CN106501520A - A kind of illicit drugs inspection reagent paper based on microfluidic capillary structure and preparation method thereof - Google Patents
A kind of illicit drugs inspection reagent paper based on microfluidic capillary structure and preparation method thereof Download PDFInfo
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure and preparation method thereof, the illicit drugs inspection reagent paper is based on fluorescence immunoassay principle, can accurate quantification determine drugs content in human saliva.The illicit drugs inspection reagent paper solves the problems, such as that conventional fluorescent immunity test strip background fluorescence is strong, the difficult uniform diffusion of saliva sample using microfluidic capillary structure, has given full play to that fluorescence immunoassay sensitivity is high, can quantitative analyses advantage.The sizing of the microfluidic capillary structure of the illicit drugs inspection reagent paper adopts the hot photoetching process of volume to volume, the method substantially reduce the preparation cost, shortens manufacturing cycle, realizes the production in enormous quantities to the illicit drugs inspection reagent paper.
Description
Technical field
The present invention relates to illicit drugs inspection reagent paper, particularly a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure and its
Preparation method.
Background technology
In recent years, the abuse of drugs has gradually been penetrated in daily life, for example take drugs after drive, in public place of entertainment
Taken drugs etc..As the perfect and citizen of structure of the law safeguards the enhancing of itself rights and interests consciousness, during illicit drugs inspection, law enforcement
The collection of person's field conduct blood and urine sample there may be certain difficulty, need other to be more easy to the sample of acquisition to substitute,
Saliva is exactly one of them.Compared with urine sample and blood sample, saliva sample possesses following advantage：
1. the collection process of saliva avoids the infringement to object privacy；
2. without the need for special collecting device；
3. sample easy purification, it is possible to decrease interference of the other impurities to assay.
Normal adults are salivated about 500～1500mL daily, are entered internal drug ingredient and are transferred to saliva from blood
The approach of liquid includes Active transport, film transport and Passive diffusion.As drugs substance has more lipotropy than its metabolite, more
Saliva is entered by fatty envelope barrier easily, therefore the object of illicit drugs inspection is mainly drugs substance in saliva.
Immunoassay is a kind of drugs screening technique general in the world at present, and which utilizes the special of antigen antibody reaction
Property and sensitivity include radioimmunoassay, RIA (RIA), EIA enzyme immunoassay detecting the trace substance in sample, immunoassay
(EIA), colloid Immuno gold analysis（CGIA）, fluorescence immunoassay（FIA）.
Radio immunoassay has that the radiation pollution of lonizing radiation, the production of test kit and reagent paper application require one
Fixed Anti-Radiation Countermeasures, waste need to be through special handlings.
Enzyme marker in EIA enzyme immunoassay is apt to deteriorate, and variation, sensitivity are low.
Colloid gold immune analysis method low cost, simple to operate, stability are high, high specificity, and suitable Site Detection is used,
Colloid gold immune analytic process can to saliva in drugs content carry out semi-quantitative analyses, but accurate quantification point can not be carried out to which
Analysis.
Fluoroimmunoassay can to saliva in drugs content carry out accurate quantification analysis.Fluoroimmunoassay
Principle is the sensitive measurability of the specificity with fluorescent technique of conjugated antigen antibody response, and the fluorescence intensity for exciting is surveyed
Amount, can quantitative analyses sample drugs content in conjunction with spectral pattern.Fluorescence immunoassay detection strip calibration tape color with to be measured
Concentration of specimens is proportionate.When in test solution, testing concentration is bigger, the bandwidth of calibration tape is wider, and color is deeper；Conversely, determinand
Concentration is less, and test bandwidth is narrower, and color is more shallow.With LED as extraneous excitation source, by the color of fluorescence immunoassay detection strip
Spectrum signal switchs to spectral signal, recycles photodiode to receive fluorescence signal, obtains the Spectral Properties of fluorescence immune chromatography strip
Linearity curve, carries out process through algorithm to the spectral pattern of strip and can determine that determinand concentration of specimens.
