CN107478613A - A kind of preparation method of a variety of drug testing chips based on SPR - Google Patents
A kind of preparation method of a variety of drug testing chips based on SPR Download PDFInfo
- Publication number
- CN107478613A CN107478613A CN201710649618.4A CN201710649618A CN107478613A CN 107478613 A CN107478613 A CN 107478613A CN 201710649618 A CN201710649618 A CN 201710649618A CN 107478613 A CN107478613 A CN 107478613A
- Authority
- CN
- China
- Prior art keywords
- variety
- chip
- drug testing
- preparation
- carboxyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of preparation method of a variety of drug testing chips based on SPR, comprise the following steps：1) carrier material is prepared；2) one layer of golden film is plated on a support material, and the carrier material of gold-plated film is immersed in sulfydryl alkanoic acid carboxyl chip is made；3) surface of carboxyl chip is fully contacted with EDC/NHS mixed liquors, activated carboxyl chip is made in the carboxylic group of activated carboxyl chip surface；4) activated carboxyl chip is fully contacted with a variety of drugs synthetic antigen solution, a variety of drugs synthetic antigens is coupled on carboxyl chip；5) by ethanolamine solutions and the carboxyl chip fully reaction sealed unreacted carboxyl site for completing a variety of drugs synthetic antigen couplings, a variety of drug testing chips are made.Not only cost is low for preparation method provided by the invention, consumptive material is few, and the detection chip prepared is simple in construction, suitable for medical disposable material, makes that the detector portability using the detection chip is strong, popularization is wide, can realize instant, the Site Detection of drugs.
Description
Technical field
The present invention relates to illicit drugs inspection technical field, more particularly to a kind of a variety of illicit drugs inspections based on SPR
The preparation method of chip.
Background technology
Surface plasma resonance (SPR) is a kind of physical optics phenomenon, is had a common boundary in the transparent medium of two kinds of different refractivities
On face (such as glass and water), when Ray Of Light incides low refractive index dielectric from high index of refraction, optical fiber will occur to reflect and reflection.
When incidence angle increases to a certain particular value, refraction angle is equal to 90 degree, and light is along the direction injection tangent with interface, entering now
Firing angle is referred to as critical angle.If incidence angle exceedes critical value, incident ray will not enter another interface, but all anti-
It is emitted back towards in incident medium, experiences total internal reflection.
In fact, although whole incident lights are reflected, a kind of electromagnetic field for being evanescent wave can pass through contacting permeation to low folding
Penetrate in rate medium, energy is exponentially decayed.If plating layer of metal film in interface, general gold-plated film or silverskin, then metal
The free electron of film surface is excited by incident light and produces electric charge vibration, and then forms surface plasma.Adjustment light enters
When firing angle or wavelength are to a certain appropriate value, surface plasma is equal with the frequency and wave number of evanescent wave, and energy just occurs in the two
Coupling, form surface plasma resonance.The total reflection condition of interface will be destroyed during resonance, and incident light energy is transferred to table
In surface plasma ripple, so as to cause intensity of reflected light drastically to decline in the air, decay total reflection phenomenon is presented.Wherein make anti-
Penetrate the incident angle of light that light is wholly absent and be referred to as resonance angle (SPR angle).Resonance angle can be with the medium of metal film surfaces
The change of refractive index and change, and the quality of molecule of the change to being incorporated in metal surface of refractive index is directly proportional.Therefore pass through
Analyze resonance angle, it is possible to obtain the information of intermolecular interaction.
SPR technique has without mark, changes sensitivity to surface characteristic and material, in real time, quickly and easily realizes automatic
The features such as change, it has been widely used in the fields such as life science, clinical diagnosis, drug screening, food security, environmental monitoring, has examined
Surveying object includes albumen, nucleic acid, hormone, toxin, agricultural chemicals, cell, microorganism etc..
Existing commercialization SPR biological sensing systems belong to laboratory large-scale instrument, bulky, complicated, week
Surrounding environment requirement is strict and expensive, seriously constrains the extensive use of SPR sensorgram system.Commonly use in current domestic laboratory
SPR drug testing chips one kind be using glass etc. as carrier material, be fabricated to prism or semi-cylindrical in configuration, and above-mentioned
Matching fluid is added dropwise in structure, gold-plated slide, slide are finally covered on the prism or semi-cylindrical in configuration for be added dropwise matching fluid
The reactant of the detection of upper integrated response；Wherein glass, slide are required to Precision Machining, and cost is high, consumptive material is more, cannot be used for one
Secondary property consumptive material.Also it is a kind of be add one layer of refractive index close in the upper surface of prism or semi-cylindrical in configuration there is certain elasticity
With the thin layer of viscosity, by gold-plated slide pressure during use.The above-mentioned effect using matching fluid or thin layer, it is provided to prevent
Influenceed between gold-plated slide and prism or semicolumn by air.
Existing SPR drug testing chips have the disadvantage that：Gold-plated slide very trouble is changed, it is necessary to be wiped with alcohol
With liquid, along with new matching fluid；Using thin layer have access times finiteness limitation, vulnerability and easily by
The influence of extraneous dust；What prism or semi-cylindrical in configuration and gold-plated slide used is all in a manner of glass is finished
Obtain, cost is high.Due to above-mentioned several big drawbacks, the instrument based on SPR drug testing chips technologies is caused not make portable
Formula, gold-plated chip is that detection chip cannot also be used as medical disposable material.
