CN109900907A - A kind of paper base sensor element and preparation method thereof for detecting cancer antigen - Google Patents
A kind of paper base sensor element and preparation method thereof for detecting cancer antigen Download PDFInfo
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Abstract
The invention discloses a kind of paper base sensor elements and preparation method thereof for detecting cancer antigen, comprising: filter paper paper base；Carboxylic carbon nano-tube, several carboxylic carbon nano-tubes are deposited on the filter paper paper base, and the carboxylic carbon nano-tube characteristic is combined with the specific antibody CA19-9 of pancreatic cancer cell.Antigen concentration information can be converted to direct measurable electric signal by the present invention, have manufacturing cost low, and precision is high, and the strong advantage of stability is, it can be achieved that economy, quick cancer antigen screening；In addition, sensor of the invention still has good detection under low CA19-9 concentration range (0~40U/ml), in early stage it can be found that cancer of pancreas.The detection time of inventive sensor only needs or so two hours, about 12 times faster than ELISA on detection efficiency.
Description
Technical field
The invention belongs to biosensor technology field and cancer detection technical fields, in particular to a kind of for detecting
Paper base sensor element of cancer antigen and preparation method thereof.
Background technique
Early discovery, early treatment are most important for it for the early detection of cancer, can significantly improve the chance for survival of patient.With
It is that a kind of grade malignancy is very high for cancer of pancreas, all highly difficult malignant tumor of digestive tract of diagnosing and treating, about 90% is
Derived from the duct adenocarcinoma of glandular tube epithelium, morbidity and mortality obviously rise in recent years.5 years survival rates are small after the onset of cancer of pancreas
It is one of highest malignant tumour of the death rate in 1%.The detection of cancer of pancreas early stage is most important to the high survival rate of guarantee, pancreas
Cancer in early detection come out be followed by it is treated almost can extend 5 years time-to-live of patient with 100%, but it is during the late stages of developmet
Drop to 31%.The mode of detection Early pancreatic carcinoma is varied at present, if detected the tumour mark object (CA19-9) in serum, surpasses
Sound wave inspection, CT scan inspection, contrast examination etc..Enzyme linked immunosorbent assay (ELISA) is the common detection side CA19-9, hospital
The repeatability of method, this method is not good enough, and the interference such as be susceptible to autoantibody, heterophil antibody, false positive easily occurs, this
Outside, when using this method, no matter instrument and manual operations, disturbing factor is more, higher cost is not easy to popularize.
Therefore, although the early detection method for cancer and its antigen is various, by detectable limit, testing cost,
The constraint of testing conditions is larger, even ELISA detection method more mature at present is remained in the presence of time-consuming (about 1 day),
It is costly, and detect the defect that need to be carried out in well-found laboratory.To sum up, existing detection technique by detection place,
The limitation such as condition, high expense is also by the economic pressures bigger to subject.
Summary of the invention
The purpose of the present invention is to provide a kind of paper base sensor element and preparation method thereof for detecting cancer antigen,
To solve above-mentioned one or more technical problems.Paper base sensor of the invention, under low CA19-9 concentration range still
With good detection；Preparation requires simply, and preparation cost is cheap；Testing conditions are of less demanding, and there is significant promote to dive
Power.
In order to achieve the above objectives, the invention adopts the following technical scheme:
It is a kind of for detecting the paper base sensor element of cancer antigen, comprising: filter paper paper base；Carboxylic carbon nano-tube, if
It does the carboxylic carbon nano-tube and is deposited on the filter paper paper base, the carboxylic carbon nano-tube is added with default tumour mark object.
Further, the filter paper paper base uses micropore filter paper.
Further, carboxylic carbon nano-tube is functionalized multi-wall carbonnanotubes, the default tumour mark that characteristic combines
Object is the specific C A19-9 antibody of pancreatic cancer cell.
It further, further include two electrodes；Two electrodes respectively with the filter paper paper base electricity that is loaded with carboxylic carbon nano-tube
Connection.
It further, further include the second substrate；The filter paper paper base is packaged between two pieces of the second substrates, and two electrodes connect
It is connected to extraction wire；Wherein, the second substrate is glass slide.