Compared with colloid gold immune analytical technology, the advantage of fluorescence immunoassay technology is as follows：
1. sensitivity：Impact of the outside noise to fluorescence analyser is less than gold colloidal；
2. label：Colloid gold immune technology only has golden label, and the label of fluorescence immunoassay skill technology has multiple, can have multiple
Select；
3. Detection results：Immunofluorence technic can realize that the accurate quantification for treating detectable substance is analyzed, and colloid gold immune technology
Semi-quantitative analyses can only be realized.
As traditional plant fiber paper has very strong background fluorescence, fluorescence immunoassay detection technique quantitative analysis results are affected
Precision, it is impossible to as the effective carrier of fluorescence immunoassay detection technique.In order to solve the problems, such as background fluorescence, present people are universal
Use polymer rather than Plant fiber is used as reagent paper base material, most representational is all-glass paper.
All-glass paper overcomes the strong problem of traditional plant fibrous paper background fluorescence, but will be applied to portable glimmering
Light immunoassay also has very big deficiency.All-glass paper does not possess water-wet behavior first, and this causes liquid in its diffusion into the surface
Difficult.Secondly, liquid cannot produce capillarity by all-glass paper, so even having carried out surface parent to all-glass paper
Aqueous treatment, it is also difficult to allow liquid to be detected to spread uniform above.If sample liquid flowing diffusion is uneven, it is meant that
In the test strips each point response time, have a strong impact on the spectral characteristic of fluorescence immune chromatography strip.
Content of the invention
In order to overcome the above-mentioned deficiencies of the prior art, the present invention devises a kind of drugs based on microfluidic capillary structure and examines
Test paper, the illicit drugs inspection reagent paper be based on fluorescence immunoassay principle, can accurate quantification determine human saliva in drugs content.
The illicit drugs inspection reagent paper solves that conventional fluorescent immunity test strip background fluorescence is strong, saliva using microfluidic capillary structure
The problem of the difficult uniform diffusion of sample, has given full play to that fluorescence immunoassay sensitivity is high, can quantitative analyses advantage.
A kind of illicit drugs inspection reagent paper based on microfluidic capillary structure includes sample region and detection zone, sample region and inspection
Survey area to be connected.The micro cylinder array being distributed in sample region and detection zone constitutes the microfluidic capillary structure, each micro- circle
Height of column is less than 150 microns, and diameter is less than 150 microns, and between adjacent micro cylinder, interval is less than 150 microns.Saliva
Liquid sample instills sample area, as each micro cylinder is uniformly arranged at equal intervals, after saliva sample and micro cylinder array contact
The identical surface tension of all directions is subject to, so as to uniformly be diffused to detection zone along micro cylinder array, and most
Zhongdao reaches detection line.Antigen in saliva sample is competitively tied with labeled antibody with isoantigen in detection line
Close, form the antigen antibody complex containing mark fluorescent dyestuff, then using extraneous photoinduction, determine the fluorescence in complex
Intensity, and then extrapolate the concentration of measured matter.