Therefore, how to provide a kind of structure simplify, a variety of drug testing chips based on SPR suitable for medical disposable material
Preparation method the problem of being those skilled in the art's urgent need to resolve.
The content of the invention
In view of this, the invention provides a kind of preparation method of a variety of illicit drugs inspection detection chips based on SPR, not only
Cost is low, consumptive material is few, and the detection chip prepared is simple in construction, suitable for medical disposable material, finally makes to apply the detection core
The detector of piece has the advantages that more portable, popularization is wider, can realize instant Site Detection.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of preparation method of a variety of drug testing chips based on SPR, comprises the following steps：
1) carrier material is prepared；
2) one layer of golden film is plated on a support material, and golden film thickness range is in 40~45nm, and the carrier material that will be coated with golden film
Material, which is immersed in sulfydryl alkanoic acid, is made carboxyl chip；
3) surface of carboxyl chip is fully contacted with EDC/NHS mixed liquors, the carboxylic group of activated carboxyl chip surface
Activated carboxyl chip is made；
4) activated carboxyl chip is fully contacted with a variety of drugs synthetic antigen solution, is coupled a variety of drugs synthetic antigens
Onto carboxyl chip；
5) ethanolamine solutions are fully reacted with completing the carboxyl chip of a variety of drugs synthetic antigen couplings, closes unreacted
Carboxyl site, be made a variety of drug testing chips.
The beneficial effects of the invention are as follows：The invention provides a kind of inexpensive a variety of drug testing chips based on SPR
Preparation method, eliminate conventional matching fluid or laminate structure, overcome technology prejudice, only on a support material plate one layer of gold
Film is that can be achieved to accurate, the efficient detection of a variety of drugs, have concurrently consumptive material it is few, it is simple in construction, used suitable for a consumptive material and
So that detector is more portable, is more conducive to the advantages that popularization.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, immersion gold in step 2)
The condition of film is：The time being incubated under conditions of 40~45 DEG C of temperature range keeps 30~35min, or under normal temperature condition
The time of incubation keeps more than 12h；Or/and the sulfydryl alkanoic acid is 11- Mercaptoundecanoic acids, the 11- Mercaptoundecanoic acids
Solution of the concentration more than 10mM is formulated as using 80% ethanol solution.
In order to improve efficiency, economize on resources, above-mentioned immersion golden film can be incubated 30min, above-mentioned normal temperature under conditions of 40 DEG C
It can be 25 degree.
Ensure under conditions of 40 DEG C incubation time under more than 30min, or normal temperature condition incubation time in more than 12h,
And the concentration of obtained 11- mercapto-undecanoics acid solution is more than 10mM, wherein the setting of corresponding time and concentration parameter, can
The preferable carboxyl chip of homogeneity is prepared on golden film surface.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, step 2) is additionally included in
The golden film on carrier material is carried out being cleaned by ultrasonic processing after gold-plated film and rinses drying process；Ultrasound condition is：Cleaning is situated between
Matter selects ethanol solution, and temperature conditionss are room temperature, and ultrasound intensity 55-60%, ultrasonic time keep more than 5min；Rinse drying
Treatment conditions are：From deionized water rinsing, dried up after flushing with nitrogen.
Ultrasound intensity is excessive, overlong time may peel off golden film, and ultrasound intensity is too small or time very short possible nothing
Method cleans up the pollutant on golden film surface, therefore it is 60% that ultrasound intensity, which may be selected, and ultrasonic time keeps 5min.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, in step 2) and step
3) also include between：Cover PDMS in carboxyl chip surface, PDMS opens up passage, passage have it is a plurality of, passage both ends be respectively into
Sample mouth and outlet, and sense channel is being made after step 3), step 4) and step 5) processing in passage.
Liquid can be controlled in the stream on golden film surface in PDMS sense channels made from carboxyl surface, you can existed with control
Which block region adds which type of sample, and passage can be coupled a variety of drugs synthetic antigens on golden film surface, so as in chip piece
It is upper that a plurality of sense channel is made, realize a variety of illicit drugs inspections.It should be noted that the carrier material for having plated golden film is i.e. usually said
Prism, prism combines with PDMS, and PDMS is provided with groove, and groove is passage, and passage is completed to repair on golden film surface
It is sense channel after decorations.
Explain：PDMS is dimethyl silicone polymer；PDMS passages：Dimethyl silicone polymer is solid in the mould of respective shapes
Change, obtain having reeded solid matter, this solid matter is bonded with smooth golden film can obtain a passage.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, EDC/ in step 3)
NHS mixed liquors are the solution of concentration 400mM EDC solution and concentration 100mM NHS solution according to isometric mixing gained.
It should be noted that：EDC:For N- (3- dimethyl aminopropyls)-N '-ethyl carbodiimide；NHS is N- hydroxysuccinimidyls
Acid imide；EDC/NHS is the mixture of above two solution.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, step 3) is to carboxyl core
The volume for the EDC/NHS mixed liquors that piece is activated is more than 200 μ L.
The volume of mixing sample is that 200 μ L are a critical values, and during more than this volume, the effect of coupling is preferable.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, to using EDC/NHS
Carboxyl chip after mixed liquor activation is passed through PBS and is rinsed, and irrigation flow rate is 20 μ L/min..
It should be noted that PBS is phosphate buffer solution, as commercially available green skies biotechnology research institute produces
PBS, above-mentioned technical proposal do not specially require to PBS volume, and for unification, the PBS for being passed through that volume is 50 μ L can be selected, and
The effect reacted under 20 μ L/min flow velocity is preferable.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, a variety of poison in step 4)
The volume of product synthetic antigen solution is more than 100 μ L.