It is a kind of for detecting the preparation method of the paper base sensor element of cancer antigen, comprising the following steps:
S1 prepares material: according to mass parts number scale, every 2 parts of functionalized multi-wall carbonnanotubes are corresponded to more than or equal to 77 parts
EDC and NHSS more than or equal to 22 parts；
S2 disperses carboxylic carbon nano-tube, EDC and NHSS in MES solution, shakes and mixes；
The S2 mixed liquor obtained is put into centrifuge centrifugation, removes supernatant after centrifugation by S3；
PBS buffer solution is added in remaining precipitating into S3 treated container in S4, neutralizes remaining EDC and NHSS, shake
It swings centrifugation and removes supernatant；This step repeated several times, then go to step S5；
S5 is added PBS first to increase the uniformity of functionalized multi-wall carbonnanotubes, is then added to CA19-9 antibody mixed
It closes in liquid, stirs so that CA19-9 antibody is added in functionalized multi-wall carbonnanotubes；
20 solution of Tween is blended in BSA by S6, is then added in the mixed liquor obtained after step S5 processing, quiet
It postpones, centrifugal treating simultaneously removes supernatant；
S7 PBS buffer solution is added in the precipitating that step S6 is obtained, and cleaning removes extra CA19-9 antibody and BSA；This step
Rapid repeated several times obtain the multi-walled carbon nanotube mixture for combining CA19-9 antibody；
S8 disperses aqueous solution for mixture obtained in step S7, is deposited on filter paper paper base, is used for after mixing
Detect the paper base sensor element of cancer antigen.
Further, step S8 is specifically included: the multi-walled carbon nanotube for combining CA19-9 antibody being sunk using filtration method
Product is on micropore filter paper.
Further, step S1 is specifically included: functionalized multi-wall carbonnanotubes 2mg, at least 4mL include the molten of 0.1M MES
Liquid, EDC powder 77mg, NHSS powder 22mg, PBS solution at least 100mL, the Tween20 solution 10uL of concentration 0.05%, concentration
The antibody-solutions 0.5mL of 1% BSA solution 100uL, concentration 0.01mg/mL.
Further, specifically includes the following steps:
(1) functionalized multi-wall carbonnanotubes for claiming 2mg, are placed in centrifuge tube；
(2) use dropper to the MES solution of dropwise addition 2mL in centrifuge tube；
(3) it puts centrifuge tube into ultrasonic oscillator, shakes 1min, reuse blending instrument and mix mixed liquor；
(4) weigh 77mg EDC and pour into centrifuge tube, clean tube wall with the MES solution of 1mL, then ultrasonic oscillation 10~
15min；
(5) weigh 22mg NHSS and pour into centrifuge tube, then plus MES solution to 4mL, then ultrasonic oscillation 40min；
(6) tube wall is rinsed with MES solution after the completion of shaking, constant volume is simultaneously put into centrifuge centrifugation, 5000r/min centrifugation
Supernatant is sucked out with dropper after centrifugation in 10min；
(7) after PBS solution to 7.5mL, then ultrasonic oscillation 1min being added into centrifuge tube, 5000r/min centrifugation
10min, after with dropper be sucked out supernatant；This step is at least repeated 5 times, and then go to step (8)；
(8) PBS solution is added to 10mL to centrifuge tube, after ultrasonic oscillation 1min, all pours into magnet rotor bottle；
(9) antibody is poured into magnet rotor bottle, temperature setting is in room temperature hereinafter, stirring diel with magnetic stirrer；
(10) after the completion of stirring, the BSA of the Tween20 and 100uL of 10uL is added into centrifuge tube with liquid-transfering gun, room temperature is quiet
It sets one hour；
(11) the centrifugal treating 10min under 5000r/min environment removes supernatant, and PBS solution is then added and is settled to
7.5mL, ultrasonic oscillation 1min；This step is at least repeated 5 times, and last time adds distilled water after being centrifuged, mixed with ultrasonic oscillation
It is even；
(12) a micropore filter paper is taken out, the rectangle of 2mm × 5mm is drawn at 8 circular symmetric positions；Filter centrifugation
It is on liquid to another filter paper in pipe, two filter paper are superimposed together, cut 8 rectangular paper based sensor elements；
(13) be respectively stained with a copper wire at 8 rectangular paper based sensor both ends with conductive silver glue, after be sealed, complete
Preparation.
