CN106568744A - Method for detecting cocaine based on SPR technology - Google Patents
Method for detecting cocaine based on SPR technology Download PDFInfo
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- CN106568744A CN106568744A CN201610876704.4A CN201610876704A CN106568744A CN 106568744 A CN106568744 A CN 106568744A CN 201610876704 A CN201610876704 A CN 201610876704A CN 106568744 A CN106568744 A CN 106568744A
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Abstract
The invention discloses a method for detecting cocaine based on an SPR technology. The method comprises that an SPR chip is coupled with an amino group on BSA through a carboxyl group activated by EDC/NHS to be used as a sensing chip, and when a to-be-measured sample passes through the surface of the sensing chip, light intensity changes of total reflected light at an interface of to-be-measured saliva and the molecular recognition sensing chip are detected by a surface plasmon resonance detector, to directly determine the content of cocaine in the saliva. The method has high detection sensitivity, greatly reduces the omission factor of detection of drug addicts, improves the work efficiency, and has important application value.
Description
Technical field
The invention belongs to illicit drugs inspection field, is related to a kind of cocaine detection method based on SPR technique.
Background technology
Cocaine is a kind of alkaloid of the White crystal extracted from Folium Cocoe, and the chemical constitution of cocaine is methyl bud
The ester that sub- alkali is formed with benzoic acid.Cocaine hydrochloric acid is medically often used as local anesthetic.It and Opium, morphine are different
It is that it is potent central nervous excitation agent and local anesthetic.Local resident chews the leaf for eating this plant, can play elimination
Fatigue, the effect for improving emotion.It can block human nerve conduction, produce local anesthesia effect, and can be by strengthening in human body
The activity of chemical substance stimulates cerebral cortex, stimulating central nervous system, show to get excited, it is active, even have attack tendency,
Short-period used will produce drug addiction.
Although methods such as existing chemical method, chromatography, spectrographic method, immunization, high performance capillary electrophoresis and ion mobility spectrometries
It is applied in illicit drugs inspection, but chemical method sensitivity is low, chromatography, high performance capillary electrophoresis and ion mobility spectrometry pretreatment process
Time is longer, needs dedicated technician and extensive costliness instrument, and spectrographic method sensitivity is low and testing sample purity is wanted
Ask higher, the immunization operating time is long.It is most widely colloid gold test paper in being applied to drug abuse detection at present, but which is with drug abuse
Personnel's urine is detection object, and its sensitivity only has 300-1000ng/ml.It is and this technology is with its high sensitivity, achievable right
In the saliva of drug addict, drugs are detected, so as to avoid the infringement to object privacy, and substantially increase work efficiency.
Surface plasma body resonant vibration (Surface Plasmon Resonance, SPR) technology is based on physical opticses phenomenon
A kind of analytical technology.When polarized light is impinged upon with resonance angle incidence on metal and dielectric interface, there is total reflection, it is incident
The part energy of light is coupled with surface plasma wave, causes surface plasma body resonant vibration, is produced light intensity and is drastically reduced
Reflected light.During using optical SPR technical measurement, a kind of functional response thing (such as part) is fixed on into sensing chip table usually
On the metal film of face, when add testing sample (receptor) and make its with sensing chip on functional response thing interact when, cause
The variations in refractive index of system, so as to cause the change of SPR optical signallings.Due to the high identity of SPR technique, high sensitivity, fast
Speed, in situ (without the need for labelling), it is convenient, real-time the advantages of, SPR sensorgram technology has been widely used in life science, medicine
The fields such as thing screening, food analyses, environmental monitoring, agricultural production.
At present, the report that cocaine detection is combined together with SPR technique is had no both at home and abroad.
The content of the invention
Present invention aims to the relatively low detection sensitivity of current colloid gold test paper, there is provided a kind of based on SPR's
In real time, quick, highly sensitive cocaine detection technique, methods described detection sensitivity are high, substantially reduce drug addict's detection
Loss, with important using value.