The μ L of volume 100 of drugs synthetic antigen are critical size, and coupling effect is without obvious poor after volume is more than 100 μ L
It is different, take critical value to save reagent cost, i.e., the volume of a variety of drugs synthetic antigen solution is preferably 100 μ L in step 4).
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, in step 4) and step
5) also include between：PBS is passed through to the carboxyl chip of coupling to be rinsed, irrigation flow rate is 20 μ L/min.
Flushing is in order to which the residual liquid of chip surface is removed, to reduce previous step liquid to next step reaction solution
Influence, PBS volume do not specially require, and in order to unified, can be passed through the PBS that volume is 50 μ L, the μ L/min of flow velocity 20 are anti-
Answer the preferable flow velocity of effect.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, closing conjunction in step 5)
Into antigen coupling carboxyl chip unreacted carboxyl groups site concrete operations for be passed through concentration be 1M, volume be 200 μ L, PH=
8.5 ethanolamine solutions, PBS is passed through again after completing and is rinsed.
The volume and concentration of monoethanolamine are to preferably close unreacted site, and are reduced as far as possible to the albumen of coupling
Influence, the residual liquid of chip surface can be removed by rinsing, to reduce shadow of the previous step liquid to next step reaction solution
Ring, PBS volume does not specially require, and for unification, can be passed through the PBS that volume is 50 μ L.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, the carrier in step 1)
Material includes glass, resin, or other meet the material of optical requirement.
Carrier material is to provide carrier for golden film, and forms a surface that can carry out SPR phenomenons, the light of glass material
Performance is good, and the preparation of resin material and price are advantageous, as long as other can meet the material of SPR phenomenons all after gold-plated film
It can be used for preparing the chip.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, a variety of poison in step 4)
The drugs of product synthetic antigen solution include but is not limited to crystal methamphetamine, cocaine, KET, morphine.
It is using the beneficial effect of such scheme：The detection chip of the present invention can be used for detecting a variety of drugs, application
It is wider, live detection immediately, high sensitivity can be carried out.
Preferably, in a kind of above-mentioned preparation method of a variety of drug testing chips based on SPR, the carrier in step 1)
Material is prepared into the structure that section is Dove prism.
It is using the beneficial effect of such scheme：The body that Dove prism structure greatly reduces material is made in carrier material
Product, because light channel structure is not related to the lower section of carrier material such as prism or semi-cylindrical in configuration.
, first will covering when a kind of a variety of drug testing chips based on SPR prepared using the present invention detect drugs
The drug testing chips of PDMS sense channels are installed on SPR instruments, secondly using 20 μ L/min flow velocity by volume as 100 μ L's
Sample to be tested flows through PDMS sense channels, records corresponding resonance pixel changing value, is then passed through volume in PDMS sense channels
The NaOH solution for being 50mM for 100 μ L, concentration, PBS of the volume as 100 μ L is finally passed through by PDMS inspections using 20 μ L/min flow velocity
Survey the regeneration that passage carries out detection chip.
It should be noted that regeneration reuses, regenerative process destroys the combination of antigen-antibody, so as to
To the chip identical regeneration chip with preparing, next sample can be detected.
Understood via above-mentioned technical scheme, compared with prior art, the present disclosure provides a kind of based on the more of SPR
The preparation method of kind drug testing chips, first carrier material are simultaneously not particularly limited and required, and need not move through strict add
Work；Secondly one layer of golden film is plated on a support material, and golden film is immersed in sulfydryl alkanoic acid and can obtain carboxyl chip overnight；Then
A variety of drug testing chips are made in activated carboxyl chip, coupling drugs synthetic antigen, closing unreacted carboxyl groups site successively.Wherein
Golden film is immersed in before sulfydryl alkanoic acid need to carry out in ethanol solution, at room temperature, ultrasound intensity 60%, ultrasonic time be
5min ultrasonic cleaning, it is necessary to explanation, is first cleaned by ultrasonic to golden film, by golden film surface clean it is clean after soak again
Bubble, combination of the thiol molecule on golden film surface is more effective when cleaning is to soak.
Existing detection chip is in sheet glass mostly by golden film direct plating, and prism is fixed in instrument, in order to be formed
SPR phenomenons need for detection chip to be placed on the surface of prism, can at this time use the matching fluid with fixed refraction, and
Use with liquid can make the application of detection chip become inconvenient, be embodied in the following aspects：When the 1st, changing detection chip
Need original matching fluid removing, and add new matching fluid, complex operation；2nd, needed during use ensure prism,
With not having bubble between liquid and chip three, the operation difficulty is big, it is necessary to professional's skilled operation；3rd, matching fluid is liquid,
Easily there is the phenomenon of position movement in detection chip after being put on prism.It is directly gold-plated on prism that above-mentioned rib can be achieved
The function of gold-plated chip three on mirror, matching fluid and sheet glass, and prism is changed into the portion of detection chip from the part of instrument
Part, the operation in detection process can be simplified using corresponding detection device, overcome technology prejudice.
The purpose of matching fluid is that a fixed refraction ring is provided between conventional die in prism (being built in instrument)
Border, directly subtract, the requirement of refractive index can not be met.And the present invention prism be on carrier material direct gold-plated film equivalent to
Part-structure in original instrument is combined with chip, the prism being built in originally in instrument and golden film are carried out again
Combination.