It is a kind of for detecting the preparation method of the paper base sensor element of cancer, comprising the following steps: by the carboxylated of 2mg
Multi-walled carbon nanotube, 0.4mmol EDC and 0.1mmol NHSS, are scattered in the MES solution of the 0.1M of 4mL；Dispersion solution exists
Water bath sonicator about 40min in Ultrasound Instrument, then be uniformly mixed with blending instrument；3000rpm is centrifuged 10min after mixing, then in removal
Clear liquid；PBS buffer solution is added in precipitating, 3000rpm is centrifuged 10min, removes supernatant, repeats PBS washing process at least 5
Extra EDC and NHSS in secondary removal precipitating；Taking concentration is the CA19-9 antibody-solutions 0.5mL of 0.01mg/mL, is added to mixing
In liquid, container is placed on magnetic stirring apparatus under room temperature and is stirred overnight, CA19-9 antibody is made to be added in the multi wall carbon of functional modification
In nanotube；The Tween20 solution that concentration is 0.05% is blended in the 1%BSA of 100 μ L, stirring one is then added into
After night in resulting solution；Mixture solution is stood to one hour at room temperature, then with centrifuge under 5000rpm environment from
Heart 10min removes supernatant, is cleaned with PBS buffer solution and precipitates and be centrifuged removal supernatant at least 5 times, to remove extra CA19-
9 antibody and BSA obtain product CA19-9 antibody-multi-walled carbon nanotube conjugate；Aqueous solution is dispersed by products therefrom, and
Water bath sonicator mixes in Ultrasound Instrument, and CA19-9 antibody-multi-walled carbon nanotube conjugate is deposited on micropore filter paper using filtration method
On, complete the preparation of paper base sensor element.
Compared with prior art, the invention has the following advantages:
In sensor of the invention, being added in multi-wall carbon nano-tube pipe surface can be with pancreatic carcinoma antigen CA19-9 specificity knot
The monoclonal antibody of conjunction, and multi-walled carbon nanotube is deposited on fixed-size test paper surface, finally connect metal electrode
Electrical testing characterization is carried out, antigen concentration information can be converted to direct measurable electric signal, and (i.e. U-I characteristic can reflect electricity
Resistance), have manufacturing cost low, precision is high, and the strong advantage of stability is, it can be achieved that economy, quick cancer antigen screening.
Specific advantage of the invention is embodied in:, maximum detectable limit very big for the biomarker detection range of tumour
It is more than ten times of existing enzyme linked immunosorbent assay (ELISA) more than 1000U/mL；In addition, the CA19-9 concentration of normal body is answered
Less than 40U/ml, sensor of the invention still has good detection under low CA19-9 concentration range (0~40U/ml), this
Mean that sensor can detecte the low concentration of CA19-9, in early stage it can be found that cancer of pancreas；Its based on principle be:
Antigen and antibody specific combines the variation that will lead to resistance, for low concentration antigen, still has and occurs in conjunction with situation, can change
Resistance value is to be measured.
In preparation method of the invention, by adding the special of pancreatic cancer cell on the multi-walled carbon nanotube surface (MWCNTs)
Property antibody CA19-9, and multi-walled carbon nanotube is deposited on fixed-size test paper surface, at low cost, precision can be produced
Height, the strong cancer antigen Concentration Testing test paper of stability can be realized economic, quick cancer antigen screening.
The detection time of inventive sensor only needs or so two hours, about 12 times faster than ELISA on detection efficiency；And
Drug dosage is relatively fewer, and the material prices such as multi-walled carbon nanotube used are cheap, advantage of lower cost, enzyme-linked exempts from existing
Epidemic disease absorption method, which is compared, is more suitable the early screening of low developed area cancer, has significant promotion potential.
Detailed description of the invention
Fig. 1 is of the invention a kind of for detecting the structural schematic diagram of the paper base sensor of cancer antigen；
Fig. 2 is of the invention a kind of for detecting the preparation method flow diagram of the paper base sensor of cancer antigen；
Fig. 3 is the preparation flow schematic diagram in preparation method of the invention with antibody multi-walled carbon nanotube；
Fig. 4 is that a kind of specific mechanism micro-explanation for detecting the paper base sensor of cancer antigen of the invention is illustrated
Figure；
Fig. 5 is the antigen concentration resistance test schematic diagram of the sensor of the invention of different batches production；Fig. 5 (a) is the
One batch resistance test schematic diagram；Fig. 5 (b) is second lot resistance test schematic diagram；Fig. 5 (c) is third batch resistance test
Schematic diagram；Fig. 5 (d) is the 4th batch resistance test schematic diagram；
Fig. 6 is the SEM electron microscope of functionalized multi-wall carbonnanotubes in the embodiment of the present invention；
Fig. 7 is the SEM electron microscope that the functionalized multi-wall carbonnanotubes after antibody are connect in the embodiment of the present invention；
Fig. 8 is the SEM electron microscope that the functionalized multi-wall carbonnanotubes after antigen-antibody are connect in the embodiment of the present invention；
In Fig. 1,1, copper conductor；2, glass slide；3, micropore filter paper；4, metal electrode；5, CA19-9 antibody-multi-wall carbon nano-tube
Pipe.
