CN104483482A - Method for manufacturing device used for detecting tumor cells - Google Patents
Method for manufacturing device used for detecting tumor cells Download PDFInfo
- Publication number
- CN104483482A CN104483482A CN201410801973.5A CN201410801973A CN104483482A CN 104483482 A CN104483482 A CN 104483482A CN 201410801973 A CN201410801973 A CN 201410801973A CN 104483482 A CN104483482 A CN 104483482A
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- China
- Prior art keywords
- cover plate
- silicon cover
- storage tank
- liquid storage
- glass substrate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
The invention belongs to the field of medical instruments, and discloses a method for manufacturing a device used for detecting tumor cells. The method comprises the following steps: a liquid storage tank and two flow channels are formed on a glass plate with an ultrasonic wave etching method, and the glass plate serves as a glass substrate; a yurt-shaped bulge coarse area is prepared on a silicon cover plate with a reaction ion etching method; two holes are prepared in the silicon cover plate with a laser punching method, the silicon cover plate is washed clean with ethyl alcohol and deionized water, and the silicon cover plate with the coarse area is prepared; the silicon cover plate is connected with the upper side of the glass substrate in a sealed mode, and the liquid storage tank corresponds to the yurt-shaped coarse area; one end of each flow channel is communicated with the liquid storage tank, and the other end of each flow channel corresponds to one of the holes. A nanometer coarse area is applied to the device used for detecting the tumor cells, the tumor cell capture efficiency is obviously improved, the detection sensitivity of the tumor cells is greatly improved and the device contributes to accurate detection of diseases.
Description
Technical field
The present invention relates to the preparation method of the device for detecting tumour cell, belonging to medical instruments field.
Background technology
At present, tumour more and more serious threat to the life and health of the mankind.It is reported, China newly sends out tumor cases every year and is about 3,120,000 examples, and wherein, whole nation people's average every day 8550, per minutely have 6 people to be diagnosed as tumour, national tumor mortality rate is 180.54/10 ten thousand, every year because the case of tumor mortality reaches 2,700,000 examples.So touch destination data, highly cause the attention of relevant departments, every medical worker is also all for reducing tumor disease and effort.The mortality ratio of tumour is in reason not except the incidence of disease, and the malignization transfer of tumour causes cancer return and the low basic reason of cure rate, and lacking effective detection to tumour cell is a major reason.
Detect tumour cell and effectively can carry out early diagnosis to tumor presence; Can assess fast and effectively the effect for the treatment of means, comparatively suitable concrete therapeutic scheme can be determined with this; Can realize, to tumor recurrence monitoring, there is important clinical value.
But traditional technical method is difficult to carry out direct-detection analysis to it.The and then development of science and technology, there has been proposed a series of isolation technics based on micrometer/nanometer material and structure to realize enrichment to tumour cell and detection.But the existing detection sensitivity based on this kind of method still awaits improving, and it is reported, utilizes this method only to have in the patient with breast cancer of five or six layers and detected tumour cell.Therefore need constantly to develop highly sensitive enrichment and detection method.
Summary of the invention
In order to overcome the deficiency of existing tumour cell detection method, it is that the device of substrate is to detect tumour cell with nano projection that the present invention prepares a kind of.
To achieve the above object of the invention, the technical solution adopted in the present invention is: for detecting the preparation method of the device of tumour cell, comprise the following steps: with ultrasound wave etching method, prepares liquid storage tank and two fluid passages on a glass, as glass substrate; Adopt reactive ion etching method on silicon cover plate, prepare the protruding rough region of yurt shape; On silicon cover plate, two holes are prepared by laser boring method, clean by ethanol and washed with de-ionized water, as the silicon cover plate with rough region; By silicon cover plate sealing-in side on the glass substrate, fluid liquid reservoir pond is corresponding with yurt rough region; One end, every bar fluid passage is communicated with liquid storage tank, and one end is corresponding with a hole.
The present invention is made up of glass substrate and silicon cover plate for the device detecting tumour cell; Described silicon cover plate is provided with the rough region be made up of yurt shape projection; Described silicon cover plate is also provided with two holes; Described glass substrate is provided with liquid storage tank and two fluid passages; Described glass substrate is positioned at the below of silicon cover plate; Described rough region is corresponding with liquid storage tank; One end, described every bar fluid passage is communicated with liquid storage tank, and one end is corresponding with a hole.
According to method of the present invention, the height of described yurt shape projection is 1.2 microns; The spacing of yurt shape projection is 0.3 micron.
According to method of the present invention, the area of described liquid storage tank is 40% of glass substrate area.
According to method of the present invention, described silicon cover plate and glass substrate are square; Two holes on described silicon cover plate are positioned at diagonal positions.
The protruding rough region of yurt on silicon cover plate catches enriched tumor cell, is surveyed area.Above-mentioned so-called rough region is corresponding with liquid storage tank refer to rough region and liquid storage tank area close, preferably unanimously; Face, face in opposite directions.