By a kind of preparation method of a variety of drug testing chips based on SPR provided by the invention, the inspection being prepared
Chip is surveyed to be not only simple in structure, and cost is low, consumptive material is few, improves the popularization and application of illicit drugs inspection technology and detector,
Simultaneously because it can be used as medical disposable material so that the use of detector is more convenient；And the present invention prepare based on the more of SPR
Kind of drug testing chips can detect crystal methamphetamine (MET), cocaine (COC), KET (KET), morphine (MOP), head-shaking pill,
The drugs such as hemp, methadone, amphetamine, C16H25NO2.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
The embodiment of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 accompanying drawings are a kind of concentration and signal to noise ratio for detecting crystal methamphetamine synthetic antigen in embodiment provided by the invention
Graph of a relation；
Fig. 2 accompanying drawings are the concentration of cocaine synthetic antigen and the pass of signal to noise ratio in a kind of detection embodiment provided by the invention
System's figure；
Fig. 3 accompanying drawings are the concentration of KET synthetic antigen and the relation of signal to noise ratio in a kind of detection embodiment provided by the invention
Figure；
Fig. 4 accompanying drawings are the concentration of morphine synthetic antigen and the relation of signal to noise ratio in a kind of detection embodiment provided by the invention
Figure；
Fig. 5 accompanying drawings are the concentrations of crystal methamphetamine and the 60th second be total in a kind of detection embodiment provided by the invention
The graph of a relation for pixel response value of shaking；
Fig. 6 accompanying drawings are a kind of concentrations and the resonance picture of the 60th second for detecting cocaine in embodiment provided by the invention
The graph of a relation of plain response；
Fig. 7 accompanying drawings are a kind of concentrations and the resonance pixel of the 60th second for detecting KET in embodiment provided by the invention
The graph of a relation of response；
Fig. 8 accompanying drawings are a kind of concentrations and the resonance pixel of the 60th second for detecting morphine in embodiment provided by the invention
The graph of a relation of response；
Fig. 9 accompanying drawings are a kind of detection for detecting the crystal methamphetamine in saliva analog sample in embodiment provided by the invention
The graph of a relation of concentration and the resonance pixel response value of the 60th second；
Figure 10 accompanying drawings are the detection of cocaine is dense in saliva analog sample in a kind of detection embodiment provided by the invention
Degree and the graph of a relation of the resonance pixel response value of the 60th second；
Figure 11 accompanying drawings for it is provided by the invention it is a kind of detection embodiment in saliva analog sample the concentrations of KET with
The graph of a relation of the resonance pixel response value of the 60th second；
Figure 12 accompanying drawings are a kind of concentrations for detecting the morphine in saliva analog sample in embodiment provided by the invention
With the graph of a relation of the resonance pixel response value of the 60th second；
Figure 13 accompanying drawings are a kind of a variety of drug testing chips structural representations based on SPR of the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
The embodiment of the invention discloses a kind of preparation method of a variety of drug testing chips based on SPR, not only step is set
Count that scientific and reasonable and simple in construction, consumptive material is few, cost can be greatly reduced, improve the popularization utilization rate of detection chip.
A kind of preparation method of a variety of drug testing chips based on SPR, comprises the following steps：
1) carrier material is prepared；
2) one layer of golden film is plated on a support material, and golden film is carried out being cleaned by ultrasonic processing and rinses drying process；Ultrasound
Condition is：Cleansing medium selects ethanol solution, and temperature conditionss are room temperature, and ultrasound intensity 60%, ultrasonic time keep 5min；Punching
Washing drying process condition is：From deionized water rinsing, dried up after flushing with nitrogen and golden film is immersed in sulfydryl alkanoic acid
Carboxyl chip is made；Immersion golden film condition be：To being cleaned by ultrasonic and rinsing condition of the golden film after drying process at 40 DEG C
Lower incubation time keeps more than 30min, or incubation time keeps more than 12h under normal temperature condition；Sulfydryl alkanoic acid is 11- sulfydryls
Hendecanoic acid, and 11- Mercaptoundecanoic acids are formulated as concentration using 80% ethanol solution and are more than 10mM；
3) surface of carboxyl chip is fully contacted with EDC/NHS mixed liquors, the carboxylic group of activated carboxyl chip surface
Activated carboxyl chip is made；EDC/NHS mixed liquors be concentration 400mM EDC solution and concentration 100mM NHS solution according to etc.
Solution obtained by volume mixture；
4) activated carboxyl chip is fully contacted with a variety of drugs synthetic antigen solution, is coupled a variety of drugs synthetic antigens
Onto carboxyl chip；
5) the carboxyl chip for completing a variety of drugs synthetic antigen couplings is fully reacted with ethanolamine solutions, concrete operations are
The ethanolamine solutions that concentration is 1M, volume is 200 μ L, PH=8.5 are passed through, close unreacted carboxyl site, a variety of poison are made
Product detection chip；The PBS that volume is 50 μ L is passed through after completing again to be rinsed.
Figure of description 13 is referred to, the invention provides a kind of a variety of drug testing chips based on SPR, including carrier
Material 1 and golden film 2；Wherein golden film 2 covers the upper end of carrier material 1.
In order to further optimize above-mentioned technical proposal, also include between step 2) and step 3)：In carboxyl chip surface
PDMS is covered, PDMS opens up passage, and passage has a plurality of, and passage both ends are respectively injection port and outlet, and passage is passing through
Sense channel is made after step 3), step 4) and step 5) processing.