Specific embodiment
Invention is further described in detail in the following with reference to the drawings and specific embodiments.
Referring to Fig. 1, the embodiment of the present invention is a kind of for detecting the paper base sensor element of cancer antigen, comprising: filter
Paper paper base, filter paper paper base use micropore filter paper 3；Several functionalized multi-wall carbonnanotubes, the carboxylic are deposited on filter paper paper base
Base multi-walled carbon nano-tube is added with the active default tumour mark object of holding；For example, CA19-9 antibody-multi-walled carbon nanotube 5,
The default tumour mark object that i.e. characteristic combines is the specific antibody CA19-9 of pancreatic cancer cell；Two metal electrodes 4 lead to respectively
Conducting resinl is crossed to be electrically connected with carboxylic carbon nano-tube.Filter paper paper base is fixed and is packaged between two pieces of glass slides 2, two with elargol
Metal electrode 4 is drawn by copper conductor 1.In sensor of the invention, pancreas is added on the multi-walled carbon nanotube surface (MWCNT)
The specific antibody CA19-9 of cancer cell, and multi-walled carbon nanotube is deposited on fixed-size test paper surface, finally connect
It connects metal electrode 4 and carries out electrical testing characterization, antigen concentration information can be converted to directly measurable electric signal, and (i.e. U-I is special
Property, can reflect resistance), have manufacturing cost low, precision is high, and the strong advantage of stability is, it can be achieved that economy, quick cancer antigen
Screening.
Fig. 2, Fig. 3 and Fig. 6 are please referred to Fig. 8, the embodiment of the present invention it is a kind of for detecting the paper base sensor of cancer
The preparation method of element, comprising:
According to mass parts number scale, functionalized multi-wall carbonnanotubes are 2 parts, and EDC should be greater than being equal to 77 parts, and NHSS should be greater than
In 22 parts；
Specifically, preparing reagent and material that preparation process needs: functionalized multi-wall carbonnanotubes 2mg, at least 4mL
(0.1M) MES solution, EDC powder 77mg, NHSS powder 22mg, PBS solution at least 100mL, Tween20 solution (concentration
0.05%) 10uL, BSA solution (concentration 1%) 100uL, filter paper two open, glass slide two panels, gel, antibody-solutions (concentration
0.01mg/mL)0.5mL。
Its preparation process the following steps are included:
1) powder, is then poured into big centrifuge tube by the carboxylic carbon nano-tube for claiming 2mg；It can also designed, designed carboxylated step
Suddenly, the binding ability of antibody and carbon nanotube can be enhanced by carboxylation.
2) with dropper to the MES solution of dropwise addition 2mL in big centrifuge tube, for improving the dispersibility of carbon nanotube.
3) it puts big centrifuge tube into ultrasonic oscillator, shakes 1min, reuse blending instrument and mix solution.
4) weigh 77mg EDC and pour into centrifuge tube, then plus 1mL MES, clean tube wall, then ultrasonic oscillation 10-
15min。
5) it weighs and 22mg NHSS is added into big centrifuge tube, for protecting EDC not hydrolyze before with antigen binding；
Again plus MES to 4mL, ultrasonic oscillation 40min later.
6) tube wall is rinsed with MES after the completion of shaking, constant volume is simultaneously put into centrifuge centrifugation (5000r/min) 10min, after centrifugation
Supernatant is sucked out with dropper.
7) PBS to 7.5mL is added into big centrifuge tube, to neutralize remaining EDC and NHSS, then ultrasonic oscillation 1min,
After the completion be centrifuged (5000r/min) 10min, after with dropper be sucked out supernatant.The step is at least repeated 5 times.