The method that cocaine of the present invention based on SPR technique is detected, is the carboxyl by SPR chips Jing after EDC/NHS activation
As sensing chip after amino on group coupling BSA, when testing sample passes through censorchip surface, using surface plasma
Resonance body detector detects that saliva to be detected and the light intensity of molecular recognition sensitive chip interface total reflection light change directly to survey
Determine the cocaine content in saliva, specifically include following steps：
Step (1)：SPR chips are placed into as sensing chip in SPR instruments, instrument temperature is set as 20～30 DEG C；
Described SPR chips are first one layer of 2～3nm of plated surface thick on the glass substrate layers of chrome, then in layers of chrome upper surface
One layer of 40～50nm of plating thick golden film；Epoxy radicals, epoxy are generated with epoxychloropropane process after golden film Surface Creation sulfydryl alkanol
Make glucosan carboxylated with bromoacetic acid process after base covalent bond glucosan, so as to obtain carboxylated dextran surface；The SPR
Carboxylic group of the chip Jing after EDC/NHS activation can be coupled the amino on BSA；
Step (2)：200～300 μ L EDC/NHS mixing is passed through toward SPR chip surfaces with the flow velocity of 10～30 μ L/min
Liquid, activates the carboxylic group of the chip surface；
Described EDC/NHS mixed liquors are the equal-volume mixed solution of EDC and NHS；Wherein EDC is N- (3- dimethylaminos
Propyl group)-N '-ethyl carbodiimide, concentration is 0.2～0.4M；NHS is N-hydroxy-succinamide, and concentration is 0.1～0.2M；
Step (3)：0.5 is passed through with the flow velocity of 10～30 μ L/min on the SPR chips after step (2) activating surface carboxyl
～2mg/mL BSA-Cocaine solution so as to be coupled on the carboxyl for activating, it is desirable to which target response value (RU) reaches 2000
～3000；
Cocaine complete antigens of the described BSA-Cocaine for synthetic, i.e., on BSA (bovine serum albumin) surface
Cocaine (cocaine) molecule has been coupled, it is available commercially；PH value is 4.5～5.5；
Step (4)：It is passed through with the flow velocity of 10～30 μ L/min on SPR chips after step (3) BSA-Cocaine is coupled
75～100 μ L ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site for activating；
The concentration of described ethanolamine hydrochloric salt solution is 1～2M；
Step (5)：30～50 μ L gradients are being passed through with the flow velocity of 10～30 μ L/min on SPR chips after step (4) process
The cocaine antibody of concentration, it is determined that suitable antibody concentration；
Described cocaine antibody is the Mus resource monoclonal antibody to BSA-Cocaine with specific recognition.
Step (6)：A series of cocaine standard substance of 30～50 μ L of variable concentrations are dense with what step (5) determined respectively
After degree antibody mixing, SPR chips are passed through successively with the flow velocity of 10～30 μ L/min, standard curve is set up；Wherein cocaine antigen
The concentration antibody equal-volume determined with step (5)；
Step (7)：After the concentration antibody equal-volume that the detected sample of 30～50 μ L is determined with step (5) is mixed, with
The flow velocity of 10～30 μ L/min is passed through SPR chips, and recording responses signal substitutes into standard curve, tries to achieve cocaine content in saliva.
Wherein, abbreviations of the SPR for surface plasma resonance.
Further, it is passed through with the flow velocity of 10～30 μ L/min after the completion of each cocaine antibody response in step (5)
NaOH solution, makes antigen-antibody dissociate, and the antibody of next concentration is added after completing chip regeneration；Described sodium hydroxide is molten
Liquid is 50～100mM.
Further, before into step (6), the most suitable concentration antibody that multiple injection step (5) determines can be carried out, is tested
The repeatability that confirmation is tested.
Further, the testing sample in step (7) can be saliva, blood, urine.
Beneficial effects of the present invention：
(1) the method for the invention is easy to operate, in real time, quickly, only antigen-antibody mixing need to be passed through chip, just can be real
When observed result.
(2) chip of the present invention prepares scheme maturation, and commercialization, purchase are convenient and reusable up to a hundred
It is secondary, it is a kind of very economical detection method.
(3) sensitivity reached by the inventive method is higher, and the time is shorter, is that other any technologies cannot all be compared.
(4) as the sensitivity of colloid gold test paper only has 300-1000ng/mL, when measuring samples content is less than its sensitivity
When, colloid gold test paper will be unable to detection, and the present invention just solves this problem.
Description of the drawings
Fig. 1 is the relation between variable concentrations antibody and SPR response signals：Antibody concentration is followed successively by 6.25 μ g/ml, 12.5 μ
g/ml、25μg/ml、50μg/ml、100μg/ml。
Regeneration schematic diagrams of the Fig. 2 for SPR chips, spr signal rapid drawdown after injection sodium hydroxide, and it is gradually brought to just primordium
Line states.
Fig. 3 is the SPR response diagrams for injecting 5 constant density antibody respectively.
In Fig. 4, a is the standard curve of cocaine detection, and b is the range of linearity of standard curve.
Specific embodiment
The present invention, but not limited to this are described in detail in detail with reference to embodiment and accompanying drawing.
The main agents information mentioned in following examples is shown in Table 1；Key instrument is shown in Table 2 with facility information.