In order to further optimize above-mentioned technical proposal, the EDC/NHS mixed liquors that step 3) is activated to carboxyl chip
For volume more than 200 μ L, flow velocity is 20 μ L/min.
In order to further optimize above-mentioned technical proposal, to being carried out using the carboxyl chip PBS after the activation of EDC/NHS mixed liquors
Rinse.
Also, the PBS that volume is 50 μ L can be passed through on the premise of unification to be rinsed.
In order to further optimize above-mentioned technical proposal, the volume of a variety of drugs synthetic antigen solution is in 100 μ L in step 4)
More than, 100 μ L can be selected.
In order to further optimize above-mentioned technical proposal, also include between step 4) and step 5)：To the carboxyl core of coupling
Piece is passed through PBS and is rinsed.
Also, the PBS that volume is 50 μ L can be passed through on the premise of unification to be rinsed.
, first will covering when a kind of a variety of drug testing chips based on SPR prepared using the present invention detect drugs
The drug testing chips of PDMS sense channels are installed on SPR instruments, secondly using 20 μ L/min flow velocity by volume as 100 μ L's
Sample to be tested flows through sense channel, records corresponding resonance pixel changing value, then sense channel be passed through volume for 100 μ L,
Concentration is 50mM NaOH solution, and PBS of the volume as 100 μ L finally is passed through into sense channel using 20 μ L/min flow velocity and examined
Survey the regeneration of chip；Wherein judge that sample to be tested is changed by the resonance pixel after detection chip according to resonance pixel changing value
Value, and the content of drugs in detection sample is further judged according to resonance pixel changing value.
The present invention provides a kind of detection method of a variety of drug testing chips based on SPR, and main agents information refers to
Table 1, key instrument refer to table 2 with facility information.
Table 1
Table 2
The detection of 4 kinds of drugs has been carried out in specific implementation, has devised 4 sense channels, 4 detections in detection chip altogether
The drugs of Air conduct measurement are respectively four kinds of crystal methamphetamine (MET), cocaine (COC), KET (KET) and morphine (MOP) drugs.
1st, preparation condition optimizes
Crystal methamphetamine synthetic antigen is coupled concentration optimization：When preparing drug testing chips, crystal methamphetamine sense channel
The crystal methamphetamine synthetic antigen concentration of interior coupling is respectively 25 μ g/ml, 50 μ g/ml, 100 μ g/ml and 200 μ g/ml.It is different even
The sense channel of connection concentration carries out crystal methamphetamine detection respectively, wherein the concentration of crystal methamphetamine antibody is in solution to be measured
100 μ g/ml, the concentration of crystal methamphetamine is respectively 0 μ g/ml and 1 μ g/ml.The resonance pixel changing value for taking the 60th second is response
Signal, as a result the signal to noise ratio under different coupling concentration is as shown in figure 1, when the concentration of crystal methamphetamine synthetic antigen is 50 μ g/ml
Noise is relatively good, therefore the more excellent concentration of crystal methamphetamine synthetic antigen is 50 μ in crystal methamphetamine sense channel preparation process
g/ml。
Cocaine synthetic antigen is coupled concentration optimization：When preparing drug testing chips, coupling in cocaine sense channel
Cocaine synthetic antigen concentration is respectively 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml and 100 μ g/ml.The detection of various concentrations is led to
Road carries out cocaine detection respectively, wherein the concentration of cocaine antibody is 100 μ g/ml in solution to be measured, the concentration of cocaine is divided
Wei not 0ng/ml and 100ng/ml.The resonance pixel changing value for taking the 60th second is response signal, as a result under different coupling concentration
Signal to noise ratio is as shown in Fig. 2 noise is relatively good when cocaine synthetic antigen concentration reaches 50 μ g/ml, therefore cocaine sense channel
The more excellent concentration of cocaine synthetic antigen is 50 μ g/ml in preparation process.
KET synthetic antigen is coupled concentration optimization：When preparing drug testing chips, the cocaine of coupling in KET sense channel
Synthetic antigen concentration is respectively 15.6 μ g/ml, 62.5 μ g/ml, 125 μ g/ml and 250 μ g/ml.The sense channel of various concentrations point
Not carry out KET detection, wherein the concentration of KET antibody is 100 μ g/ml in solution to be measured, the concentration of KET be respectively 0ng/ml and
100ng/ml.The resonance pixel changing value for taking the 60th second is response signal, as a result noise such as Fig. 3 institutes under different coupling concentration
Show, noise is relatively good when KET synthetic antigen concentration is more than 62.5 μ g/ml, therefore KET synthesizes in KET sense channel preparation process
The more excellent concentration of antigen is 62.5 μ g/ml.
Morphine synthetic antigen is coupled concentration optimization：When preparing drug testing chips, the morphine of coupling in morphine sense channel
Synthetic antigen concentration is respectively 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml and 100 μ g/ml.The sense channel difference of various concentrations
Carry out morphine detection, wherein the concentration of morphine Abs be 100 μ g/ml in solution to be measured, the concentration of morphine be respectively 0ng/ml with
10ng/ml.The resonance pixel changing value for taking the 60th second is response signal, as a result noise such as Fig. 4 institutes under different coupling concentration
Show, noise is relatively good when morphine synthetic antigen concentration reaches 50 μ g/ml, therefore morphine synthesizes in morphine sense channel preparation process
The more excellent concentration of antigen is 50 μ g/ml.