8) PBS to 10mL is added, more PBS, which can also be added, makes carbon nanotube dispersion more evenly, ultrasonic oscillation 1min,
All pour into magnet rotor bottle；Centrifugation tube wall is cleaned with PBS, washing lotion pours into magnetic agitation bottle.
9) it takes out antibody and thaws at room temperature；It pours antibody into magnet rotor bottle, and is flushed out remaining antibody with PBS
Come.
10) diel is stirred with magnetic stirrer, temperature setting is at 20 degree hereinafter, CA19-9 is made to be added in functional modification
In multi-walled carbon nanotube.
11) after the completion of stirring, the BSA of the Tween20 and 100Ul of 10uL is added into big centrifuge tube with liquid-transfering gun；Wherein,
Tween20 is nonionic surface active agent, and for removing the binding site of CA19-9 antibody, BSA is used to preventing all non-
Feature combines；Then it is stored at room temperature one hour.
12) it is centrifuged 10min (5000r/min), removes supernatant, PBS is then added and is settled to 7.5mL, ultrasonic oscillation
1min, to remove extra CA19-9 antibody and BSA.The step is at least repeated 5 times, and last time adds water after being centrifuged, and uses ultrasonic wave
Concussion mixes.
13) filter paper is taken out, the rectangle of 2mm × 5mm is drawn at 8 circular symmetric positions.
14) it filters on the liquid to another filter paper in big centrifuge tube.
15) two filter paper of step 13) He step 14) are superimposed together, cut 8 rectangular paper based sensor members
Part.
16) be respectively stained with a copper wire at 8 rectangular paper based sensor both ends with conductive silver glue, after be sealed, complete
Preparation.
The present invention by the multi-walled carbon nanotube surface (MWCNTs) add pancreatic cancer cell specific antibody CA19-9,
And be deposited into fixed-size test paper surface, it finally connects metal electrode and carries out electrical testing characterization, antigen is dense
Degree information is converted to direct measurable electric signal (the U-I characteristic that can reflect resistance), produces at low cost, precision height, stable
The strong cancer antigen Concentration Testing test paper of property is, it can be achieved that economy, quick cancer antigen screening.The present invention has the advantage that
Very big for the biomarker detection range of tumour, it is existing Enzyme-linked Immunosorbent Assay that maximum detection limit, which is more than 1000U/mL,
More than ten times of method (ELISA), and importantly, the CA19-9 concentration of normal body should be less than 40U/ml, this sensor is low
Still there is good detection, it means that sensor can detecte CA19-9's under CA19-9 concentration range (0~40U/ml)
Low concentration, in early stage it can be found that cancer of pancreas.Detection time only needs or so two hours, compares on detection efficiency
About 12 times fastly of ELISA, and the material prices such as multi-walled carbon nanotube used are cheap, compared with existing enzyme linked immunosorbent assay
It is more suitable the early screening of low developed area cancer.
Referring to Fig. 4, use process and mechanism explanation of the invention are as follows:
1) before filter paper is not completely dried, the initial resistance value R of paper base sensor is first measured and recorded0。
2) 10uL antigen is added dropwise on paper base sensor；Add herein according to the preset vol for preparing antigen in paper base sensor
Enter.
3) in 36 DEG C~38 DEG C Environment cultivations antigen-antibody is combined；It is dry to remove extra water；For example, sealing after
37 DEG C of cultivation 1.5h, then open lid in air in drying box, and lid is left unlocked or unlatched, dry 30min.
4) the paper base sensor resistance R after measurement antigen-antibody combines.
5) R/R is calculated0Value, antigen concentration can be obtained by the value and the linear relationship of antigen concentration, can be detected out
Tumour mark object concentration in serum.
The embodiment of the present invention it is a kind of for detecting the preparation method of the paper base sensor element of cancer, including following step
Rapid: the functionalized multi-wall carbonnanotubes for preparing 2mg (are bought in Shanghai Mike's woods biochemical technology Co., Ltd, internal diameter 5-12nm, outside
Diameter 30-50nm, length < 10nm), 0.4mmolEDC and 0.1mmolNHSS is added, and be scattered in the MES solution of the 0.1M of 4mL
In.Disperse solution water bath sonicator about 40min in Ultrasound Instrument (new sesame SB-3200OTD), then with blending instrument (SCI LOGEX
ICC8 it) is uniformly mixed.With centrifuge (ordinary instrument TD6) centrifugation 10min (3000rp/m) after mixing, supernatant is then removed.