It is another object of the present invention to being to provide a kind of preparation of the illicit drugs inspection reagent paper based on microfluidic capillary structure
Method：
1. antibody-solutions prepare：380mg sodium borate is taken in 50ml centrifuge tubes, sequentially add 1g sucrose, 50mg sodium azides and
The drugs monoclonal antibody powder of 1g.Phosphate buffer is added toward centrifuge tube and is settled to 50mL.Slowly shake up, room temperature is deposited
Storage；
2. the coupling of label and antibody：Add pH=9.0,0.5mol/L carbonate buffer solution 4ml.Under 25 DEG C of magnetic agitation, by
It is added dropwise to Fluorescein isothiocyanates of the 1ml containing 2mg（FITC）Solution.1h is stirred at 25 DEG C, at 4 DEG C, continues stirring 4h；
3. the antibody purification being fluorescently labeled：Antibody-solutions after by labelling are loaded on bag filter, dialyse in the tap water of flowing
About 10min, then moves in the phosphate buffer of pH=7.4, dialyses at 4 DEG C, until extracellular fluid dialysis are not under uviol lamp
Till existing fluorescence；
4. reagent paper microfluidic capillary structure sizing：The hot photoetching forming principle of volume to volume is based on, by negative for the SU-8 of slush state light
Photoresist is uniformly coated on solid-state pet film, is suppressed via micro-nano mould, slush state
Micro- cylindrical cavity array on the full micro-nano mould of SU-8 fillings.Ultraviolet light is carried out to which simultaneously, by slush state
The negative photoresists of SU-8 are solidificated on pet film, are formed the detection with microfluidic capillary structure and are tried
Paper；
5. reagent paper adds and covers hydrophilic coating：The reagent paper of sizing is put into ultrasound 20min removal tables in the mixed liquor of dehydrated alcohol and water
Face oils and fatss, use a large amount of deionized water rinsings, are put in drying baker and dry.Then by reagent paper immersion concentration be 10% poly- carbonization
30～60s in diimine solution, reagent paper is lifted out from polycarbodiimide solution with the speed of 6～10cm/min, room
Temperature is lower to dry 15～30min.Reagent paper is immersed 30～60s in certain density polymethyl vinyl ether copolymerization maleic acid solution,
Solidify at a certain temperature.By a period of time in the NaHCO3 solution of reagent paper immersion 20%, with a large amount of deionized water eluting, dry
Stand-by；
6. the antigen-antibody solidification in detection paper area：Labeled drugs monoclonal antibody solution is uniformly applied to detection
Area, dries 16 hours at 37 DEG C.The detection line position that antigenic solution is evenly coated in detection zone with liquid-transfering gun, between detection line between
Away from for 8mm.Reagent paper dries 30min in freezer dryer, takes out standby after 4 DEG C of storages.
After saliva sample instills reagent paper sample area, uniformly spread to detection zone.When the drugs concentration contained in saliva sample
Gao Shi, the drugs monoclonal antibody that major part is fluorescently labeled form complex with the enough drugs antigen of saliva sample mesopodium and gather
Accumulate, remaining drugs monoclonal antibody marked on a small quantity is reacted with fixed drugs antigen in detection line, outside
Under boundary's photoinduction, the fluorescence intensity in detection line is weaker.If conversely, the drugs concentration hour contained in saliva sample, quilt on a small quantity
Drugs antigen-reactive in fluorescently-labeled drugs monoclonal antibody and saliva sample, the drugs monoclonal after major part is labeled
In antibody and detection line, fixed drugs antigen carries out reaction and forms complex building up, under extraneous photoinduction, detection line
On fluorescence intensity stronger.Fluorescence signal is received using photodiode, measurement obtains the fluorescence intensity in detection line, according to examination
The spectral pattern of paper slip, can analyze and obtain drugs concentration.
A kind of illicit drugs inspection reagent paper based on microfluidic capillary structure of the present invention has following benefit：
1., due to the presence of reagent paper microfluidic capillary structure, after saliva sample and micro cylinder array contact, it is subject to all directions equal
Deng surface tension, can uniformly diffuse to whole detection zone；
2. reagent paper employs polyethylene terephthalate for base material, and micro cylinder array is made up of the negative photoresists of SU-8, no
Background fluorescence can be produced, the degree of accuracy of fluorescence immunoassay quantitative analyses is not affected；
3. reagent paper adopt fluorescence immunoassay detection technique, can to saliva in drugs content carry out accurate quantification analysis.