Preferably, fluid passage comprises level and smooth runner and fishbone runner.Fishbone runner can mix inflow fluid wherein well.
Glass substrate is combined with the sealing-in of silicon cover plate, liquid storage tank on glass substrate and fluid passage form enclosed construction, and the liquid storage tank that glass substrate is formed can be used for depositing reagent, and passage is used for the circulation of fluid, need on silicon cover plate, to arrange aperture, as the gateway of fluid; The aperture of the preferred aperture of the present invention is 1 millimeter.
In preparation for detecting tumour cell device disclosed by the invention:
According to liquid storage tank, the fluid passage of design, draw out required figure, make diaphragm and be attached on glass; With ultrasound wave etching method, prepare liquid storage tank and fluid passage on a glass; According to design, adopt reactive ion etching method on silicon cover plate, prepare the protruding rough region of yurt shape; On silicon cover plate, two apertures are prepared by laser boring method; Finally use ethanol and washed with de-ionized water clean; For the silicon cover plate with rough region; By silicon cover plate sealing-in side on the glass substrate, fluid liquid reservoir pond is corresponding with yurt rough region; One end of fluid passage is corresponding with aperture, is respectively fluid intake and outlet.Ultrasound wave etching method, reactive ion etching method, laser boring method and sealing-in belong to prior art, and those skilled in the art can selective reagent and Parameter Conditions as required.
Due to the utilization of technique scheme, the present invention has the following advantages:
Nanometer rough region is applied to for detecting in the device of tumour cell by the present invention, considerably improves the efficiency catching tumour cell; Greatly improve the sensitivity that tumour cell detects; Be conducive to the accurate detection of illness.
Accompanying drawing explanation
Fig. 1 is the structural representation for detecting tumour cell device;
Fig. 2 is the structural representation of embodiment one silicon cover plate;
Fig. 3 is the structural representation of embodiment one glass substrate;
Wherein, 1, glass substrate, 2, silicon cover plate, 21, fluid intake, 22, fluid egress point, 23, the protruding rough region of yurt, 11, liquid storage tank, 12, fluid passage, 13, fluid passage;
Fig. 4 is known number object tumour cell and plot of light intensity tumour cell being detected with device of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
Embodiment one
Accompanying drawing 1 is the structural representation detecting tumour cell device, is made up of the silicon cover plate 2 of glass substrate 1 and sealing-in side on the glass substrate.
Accompanying drawing 2 is the structural representation of silicon cover plate, and silicon cover plate is provided with fluid intake 21 and outlet 22, is also provided with the protruding rough region 23 of yurt.
Accompanying drawing 3 is the structural representation of glass substrate, is provided with liquid storage tank 11, fluid egress point and entrance by fluid passage 12,13 respectively with liquid storage tank.Fluid passage comprises level and smooth runner and fishbone runner.The fluid flowed in fishbone runner can mix well.
Detect the preparation of tumour cell device:
According to liquid storage tank, the fluid passage of design, draw out required figure, make diaphragm and be attached on glass; With ultrasound wave etching method, prepare liquid storage tank and fluid passage on the glass substrate; According to design, adopt reactive ion etching method on silicon cover plate, prepare the protruding rough region of yurt shape; On silicon cover plate, two apertures are prepared by laser boring method; Finally use ethanol and washed with de-ionized water clean; For the silicon cover plate with rough region region; By silicon cover plate sealing-in side on the glass substrate, fluid liquid reservoir pond is corresponding with yurt rough region; One end of fluid passage is corresponding with aperture, is respectively fluid intake and outlet.By clean silicon cover plate and glass baseplate surface plasma oxidation process 1 minute, then correspondence is combined with each other, and processes 22 hours, realize irreversible sealing-in in 124 DEG C of baking ovens.
Liquid storage tank size is 1.3 cm x 1.4 cm x 2 millimeters; Fluid passage diameter is 1 millimeter, and corresponding with aperture, fluid does not leak; The height of yurt shape projection is 1.2 microns; The spacing of yurt shape projection is 0.3 micron.
Sample detection:
By erythrocyte splitting in blood sample, isolate leucocyte and tumour cell; Add the electrochemiluminescence material (terpyridyl ruthenium compound) that a certain amount of antibody (tumour cell detected as required selects suitable antibody) marks afterwards, hatch altogether, centrifugal segregation, in conjunction with the electrochemiluminescence material of tumour cell, obtains cell suspending liquid; Cell suspension is added the entrance detecting tumour cell device with constant flow velocity, at the uniform velocity enter in liquid storage tank through fluid passage.Under the effect of the protruding coarse line of yurt, the tumour cell in conjunction with the chemiluminescent material that powers on firmly is caught by rough region; After cell suspending liquid has added, the tumour cell caught can elute, electrochemiluminescsystem system is utilized to detect the tumour cell of catching, cycle potentials scanning can be carried out, or adopt pulse potential pattern, or employing potentiostatic mode, can obtain electrochemical luminescence signals, is calculated the capture rate of tumour cell by electrochemiluminescence intensitometer.