After by PBS buffer solution be added precipitating in, be centrifuged 10min (3000rp/m), remove supernatant.Repeat above-mentioned PBS washing
Process is washed till EDC and NHSS extra in few 5 removals precipitating.By CA19-9 antibody 0.5mL, (0.01mg/mL is bought in Shanghai
Ling Chao Biotechnology Co., Ltd), it is added in mixed liquor, bottle is placed on magnetic stirring apparatus under room temperature and is stirred overnight, is made
CA19-9 is added in the multi-walled carbon nanotube of functional modification.
20 solution of Tween that concentration is 0.05% is blended in the 1%BSA of 100 μ L, stirring one is then added into
After night in resulting solution.After object solution to be mixed stands one hour at room temperature, it is centrifuged with centrifuge (ordinary instrument TD6)
10min (5000rp/m), removes supernatant, cleaning precipitating and being centrifuged with PBS buffer solution remove for 5 times extra CA19-9 antibody and
BSA.The resulting product of the product of this process is called CA19-9 antibody-multi-walled carbon nanotube.
Aqueous solution is dispersed by products therefrom, and water bath sonicator mixes in Ultrasound Instrument (new sesame SB-3200 OTD).Using
CA19-9 antibody-multi-walled carbon nanotube is deposited on micropore filter paper by injection filtration method, and carbon nanotube cake filter paper is cut into 5mm
X2mm specification (filter paper should be cut from center to circumference), to prepare our paper base sensor element.
A kind of paper base sensor testing method of the embodiment of the present invention, comprising: include the CA19-9 of PBS solution by 100 μ L
Drop should retain a piece of sensor unit that CA19-9 is not added dropwise and compare, be placed on the plastics with sealing cover on filter paper unit
37 DEG C of holding 1.5h in ware dry 30min in air.
At ambient conditions, the measurement for carrying out U-I characteristic, measures the resistance value of the sensor unit, and CA19- is not added
The ratio between 9 resistance value of sensor unit should meet following linear relationship:
Antigen concentration (U/mL)=90.513 (R/R0)-87.636
According to this measurement result, antigen concentration be added dropwise can be calculated.In the case where reagent is slightly distinguished, line
Sexual intercourse function value may be slightly different, therefore in order to be more accurate, in actual use, it is proposed that first by preparation process production first
It criticizes sample and calculates functional relation, then detected.
Referring to Fig. 5, the performance verification of paper base sensor of the invention:
It actually considers in paper base sensor performance, has made 4 batches of products altogether.Once 3 have been manufactured from first batch
Identical sensing element, and resistance test has been carried out in 3 CA19-9 levels；Once 6 phases have been manufactured from second batch
Same sensing element, and resistance test has been carried out in 6 CA19-9 levels；Once from third batch manufactured 5 it is identical
Sensing element, and carried out resistance test in 5 CA19-9 levels；Once from the 4th batch manufactured 6 it is identical
Sensing element, and resistance test has been carried out in 6 CA19-9 levels.This is a kind of side for checking sensor performance well
Method, because the sensor of preparation should not be dried, until CA19-9 detection process keeps bioactivity.Scheme (a), (b), (c),
(d) changing in the approximately linearly CA19-9 concentration within the scope of 0~1000U/ml for resistance is shown to change.This linearity
Test may be used as the internal standard of detection, to confirm detectability of the identical batch of sensing element under different detection environment.
The above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof, although referring to above-described embodiment pair
The present invention is described in detail, those of ordinary skill in the art still can to a specific embodiment of the invention into
Row modification perhaps equivalent replacement these without departing from any modification of spirit and scope of the invention or equivalent replacement, applying
Within pending claims of the invention.
Claims (10)
1. a kind of for detecting the paper base sensor element of cancer antigen characterized by comprising
Filter paper paper base；
Carboxylic carbon nano-tube, several carboxylic carbon nano-tubes are deposited on the filter paper paper base, the carboxylated carbon nanometer
Pipe is added with default tumour mark object.
2. according to claim 1 a kind of for detecting the paper base sensor element of cancer antigen, which is characterized in that described
Filter paper paper base uses micropore filter paper.