The illicit drugs inspection reagent paper completes the sizing of microfluidic capillary structure based on the hot photoetching process of volume to volume, by slush
Micro-nano mould compacting of the negative photoresist of the SU-8 of state via customization, under the irradiation of extraneous ultraviolet source, can fast setting,
Form micro cylinder array, i.e., the microfluidic capillary structure of described reagent paper.The method can substantially reduce being prepared into for the reagent paper
This, shortens manufacturing cycle, realizes the production in enormous quantities to the reagent paper.
Description of the drawings
Fig. 1 is a kind of illicit drugs inspection test paper structure figure based on microfluidic capillary structure.
Fig. 2 is the illicit drugs inspection reagent paper microfluidic capillary structural representation.
Fig. 3 is the top view of the illicit drugs inspection reagent paper.
Fig. 4 is the hot photoetching setting process flow chart of volume to volume of the illicit drugs inspection reagent paper microfluidic capillary structure.
Specific embodiment
Illicit drugs inspection reagent paper of the present invention is based on fluorescence immunoassay principle, drugs can contain in quantifying sialic
Amount.Test paper structure is as shown in figure 1, reagent paper substrate 1 is made up of polyethylene terephthalate.Reagent paper includes detection zone 2 and adopts
Sample area 3, detection zone 2 are connected with sample region 3.4 array of micro cylinder being distributed in detection zone 2 and sample region 3 constitutes reagent paper
Microfluidic capillary structure.
As shown in Fig. 2 each 4 height of micro cylinder in microfluidic capillary structure is 30 microns, a diameter of 30 microns,
At intervals of 30 microns between adjacent micro cylinder, each micro cylinder surface is covered with one layer of hydrophilic figure layer 7.
Saliva collector instills the saliva sample that collects in sample region 3, saliva sample and micro cylinder array contact
The surface tension for being subject to all directions impartial afterwards, to 2 uniform diffusion of detection zone.Methamphetamine detection line 5 is successively reached
With morphine detection line 6.As shown in figure 3, being spaced 8mm between methamphetamine detection line 5 and morphine detection line 6.
A kind of preparation method of fluorescence immunoassay Test paper based on microfluidic capillary structure is as follows：
1. antibody-solutions prepare：381mg sodium borate is taken in 50ml centrifuge tubes, sequentially add 1g sucrose, 50mg sodium azides and
1g methamphetamines monoclonal antibody and 1g morphine monoclonal antibody powder.Then phosphate buffer is added simultaneously toward centrifuge tube
It is settled to 50mL.Slowly shake up, room temperature is stored；
2. the coupling of label and antibody：Add pH=9.0,0.5mol/L carbonate buffer solution 4ml.Under 25 DEG C of magnetic agitation, by
It is added dropwise to Fluorescein isothiocyanates of the 1ml containing 2mg（FITC）Solution.1h is stirred at 25 DEG C, at 4 DEG C, continues stirring 4h；
3. the antibody purification being fluorescently labeled：Antibody-solutions after by labelling are loaded on bag filter, dialyse in the tap water of flowing
About 10min, then moves in the phosphate buffer of pH=7.4, dialyses at 4 DEG C, until extracellular fluid dialysis are not under uviol lamp
Till existing fluorescence；
4. reagent paper microfluidic capillary structure sizing：The hot photoetching forming principle of volume to volume is based on, as shown in Figure 4.8 for unreel module, 9
It is winding module for topper module, 10 for sizing module, 11.Pet film 12 is with unreeling module 8
With the winding rotation of module 11 and slowly moving, while having driven the slow rotation of micro-nano module 14.Liquid-transfering gun 13 will be partly
The negative photoresists of the SU-8 of melting state are uniformly coated on slow mobile pet film 12, via micro-
Nano die 14 is suppressed, the micro- cylindrical cavity array in the SU-8 fillings completely micro-nano model 14 of slush state, each micro- cylinder
Hole diameter is 30 microns, depth is 30 microns, at intervals of 30 microns between adjacent micro cylinder hole.In ultraviolet source 15
Under irradiation, the negative photoresists of SU-8 of slush state are solidificated on pet film 12, are formed with micro-
