Summary of the invention
The purpose of the present invention is to provide a kind of detection method of paper base sensor based on specific C A19-9 antibody, with
Solve above-mentioned one or more technical problems.Paper base sensor of the invention still has under low CA19-9 concentration range
There is good detection;Testing conditions of the present invention are of less demanding, and detection speed is quick, have significant promotion potential.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A kind of detection method of the paper base sensor based on specific C A19-9 antibody, comprising the following steps:
Step 1, before the filter paper paper base of paper base sensor is not completely dried, pass through two electrode measurement paper base sensors
Initial resistance value R0, and record;
Step 2, test serum is added dropwise in the biosensor component of paper base sensor;
Step 3, by step 2, treated that paper base sensor is sealed in drying box cultivates preset time, so that blood to be measured
Antigen in clear is combined with the antibody in biosensor component, is then dried in air;
Step 4, the resistance value R of measurement obtaining step 3 treated paper base sensor;
Step 5, R/R is calculated0Ratio;Show that antigen is dense according to the linear relationship of the ratio of calculating and antigen concentration
Degree completes the tumour mark object Concentration Testing in test serum.
Further, step 3 specifically includes: in drying box, making antigen-antibody knot in 36 DEG C~38 DEG C Environment cultivations
It closes;It is dry to remove extra water.
Further, in step 5, R/R0Ratio and antigen concentration are linear relationship.
Further, acquisition is prepared according to the following steps in the paper base sensor in step 1:
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, in step 1, the paper base sensor includes: filter paper paper base, biosensor component and two electricity
Pole;
The biosensor component is deposited on the filter paper paper base;The biosensor component includes: carboxylated
Carbon nanotube;The carboxylic carbon nano-tube surface characteristics is combined with the specific C A19-9 antibody of pancreatic cancer cell;Two
Electrode is electrically connected with the biosensor component respectively.
Further, the filter paper paper base uses micropore filter paper.
Further, carboxylic carbon nano-tube is functionalized multi-wall carbonnanotubes.
Further, electrode is metal electrode;Two metal electrodes pass through conductive adhesive in biosensor member respectively
On part.
Further, further includes: the second substrate;The filter paper paper base is packaged between two pieces of the second substrates, two electrodes
It is connected with extraction wire.
Further, the second substrate is glass slide;The extraction wire is copper conductor;The filter paper paper base passes through silver
It is adhesive on glass slide.
Compared with prior art, the invention has the following advantages:
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.
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.
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 biosensor component of cancer, comprising: filter paper paper
Base, filter paper paper base use micropore filter paper 3, and the filter opening of micropore filter paper can be less than or equal to 1.8um;If being deposited on filter paper paper base
Dry functionalized multi-wall carbonnanotubes, the functionalized multi-wall carbonnanotubes are added with the active default tumour mark object of holding;Such as
For CA19-9 antibody-multi-walled carbon nanotube 5, i.e., the default tumour mark object that characteristic combines is the specific antibody of pancreatic cancer cell
CA19-9;Two metal electrodes 4 are electrically connected by conducting resinl with carboxylic carbon nano-tube respectively.Filter paper paper base is fixed simultaneously with elargol
It is packaged between two pieces of glass slides 2, two metal electrodes 4 are drawn by copper conductor 1.In sensor of the invention, in multi wall carbon
The nanotube surface (MWCNTs) is added with the specific antibody CA19-9 of pancreatic cancer cell, and multi-walled carbon nanotube is deposited on tool
There is fixed-size test paper surface, finally connects metal electrode 4 and carry out electrical testing characterization, antigen concentration information can be converted
For direct measurable electric signal (i.e. U-I characteristic can reflect resistance), have manufacturing cost low, precision is high, strong excellent of stability
Point is, it can be achieved that economic, quick cancer antigen screening.
Fig. 2, Fig. 3 and Fig. 6 are please referred to Fig. 8, a kind of paper based on specific C A19-9 antibody of the embodiment of the present invention
The preparation method of based sensor, 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, NHSS should be greater than being equal to 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 are superimposed together, cut 8 rectangular paper based sensor elements.
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 (MWCNT) add pancreatic cancer cell specific C A19-9 antibody,
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, a kind of detection of paper base sensor based on specific C A19-9 antibody of the embodiment of the present invention is anti-
The method of original content, comprising:
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, in 37 DEG C of rings
Cultivate at least 1.5h in border;Dry at least 30min in air;Specifically, sealing after in drying box 37 DEG C of cultivation 1.5h, then
Lid is opened in air, 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 biosensor component 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 0.1mol of 4mL
In.Disperse solution water bath sonicator about 40min in Ultrasound Instrument (new sesame SB-3200 OTD), then with blending instrument (SCI LOGEX
ICC 8) 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-3200OTD).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
× 2mm specification (filter paper should be cut from center to circumference), to prepare our biosensor component.
A kind of paper base sensor detecting 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.