3. according to claim 1 a kind of for detecting the paper base sensor element of cancer antigen, which is characterized in that carboxyl
Carbon nano tube is functionalized multi-wall carbonnanotubes, and the default tumour mark object that characteristic combines is the specificity of pancreatic cancer cell
CA19-9 antibody.
4. according to claim 1 a kind of for detecting the paper base sensor element of cancer antigen, which is characterized in that also wrap
Include two electrodes；Two electrodes are electrically connected with the filter paper paper base for being loaded with carboxylic carbon nano-tube respectively.
5. according to claim 4 a kind of for detecting the paper base sensor element of cancer antigen, which is characterized in that also wrap
Include the second substrate；
The filter paper paper base is packaged between two pieces of the second substrates, and two electrodes are connected with extraction wire；
Wherein, the second substrate is glass slide.
6. a kind of for detecting the preparation method of the paper base sensor element of cancer antigen, which comprises the following steps:
S1 prepares material: according to mass parts number scale, the corresponding EDC more than or equal to 77 parts of every 2 parts of functionalized multi-wall carbonnanotubes with
And the NHSS more than or equal to 22 parts；
S2 disperses carboxylic carbon nano-tube, EDC and NHSS in MES solution, shakes and mixes；
The S2 mixed liquor obtained is put into centrifuge centrifugation, removes supernatant after centrifugation by S3；
S4 is added PBS buffer solution in remaining precipitating into S3 treated container, neutralizes remaining EDC and NHSS, shake from
The heart simultaneously removes supernatant；This step repeated several times, then go to step S5；
S5 is added PBS first to increase the uniformity of functionalized multi-wall carbonnanotubes, CA19-9 antibody is then added to mixed liquor
In, it stirs so that CA19-9 antibody is added in functionalized multi-wall carbonnanotubes；
20 solution of Tween is blended in BSA by S6, is then added in the mixed liquor obtained after step S5 processing, after standing,
Centrifugal treating simultaneously removes supernatant；
S7 PBS buffer solution is added in the precipitating that step S6 is obtained, and cleaning removes extra CA19-9 antibody and BSA；This step weight
Again several times, the multi-walled carbon nanotube mixture for combining CA19-9 antibody is obtained；
S8 disperses aqueous solution for mixture obtained in step S7, is deposited on filter paper paper base after mixing, obtains for detecting
The paper base sensor element of cancer antigen.
7. it is according to claim 6 a kind of for detecting the preparation method of the paper base sensor element of cancer antigen, it is special
Sign is that step S8 is specifically included: the multi-walled carbon nanotube for combining CA19-9 antibody being deposited on micropore filter using filtration method
On paper.
8. according to claim 6 a kind of for detecting the preparation method of the paper base sensor element of cancer, feature exists
In step S1 is specifically included: functionalized multi-wall carbonnanotubes 2mg, at least 4mL include the solution of 0.1M MES, EDC powder
77mg, NHSS powder 22mg, PBS solution at least 100mL, Tween20 the solution 10uL, the BSA of concentration 1% of concentration 0.05% are molten
The antibody-solutions 0.5mL of liquid 100uL, concentration 0.01mg/mL.
9. according to claim 8 a kind of for detecting the preparation method of the paper base sensor element of cancer, feature exists
In, specifically includes the following steps:
(1) functionalized multi-wall carbonnanotubes for claiming 2mg, are placed in centrifuge tube；
(2) use dropper to the MES solution of dropwise addition 2mL in centrifuge tube；
(3) it puts centrifuge tube into ultrasonic oscillator, shakes 1min, reuse blending instrument and mix mixed liquor；
(4) weigh 77mg EDC and pour into centrifuge tube, clean tube wall with the MES solution of 1mL, then ultrasonic oscillation 10~
15min；
(5) weigh 22mg NHSS and pour into centrifuge tube, then plus MES solution to 4mL, then ultrasonic oscillation 40min；
(6) tube wall is rinsed with MES solution after the completion of shaking, constant volume is simultaneously put into centrifuge centrifugation, and 5000r/min is centrifuged 10min, from
Supernatant is sucked out with dropper after the heart；
(7) after PBS solution to 7.5mL, then ultrasonic oscillation 1min being added into centrifuge tube, 5000r/min is centrifuged 10min, knot
Supernatant is sucked out in Shu Houyong dropper；This step is at least repeated 5 times, and then go to step (8)；
(8) PBS solution is added to 10mL to centrifuge tube, after ultrasonic oscillation 1min, all pours into magnet rotor bottle；
(9) antibody is poured into magnet rotor bottle, temperature setting is in room temperature hereinafter, stirring diel with magnetic stirrer；
(10) after the completion of stirring, the BSA of the Tween20 and 100uL of 10uL is added into centrifuge tube with liquid-transfering gun, is stored at room temperature one
Hour；
(11) the centrifugal treating 10min under 5000r/min environment removes supernatant, and PBS solution is then added and is settled to 7.5mL,
Ultrasonic oscillation 1min；This step is at least repeated 5 times, and last time adds distilled water after being centrifuged, and is mixed with ultrasonic oscillation；
(12) a micropore filter paper is taken out, the rectangle of 2mm × 5mm is drawn at 8 circular symmetric positions；It filters in centrifuge tube
Liquid to another filter paper on, two filter paper are superimposed together, cut 8 rectangular paper based sensor elements；
(13) be respectively stained with a copper wire at 8 rectangular paper based sensor both ends with conductive silver glue, after be sealed, complete system
It is standby.