Fluid capillary structure Test paper；
5. reagent paper adds and covers hydrophilic coating：The reagent paper of sizing is put into ultrasound 20min removal tables in the mixed liquor of dehydrated alcohol and water
Face oils and fatss, use a large amount of deionized water rinsings, are put in drying baker and dry.Then by reagent paper 1 immerse the poly- carbonization that concentration is 10%
30～60s in diimine solution, reagent paper is lifted out from polycarbodiimide solution with the speed of 6～10cm/min, room
Temperature is lower to dry 15～30min.Reagent paper is immersed 30～60s in certain density polymethyl vinyl ether copolymerization maleic acid solution,
Solidify at a certain temperature.By a period of time in the NaHCO3 solution of reagent paper immersion 20%, with a large amount of deionized water eluting, dry
Stand-by；
6. the antigen-antibody solidification in detection paper area：Methamphetamine monoclonal antibody and morphine monoclonal antibody by labelling
Mixed solution is uniformly applied on the detection zone 2 of reagent paper, 37 DEG C of dryings 16 hours.With liquid-transfering gun by methamphetamine-BSA
Solution is evenly coated in the position of methamphetamine detection line 5, the position that morphine-BSA solution is evenly coated in morphine detection line 6
Put.30min is dried in reagent paper freezer dryer, is taken out standby after 4 DEG C of storages.
When saliva sample instills sample region 3, labeled methamphetamine monoclonal antibody and morphine monoclonal antibody,
As saliva sample is uniformly spread between micro cylinder array 4.When methamphetamine in saliva sample and morphine concentration little
When, a small amount of labeled methamphetamine monoclonal antibody and morphine monoclonal antibody in diffusion process with saliva in first
Base amphetaminess antigen and morphine antigen binding, when saliva sample reaches the methyl An Feita for being fixed with methamphetamine-BSA
Life detection line 5 and be fixed with morphine-BSA morphine p-wire 6 when, the methamphetamine being fluorescently labeled in a large number and morphine list
Methamphetamine antigen and morphine antigen binding in clonal antibody and detection line is built up for polymer, in extraneous LED
Deng irradiation under, fluorescent effect is strong；When methamphetamine in saliva sample and big morphine concentration, a large amount of labeled methyl
Amphetaminess monoclonal antibody and morphine monoclonal antibody in diffusion process with saliva in methamphetamine antigen and
Coffee antigen binding is built up, when saliva sample reaches the methamphetamine detection line 5 for being fixed with methamphetamine-BSA
Be fixed with morphine-BSA morphine p-wire 6 when, the methamphetamine that is fluorescently labeled on a small quantity of residue and morphine monoclonal
Methamphetamine antigen and morphine antigen binding on antibody and detection line is closed, under the irradiation of extraneous LED etc., fluorescent effect
Weak.
Methamphetamine antigen, morphine antigen in saliva sample be fixed on the methamphetamine in detection line
Antigen, morphine antigenic competition combine a certain amount of methamphetamine and morphine monoclonal antibody.Irradiation in extraneous LED
Under, fluorescence signal is received using photodiode.Determine methamphetamine detection line 5 and the fluorescence in morphine detection line 6 is strong
Degree, is compared with the spectral pattern of reagent paper, can so that quantitative extrapolate in saliva contained methamphetamine and
The concentration of coffee.
Although being described to illustrative specific embodiment of the invention above, in order to the technology of the art
Personnel understand the present invention, it should be apparent that the invention is not restricted to the scope of specific embodiment, the common skill to the art
For art personnel, as long as various change is in appended claim restriction and the spirit and scope of the present invention for determining, these
Change is it will be apparent that all utilize the innovation and creation of present inventive concept in the row of protection.
Claims (8)
1. a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure, is poisoned using fluoroimmunoassay quantifying sialic
Product content, it is characterised in that the illicit drugs inspection reagent paper is based on microfluidic capillary structure.