In blood sample, placed the breast cancer cell of known different number, detect the signal of tumour cell according to method.Accompanying drawing 4 is the electrochemiluminescence intensity maps that placed the known breast cancer cell electrochemiluminescence intensity of different number and remove to detect tumour cell with device of the present invention in blood sample.Can see that the electrochemiluminescence intensity of the signal that device of the present invention captures and known number object cell has situation about significantly matching, prove that device of the present invention has very high seizure and detection efficiency.
Claims (5)
1. for detecting the preparation method of the device of tumour cell, it is characterized in that, comprising the following steps: with ultrasound wave etching method, prepare liquid storage tank and two fluid passages on a glass, as glass substrate; Adopt reactive ion etching method on silicon cover plate, prepare the protruding rough region of yurt shape; On silicon cover plate, two holes are prepared by laser boring method, clean by ethanol and washed with de-ionized water, as the silicon cover plate with rough region; By silicon cover plate sealing-in side on the glass substrate, fluid liquid reservoir pond is corresponding with yurt rough region; One end, every bar fluid passage is communicated with liquid storage tank, and one end is corresponding with a hole.
2. according to right 1 for detecting the preparation method of the device of tumour cell, it is characterized in that: the height of described yurt shape projection is 1.2 microns; The spacing of yurt shape projection is 0.3 micron.
3. according to right 1 for detecting the preparation method of the device of tumour cell, it is characterized in that: the area of described liquid storage tank is 40% of glass substrate area.
4. according to right 1 for detecting the preparation method of the device of tumour cell, it is characterized in that: described silicon cover plate and glass substrate are square.
5. according to right 4 for detecting the preparation method of the device of tumour cell, it is characterized in that: two holes on described silicon cover plate are positioned at diagonal positions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410801973.5A CN104483482A (en) | 2014-12-22 | 2014-12-22 | Method for manufacturing device used for detecting tumor cells |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410801973.5A CN104483482A (en) | 2014-12-22 | 2014-12-22 | Method for manufacturing device used for detecting tumor cells |
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| CN104483482A true CN104483482A (en) | 2015-04-01 |
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| CN201410801973.5A Pending CN104483482A (en) | 2014-12-22 | 2014-12-22 | Method for manufacturing device used for detecting tumor cells |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105352875A (en) * | 2015-09-28 | 2016-02-24 | 周辉 | Unmarked tumor cell detecting and diagnosing device and detecting and diagnosing method thereof |
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| WO2010080978A2 (en) * | 2009-01-08 | 2010-07-15 | The General Hospital Corporation | Pre-depletion of leukocytes in whole blood samples prior to the capture of whole blood sample components |
| CN102732415A (en) * | 2012-04-24 | 2012-10-17 | 武汉介观生物科技有限责任公司 | High-efficiency rare-cell-capturing integrated chip, manufacturing method thereof, and application thereof |
| CN103266050A (en) * | 2013-06-04 | 2013-08-28 | 上海市东方医院 | Microfluidic chip for sorting and application thereof |
| WO2014065861A1 (en) * | 2012-10-26 | 2014-05-01 | The Trustees Of The University Of Pennsylvania | Compositions, methods and microfluidics device for telomerase based in vitro diagnostic assays for detecting circulating tumor cells (ctc) |
| CN104073428A (en) * | 2014-07-09 | 2014-10-01 | 北京大学 | Cell separating micro-structural system |
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2014
- 2014-12-22 CN CN201410801973.5A patent/CN104483482A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010080978A2 (en) * | 2009-01-08 | 2010-07-15 | The General Hospital Corporation | Pre-depletion of leukocytes in whole blood samples prior to the capture of whole blood sample components |
| CN102732415A (en) * | 2012-04-24 | 2012-10-17 | 武汉介观生物科技有限责任公司 | High-efficiency rare-cell-capturing integrated chip, manufacturing method thereof, and application thereof |
| WO2014065861A1 (en) * | 2012-10-26 | 2014-05-01 | The Trustees Of The University Of Pennsylvania | Compositions, methods and microfluidics device for telomerase based in vitro diagnostic assays for detecting circulating tumor cells (ctc) |
| CN103266050A (en) * | 2013-06-04 | 2013-08-28 | 上海市东方医院 | Microfluidic chip for sorting and application thereof |
| CN104073428A (en) * | 2014-07-09 | 2014-10-01 | 北京大学 | Cell separating micro-structural system |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105352875A (en) * | 2015-09-28 | 2016-02-24 | 周辉 | Unmarked tumor cell detecting and diagnosing device and detecting and diagnosing method thereof |
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Application publication date: 20150401 |