2nd, the Detection results in PBS
The detection chip of four kinds of drugs can be detected by being prepared according to the condition after optimization, with the PBS containing different drugs concentration
The Detection results of chip are analyzed as detection sample.
The Detection results of crystal methamphetamine：The concentration of crystal methamphetamine antibody is 100 μ g/ml in solution to be measured, methyl
The concentration of amphetamine is respectively 0ng/ml, 0.1ng/ml, 1ng/ml, 10ng/ml, 100ng/ml and 1 μ g/ml, is total within the 60th second
Pixel response value of shaking is as shown in figure 5, drug testing chips crystal methamphetamine prepared in PBS solution as can be seen from Figure
Detection limit be less than 10ng/ml.
The Detection results of cocaine：The concentration of cocaine antibody is 100 μ g/ml in solution to be measured, the concentration of cocaine
Respectively 0ng/ml, 1ng/ml, 10ng/ml, 100ng/ml, 1 μ g/ml and 10 μ g/ml, the resonance pixel response value of the 60th second is such as
Shown in Fig. 6, the detection limit of drug testing chips cocaine prepared in PBS solution is less than 100ng/ as can be seen from Figure
ml。
The Detection results of KET：The concentration of KET antibody is 100 μ g/ml in solution to be measured, and the concentration of KET is respectively 0ng/
Ml, 1ng/ml, 10ng/ml, 100ng/ml, 1 μ g/ml and 10 μ g/ml, the resonance pixel response value of the 60th second as shown in fig. 7, by
The detection limit of it can be seen from the figure that drug testing chips KET prepared in PBS solution is less than 1ng/ml.
The Detection results of morphine：The concentration of morphine Abs is 100 μ g/ml in solution to be measured, and the concentration of morphine is respectively
0ng/ml, 100ng/ml, 300ng/ml, 400ng/ml, 500ng/ml and 1 μ g/ml, the resonance pixel response value of the 60th second is as schemed
Shown in 8, the detection limit of drug testing chips morphine prepared in PBS solution is less than 300ng/ml as can be seen from Figure.
3rd, Detection results in saliva analog sample
The detection chip of four kinds of drugs can be detected by being prepared according to optimization postcondition, detect the saliva simulation of different drugs concentration
The response of sample, the wherein preparation method of saliva analog sample are that the drugs of various concentrations are added in common saliva sample
Standard items.
The Detection results of crystal methamphetamine：The concentration of crystal methamphetamine antibody is 100 μ g/ml in solution to be measured, methyl
The concentration of amphetamine is respectively 0ng/ml, 10ng/ml, 100ng/ml and 1 μ g/ml, the 60th second resonance pixel response value such as Fig. 9
Shown, the detection limit of drug testing chips p-Methylamphetamine prepared in saliva analog sample is low as can be seen from Figure
In 10ng/ml.
The Detection results of cocaine：The concentration of cocaine antibody is 100 μ g/ml in solution to be measured, the concentration of cocaine
Respectively 0ng/ml, 1ng/ml, 10ng/ml, 100ng/ml, 1 μ g/ml and 10 μ g/ml, the resonance pixel response value of the 60th second is such as
Shown in Figure 10, the detection limit of drug testing chips cocaine prepared in saliva analog sample is less than as can be seen from Figure
100ng/ml。
The Detection results of KET：The concentration of KET antibody is 100 μ g/ml in solution to be measured, and the concentration of KET is respectively 0ng/
Ml, 100ng/ml and 1 μ g/ml, the resonance pixel response value of the 60th second is as shown in figure 11, is simulated as can be seen from Figure in saliva
The detection limit of prepared drug testing chips KET is less than 1 μ g/ml in sample.
The Detection results of morphine：The concentration of morphine Abs is 100 μ g/ml in solution to be measured, and the concentration of morphine is respectively
0ng/ml, 100ng/ml, 200ng/ml, 300ng/ml, 500ng/ml and 1 μ g/ml, the resonance pixel response value of the 60th second is as schemed
Shown in 12, the detection limit of drug testing chips morphine prepared in saliva analog sample is less than as can be seen from Figure
200ng/ml。
It should be noted that if other drugs have corresponding antigen-antibody can be detected in the same way,
Such as head-shaking pill, hemp, methadone, amphetamine, C16H25NO2 etc..
Each embodiment is described by the way of progressive in this specification, what each embodiment stressed be and other
The difference of embodiment, between each embodiment identical similar portion mutually referring to.For device disclosed in embodiment
For, because it is corresponded to the method disclosed in Example, so description is fairly simple, related part is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments, professional and technical personnel in the field are enable to realize or using the present invention.
A variety of modifications to these embodiments will be apparent for those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, it is of the invention
The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one
The most wide scope caused.
Claims (10)
1. a kind of preparation method of a variety of drug testing chips based on SPR, it is characterised in that comprise the following steps：
1) carrier material is prepared；
2) one layer of golden film is plated on a support material, and golden film thickness range soaks in 40~45nm, and by the carrier material for being coated with golden film
Carboxyl chip is made in bubble in sulfydryl alkanoic acid；
3) surface of carboxyl chip is fully contacted with EDC/NHS mixed liquors, the carboxylic group of activated carboxyl chip surface is made
Activated carboxyl chip；
4) activated carboxyl chip is fully contacted with a variety of drugs synthetic antigen solution, a variety of drugs synthetic antigens is coupled to carboxylic
On base chip；
5) ethanolamine solutions are fully reacted with completing the carboxyl chip of a variety of drugs synthetic antigen couplings, closes unreacted carboxylic
Base site, a variety of drug testing chips are made.