2. a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure as claimed in claim 1, it is characterised in that described micro-
Fluid capillary structure is made up of one group of micro cylinder array.
3. a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure as claimed in claim 1 or 2, it is characterised in that institute
State micro cylinder and be highly not more than 150 microns, diameter is not more than 150 microns, interval no more than 200 is micro- between adjacent micro cylinder
Rice.
4. a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure as claimed in claim 1 or 2, it is characterised in that institute
State micro cylinder to be made up of the negative photoresists of SU-8.
5. a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure as claimed in claim 1 or 2, it is characterised in that institute
State.
6. a kind of illicit drugs inspection reagent paper based on microfluidic capillary structure as claimed in claim 1, it is characterised in that the poison
Product examine test paper base material is made up of polyethylene terephthalate.
7. a kind of preparation method of the illicit drugs inspection reagent paper based on microfluidic capillary structure, it is characterised in that comprise the following steps：
A) antibody-solutions prepare；
B) coupling of label and antibody；
C) antibody purification being fluorescently labeled；
D) reagent paper microfluidic capillary structure sizing；
E) reagent paper adds and covers hydrophilic coating；
F) the antigen-antibody solidification in detection paper area.
8. a kind of preparation method of the illicit drugs inspection reagent paper based on microfluidic capillary structure as claimed in claim 6, its feature
It is, the reagent paper microfluidic capillary structure fixating shape step described in step d) is based on the hot photoetching forming principle of volume to volume, by slush
The negative photoresists of the SU-8 of state are uniformly coated on pet film, are suppressed via micro-nano mould, half
Micro- cylindrical cavity array in the SU-8 fillings completely micro-nano model of melting state, under the irradiation of ultraviolet source, slush shape
The negative photoresists of the SU-8 of state are solidificated on pet film, form the examination with microfluidic capillary structure
Paper.
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| CN106950072A (en) * | 2017-02-27 | 2017-07-14 | 深圳中物安防科技有限公司 | A kind of special sampling paper of drugs and preparation method thereof |
| CN108844922A (en) * | 2018-05-17 | 2018-11-20 | 浙江诺迦生物科技有限公司 | The rapid detection method of drugs in a kind of hair |
| CN109270057A (en) * | 2018-10-17 | 2019-01-25 | 无限极（中国）有限公司 | A kind of kit and its preparation method and application detecting skin-lightening cosmetic effect |
| CN111024669A (en) * | 2019-12-27 | 2020-04-17 | 南京工业大学 | Microfluid chip based on multilevel ordered structure photonic crystal paper and preparation method thereof |
| WO2020147203A1 (en) * | 2019-01-15 | 2020-07-23 | 京东方科技集团股份有限公司 | Detection chip and use method therefor, and reaction system |
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| CN106950072A (en) * | 2017-02-27 | 2017-07-14 | 深圳中物安防科技有限公司 | A kind of special sampling paper of drugs and preparation method thereof |
| CN108844922A (en) * | 2018-05-17 | 2018-11-20 | 浙江诺迦生物科技有限公司 | The rapid detection method of drugs in a kind of hair |
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| US11607682B2 (en) | 2019-01-15 | 2023-03-21 | Beijing Boe Technology Development Co., Ltd. | Detection chip, using method for the same, and reaction system |
| US11654435B2 (en) | 2019-03-29 | 2023-05-23 | Boe Technology Group Co., Ltd. | Detection chip, method for operating detection chip, and reaction system |
| CN111024669A (en) * | 2019-12-27 | 2020-04-17 | 南京工业大学 | Microfluid chip based on multilevel ordered structure photonic crystal paper and preparation method thereof |
| CN113341130A (en) * | 2021-05-31 | 2021-09-03 | 湖北美宝生物科技股份有限公司 | Early pregnancy test paper |
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Application publication date: 20170315 |