10. a kind of for detecting the preparation method of the paper base sensor element of cancer, which comprises the following steps: will
Functionalized multi-wall carbonnanotubes, 0.4mmol EDC and the 0.1mmol NHSS of 2mg, is scattered in the MES solution of the 0.1M of 4mL；
Disperse solution water bath sonicator about 40min in Ultrasound Instrument, then is uniformly mixed with blending instrument；3000rpm is centrifuged 10min after mixing,
Then supernatant is removed；PBS buffer solution is added in precipitating, 3000rpm is centrifuged 10min, removes supernatant, repeats PBS and washes
Wash EDC and NHSS extra in process at least 5 times removal precipitatings；Taking concentration is the CA19-9 antibody-solutions of 0.01mg/mL
0.5mL is added in mixed liquor, and container is placed on magnetic stirring apparatus under room temperature and is stirred overnight, and CA19-9 antibody is made to be added in function
In the multi-walled carbon nanotube of energyization modification；The Tween20 solution that concentration is 0.05% is blended in the 1%BSA of 100 μ L, so
After be added into and be stirred overnight in rear resulting solution；Mixture solution is stood to one hour at room temperature, then uses centrifuge
It is centrifuged 10min under 5000rpm environment, removes supernatant, is cleaned with PBS buffer solution and precipitates and be centrifuged removal supernatant at least 5
It is secondary, to remove extra CA19-9 antibody and BSA, obtain product CA19-9 antibody-multi-walled carbon nanotube conjugate；By products therefrom
It is scattered in aqueous solution, and water bath sonicator mixes in Ultrasound Instrument, is combined CA19-9 antibody-multi-walled carbon nanotube using filtration method
Object is deposited on micropore filter paper, completes the preparation of paper base sensor element.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103995031A (en) * | 2014-06-10 | 2014-08-20 | 江南大学 | Preparation method and detection method of plant virus paper detection sensor |
| WO2016161255A1 (en) * | 2015-04-01 | 2016-10-06 | The Board Of Trustees Of The University Of Illinois | Analyte sensing for eye injuries and conditions |
| CN108709914A (en) * | 2018-05-28 | 2018-10-26 | 华东理工大学 | A kind of method of quick detection alpha-fetoprotein |
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2019
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| CN103995031A (en) * | 2014-06-10 | 2014-08-20 | 江南大学 | Preparation method and detection method of plant virus paper detection sensor |
| WO2016161255A1 (en) * | 2015-04-01 | 2016-10-06 | The Board Of Trustees Of The University Of Illinois | Analyte sensing for eye injuries and conditions |
| CN108709914A (en) * | 2018-05-28 | 2018-10-26 | 华东理工大学 | A kind of method of quick detection alpha-fetoprotein |
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| SUNGKYUNG JI等: "Detection of early stage prostate cancer by using a simple carbon nanotube@paper biosensor", 《BIOSENSORS AND BIOELECTRONICS》 * |
| YANG WANG等: "Electrochemical integrated paper-based immunosensor modified with multi-walled carbon nanotubes nanocomposites for point-of-care testing of 17β-estradiol", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 毛宇湘等: "《实用中西医消化病诊疗学》", 31 January 2018, 科学技术文献出版社 * |
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Application publication date: 20190618 |