A kind of 2. preparation method of a variety of drug testing chips based on SPR according to claim 1, it is characterised in that
The condition of immersion golden film is in step 2)：The time being incubated under conditions of 40~45 DEG C of temperature range keeps 30~35min, or
The time that person is incubated under normal temperature condition keeps more than 12h.
A kind of 3. preparation method of a variety of drug testing chips based on SPR according to claim 1, it is characterised in that
Step 2) also includes the golden film on carrier material is carried out being cleaned by ultrasonic processing after gold-plated film and rinses drying process；Ultrasound
Condition is：Cleansing medium selects ethanol solution, and temperature conditionss are room temperature, and ultrasound intensity 55-60%, ultrasonic time keep 5min
More than；Rinsing drying process condition is：From deionized water rinsing, dried up after flushing with nitrogen.
4. a kind of preparation method of a variety of drug testing chips based on SPR according to any one of claims 1 to 3, its
It is characterised by, also includes between step 2) and step 3)：PDMS is covered in carboxyl chip surface, PDMS opens up passage, passage
Have a plurality of, passage both ends are respectively injection port and outlet, and passage is handling it by step 3), step 4) and step 5)
After sense channel is made.
A kind of 5. preparation method of a variety of drug testing chips based on SPR according to claim 1, it is characterised in that
EDC/NHS mixed liquors are concentration 400mM EDC solution and concentration 100mM NHS solution according to isometric mixing institute in step 3)
The solution obtained.
A kind of 6. preparation method of a variety of drug testing chips based on SPR according to claim 1, it is characterised in that
The volume for the EDC/NHS mixed liquors that step 3) is activated to carboxyl chip is more than 200 μ L.
A kind of 7. preparation method of a variety of drug testing chips based on SPR according to claim 6, it is characterised in that
It is rinsed to being passed through PBS using the carboxyl chip after the activation of EDC/NHS mixed liquors, irrigation flow rate is 20 μ L/min.
A kind of 8. preparation method of a variety of drug testing chips based on SPR according to claim 1, it is characterised in that
The volume of a variety of drugs synthetic antigen solution is more than 100 μ L in step 4).
A kind of 9. preparation method of a variety of drug testing chips based on SPR according to claim 1, it is characterised in that
Also include between step 4) and step 5)：PBS is passed through to the carboxyl chip of coupling to be rinsed, irrigation flow rate is 20 μ L/
min。
A kind of 10. preparation side of a variety of drug testing chips based on SPR according to any one of claim 1-3,5-9
Method, it is characterised in that in step 5) concrete operations in the unreacted carboxyl groups site of the carboxyl chip of closing synthetic antigen coupling be
The ethanolamine solutions that concentration is 1M, volume is 200 μ L, PH=8.5 are passed through, being passed through PBS again after completing is rinsed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710649618.4A CN107478613A (en) | 2017-08-02 | 2017-08-02 | A kind of preparation method of a variety of drug testing chips based on SPR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710649618.4A CN107478613A (en) | 2017-08-02 | 2017-08-02 | A kind of preparation method of a variety of drug testing chips based on SPR |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107478613A true CN107478613A (en) | 2017-12-15 |
Family
ID=60598457
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710649618.4A Pending CN107478613A (en) | 2017-08-02 | 2017-08-02 | A kind of preparation method of a variety of drug testing chips based on SPR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107478613A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113281309A (en) * | 2021-05-17 | 2021-08-20 | 深圳市罗湖区人民医院 | Chlorpyrifos, carbendazim and atrazine three-in-one detection method |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1237705A (en) * | 1999-05-28 | 1999-12-08 | 清华大学 | Real-time phase detection analysis method and system for biomolecular interaction |
| CN101251541A (en) * | 2008-04-11 | 2008-08-27 | 清华大学 | Unmarked phage display protein chip and its preparation and detection method |
| CN102243174A (en) * | 2011-04-14 | 2011-11-16 | 中国科学院半导体研究所 | Phase detection-based surface plasma resonance sensing device |
| CN102520161A (en) * | 2012-01-05 | 2012-06-27 | 中南大学 | Biosensor chip, kit and detection method for SPR (Surface Plasmon Resonance) dual-channel method to detect specific prostate antigen |
| CN102706835A (en) * | 2012-05-14 | 2012-10-03 | 中央民族大学 | Sensing chip of dual-detecting biochemical sensing detector and preparation method thereof |
| CN103675053A (en) * | 2013-09-27 | 2014-03-26 | 中国科学院电子学研究所 | Local electrochemical imaging test system based on surface plasma resonance |
| CN103884682A (en) * | 2014-03-20 | 2014-06-25 | 暨南大学 | Surface plasma resonance biochip, and preparation method and application thereof |
| CN104359870A (en) * | 2014-11-28 | 2015-02-18 | 北京中龙益诚科技有限公司 | Preparation method of surface plasma resonance (SPR) biosensor chip |
