 | 1387 | 2768.5 | 5213.7 |
Table 7:CEA standard substance and fluorescence intensity synopsis
| AFP | STD0 | STD1 | STD2 | STD 3 | STD 4 |
| C(U/ml) | 0 | 2 | 10 | 50 | 200 |
| Fluorescence intensity F | 3.7 | 666.5 | 1387 | 2768.5 | 5213.7 |
4. the mensuration of quality-control product
Prepare the quality-control product of each tumor markers with PBS-TBN, then detect with this utility model, the results are shown in Table 8, in fact
Measured value is good with theoretical value accordance.
Table 8: the measurement result of tumor mark quality control product
The all documents mentioned in the application are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after having read above-mentioned teachings of the present utility model, people in the art
This utility model can be made various changes or modifications by member, and these equivalent form of values fall within the application appended claims institute equally
The scope limited.
Claims (8)
1., for a flow cell for detection, the adding mouth including capillary body, being positioned at capillary tube one end, to be positioned at capillary tube another
The flow export of one end and be positioned at the filter post within capillary tube and trapping column.
2. flow cell as claimed in claim 1, it is characterised in that filter post described capillary tube within and trapping column with filter post-
Sequence interval arrangement for the trapping column of Acquisition Detection thing.
3. flow cell as claimed in claim 1, it is characterised in that the flow export end of described capillary tube is additionally provided with clamping, supports
And fixing device.
4. flow cell as claimed in claim 1, it is characterised in that described trapping column number is n, and described filter post number is n+1,
Wherein, n is the positive integer more than or equal to 1.
5. flow cell as claimed in claim 1, it is characterised in that described microcapillary tube height L1For 50-200mm, and internal diameter
d1For 0.5-2mm.
6. flow cell as claimed in claim 1, it is characterised in that described filter column length L3For 3-5mm.
7. flow cell as claimed in claim 1, it is characterised in that the aperture d of described filter post3For 5-20 μm.
8. flow cell as claimed in claim 1, it is characterised in that described capture column length L4For 1-3mm.
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|---|---|---|---|
| CN201620587927.4U CN205861533U (en) | 2016-06-16 | 2016-06-16 | A kind of flow cell |
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| CN201620587927.4U CN205861533U (en) | 2016-06-16 | 2016-06-16 | A kind of flow cell |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406774A (en) * | 2018-10-27 | 2019-03-01 | 东北师范大学 | A kind of novel signal amplification-capillary chemistry electrochemiluminescent immunoassay sensor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406774A (en) * | 2018-10-27 | 2019-03-01 | 东北师范大学 | A kind of novel signal amplification-capillary chemistry electrochemiluminescent immunoassay sensor |
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Granted publication date: 20170104 Termination date: 20190616 |
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