| CN105300932A (en) * | 2015-11-13 | 2016-02-03 | 上海出入境检验检疫局工业品与原材料检测技术中心 | Surface plasma resonance rapid detection method of dibutyl phthalate |
| CN106568744A (en) * | 2016-10-08 | 2017-04-19 | 浙江警察学院 | Method for detecting cocaine based on SPR technology |
| CN106568743A (en) * | 2016-10-08 | 2017-04-19 | 浙江警察学院 | SPR technique-based ketamine (K powder) detection method |
| CN106841121A (en) * | 2017-04-12 | 2017-06-13 | 清华大学深圳研究生院 | A kind of SPR biochemical sensors based on ridge optical waveguide |
| CN106896086A (en) * | 2016-10-08 | 2017-06-27 | 浙江警察学院 | A kind of morphine detection method based on SPR technique |
-
2017
- 2017-08-02 CN CN201710649618.4A patent/CN107478613A/en active Pending
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1237705A (en) * | 1999-05-28 | 1999-12-08 | 清华大学 | Real-time phase detection analysis method and system for biomolecular interaction |
| CN101251541A (en) * | 2008-04-11 | 2008-08-27 | 清华大学 | Unmarked phage display protein chip and its preparation and detection method |
| CN102243174A (en) * | 2011-04-14 | 2011-11-16 | 中国科学院半导体研究所 | Phase detection-based surface plasma resonance sensing device |
| CN102520161A (en) * | 2012-01-05 | 2012-06-27 | 中南大学 | Biosensor chip, kit and detection method for SPR (Surface Plasmon Resonance) dual-channel method to detect specific prostate antigen |
| CN102706835A (en) * | 2012-05-14 | 2012-10-03 | 中央民族大学 | Sensing chip of dual-detecting biochemical sensing detector and preparation method thereof |
| CN103675053A (en) * | 2013-09-27 | 2014-03-26 | 中国科学院电子学研究所 | Local electrochemical imaging test system based on surface plasma resonance |
| CN103884682A (en) * | 2014-03-20 | 2014-06-25 | 暨南大学 | Surface plasma resonance biochip, and preparation method and application thereof |
| CN104359870A (en) * | 2014-11-28 | 2015-02-18 | 北京中龙益诚科技有限公司 | Preparation method of surface plasma resonance (SPR) biosensor chip |
| CN105300932A (en) * | 2015-11-13 | 2016-02-03 | 上海出入境检验检疫局工业品与原材料检测技术中心 | Surface plasma resonance rapid detection method of dibutyl phthalate |
| CN106568744A (en) * | 2016-10-08 | 2017-04-19 | 浙江警察学院 | Method for detecting cocaine based on SPR technology |
| CN106568743A (en) * | 2016-10-08 | 2017-04-19 | 浙江警察学院 | SPR technique-based ketamine (K powder) detection method |
| CN106896086A (en) * | 2016-10-08 | 2017-06-27 | 浙江警察学院 | A kind of morphine detection method based on SPR technique |
| CN106841121A (en) * | 2017-04-12 | 2017-06-13 | 清华大学深圳研究生院 | A kind of SPR biochemical sensors based on ridge optical waveguide |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113281309A (en) * | 2021-05-17 | 2021-08-20 | 深圳市罗湖区人民医院 | Chlorpyrifos, carbendazim and atrazine three-in-one detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9829434B2 (en) | Optical detection system for liquid samples | |
| JP3823053B2 (en) | Detection automation equipment for medical sites with full sample processing capability | |
| CN105233892B (en) | Magnetic microparticle chemiluminescence double layer micro fluidic chip for whole blood sample detection | |
| CN105241871B (en) | A kind of magnetic microparticle chemiluminescence micro-fluidic chip for whole blood sample detection | |
| CN105259163B (en) | The direct chemiluminescence micro-fluidic chip of magnetic particle for whole blood sample detection | |
| CN108344714B (en) | Biodetector based on interference effect of ordered porous nanostructured thin film and method for detecting biomolecules | |
| JPH04501606A (en) | Sensor units and their use in biosensor systems | |
| CN119425822A (en) | A microfluidic chip for bedside diagnosis | |
| CN101278186A (en) | Biosensors with improved sensitivity | |
| CN205449995U (en) | Magnetic particle chemiluminescence micro -fluidic chip | |
| JPH03506078A (en) | Equipment used in chemical test methods | |
| CN104330553B (en) | A kind of unmarked chemiluminescence immunoassay sensor and immune analysis method thereof | |
| CN110468026B (en) | Microfluidic chip for optical fiber photodynamic cell manipulation | |
| CN108061727A (en) | A kind of thyroglobulin quantitative testing test paper item and preparation method thereof | |
| CN109444412A (en) | A kind of pet d-dimer detection kit for very small chemical luminescence immunoassay system | |
| CN108051588A (en) | For the antibody fixing means on the micro-fluidic chip of whole blood sample separation detection | |
| Krupin et al. | Detection of small molecules using long-range surface plasmon polariton waveguides | |
| CN117623212A (en) | Visualized microfluidic chip based on liquid metal and its preparation method and application | |
| CN107478613A (en) | A kind of preparation method of a variety of drug testing chips based on SPR | |
| CN101561393A (en) | Method for detecting human serum tumor marker by surface plasmon resonance (SPR) technology and chip used by same | |
| CN1323296C (en) | Silane-crosslinked chitosan membrane-based flow injection chemiluminescence immunoassay cell and preparation method | |
| CN109211866A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection CK-MB | |
| CN210465451U (en) | Colloidal gold accelerated reaction device | |
| CN1424584A (en) | Magnetic separated immunoreaction optical inspecting device and method | |
| CN109939751A (en) | A kind of micro-fluidic chip of whole blood test, detection device and its detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171215 |