Table 1
Table 2
The CM5 chips that the SPR chips adopted by following examples are produced for GE companies, the surface of the chip is carboxy methylation
Glucosan, the amino being easily coupled on BSA；The SPR instruments for being used are the Biacore3000 of GE companies production；Colloid gold test paper
Purchased from Abon Biopharm (Hangzhou) Co., Ltd..Embodiment 1：
(1) SPR instruments are opened, unlocks SPR chip pockets, SPR chips are placed in slot, chip pocket is locked；SPR
Instrument temperature is set as 25 DEG C；
(2) with the flow velocity of 20 μ L/min toward EDC the and NHS equal-volume ratios mixing that 250 μ L of injection are passed through in SPR chip surfaces
Liquid, activates the carboxylic group of the chip surface；Wherein EDC concentration is 0.4M, and NHS concentration is 0.1M；
(3) BSA- of the pH5.0 of 1mg/mL is passed through on the SPR chips after activating surface carboxyl with the flow velocity of 20 μ L/min
Cocaine solution so as to be coupled on the carboxyl for activating, it is desirable to which target response value (RU) reaches 3000；
(4) in the ethanolamine for being coupled on the SPR chips of BSA-Cocaine the 1M that 100 μ L are passed through with the flow velocity of 20 μ L/min
HCI solution, to close the unreacted carboxyl site for activating；
(5) 6.25 μ g/mL, 12.5 μ g/mL, the 25 μ g/mL of 40 μ L, 50 μ g/mL, 100 μ are passed through with the flow velocity of 20 μ L/min
The cocaine antibody of g/mL gradient concentrations, as a result as shown in figure 1, as a result showing the antibody concentration of 6.25 μ g/mL and 12.5 μ g/mL
Under RU values it is too small, mark song gradient is narrower, reduce sensitivity；RU values under the antibody concentration of 50 μ g/mL and 100 μ g/mL are higher,
But can expend excessive antibody, be unfavorable for cost control, under Integrated comparative, the antibody concentration of 25 μ g/mL is the most suitable, really
It is 25 μ g/mL to determine the antibody concentration in follow-up test,；
(6) NaOH solution of 10 μ L 50mM is passed through after the completion of each example reaction with the flow velocity of 20 μ L/min, resists antigen
Body dissociates, and the antibody of next concentration is added after completing chip regeneration, as shown in Fig. 2 initial baseline is 2301RU, after regeneration
Baseline returns to 2297RU, and CV (coefficient of variation) is only 0.12%, shows that chip regeneration effect is preferable；
(7) it is passed through the 40 μ L of antibody totally 5 times of 25 μ g/mL with the flow velocity of 20 μ L/min, the repeatability of confirmatory experiment, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 948.4RU, and 960.3RU, 946.8RU, 975.4RU, 981.6RU, CV are only
1.63%, show the repeatability tested preferably；
(8) by a series of cocaine standard substance of 40 μ L of variable concentrations (2000ng/mL, 1000ng/mL, 500ng/mL,
250ng/mL, 125ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/
ML, 1.95ng/mL, 0ng/mL) arrive according to being passed through 40 μ L successively with the flow velocity of 20 μ L/min after mixing with 25 μ g/mL antibody equal-volumes
SPR chips obtain SPR response signals, set up standard curve, as a result as shown in Fig. 4 (a) and Fig. 4 (b), in 7.81～1000ng/
In the range of ml, the concentration of cocaine is linear with SPR response signals, and minimal detectable concentration is in 7.81-15.63ng/ml models
Enclose, when cocaine concentration more than 1000ng/ml or less than 7.81ng/ml when, the concentration of cocaine is no longer linear with spr signal
Relation.
(9) basic condition of people's (first) to be detected：Sex man, at the age 21, its urine Jing colloid gold test paper testing result is
Cocaine is positive.
After by the saliva 12000pm of first centrifugation 10min, take 40 μ L sample mix with isopyknic antibody after, with 20 μ L/
The flow velocity of min is passed through 30 μ L to SPR chips successively, and recording responses signal substitutes into standard curve, tries to achieve cocaine content in saliva
For 547.18ng/mL.
The verity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and combine that to measure cocaine content in first saliva be 551.65ng/mL, it is consistent with the result that the present invention is measured.
Embodiment 2
(1) SPR instruments are opened, unlocks SPR chip pockets, SPR chips are placed in slot, chip pocket is locked；SPR
Instrument temperature is set as 25 DEG C；
(2) it is passed through 100 μ L0.2M EDCs and 100 μ L0.1M NHSs toward SPR chip surfaces with the flow velocity of about 10 μ L/min
Mixed liquor, activates the carboxylic group of the chip surface；
(3) 0.5mg/mL is passed through with the flow velocity of 10 μ L/min on the SPR chips after step (2) activating surface carboxyl
BSA-Cocaine solution (pH value is 4.5) so as to be coupled on the carboxyl for activating, it is desirable to which target response value (RU) reaches about
2000；
(4) 75 μ L 2M second are passed through with the flow velocity of 10 μ L/min on the SPR chips after step (3) BSA-Cocaine couplings
Alcohol amide hydrochloride, to close the unreacted carboxyl site for activating；
(5) 6.25 μ g/mL, 12.5 μ g/mL, the 25 μ g/mL of 30 μ L, 50 μ g/mL, 100 μ are passed through with the flow velocity of 10 μ L/min
The cocaine antibody of g/mL gradient concentrations, as a result as shown in figure 1, as a result showing the antibody concentration of 6.25 μ g/mL and 12.5 μ g/mL
Under RU values it is too small, mark song gradient is narrower, reduce sensitivity；RU values under the antibody concentration of 50 μ g/mL and 100 μ g/mL are higher,
But can expend excessive antibody, be unfavorable for cost control, under Integrated comparative, the antibody concentration of 25 μ g/mL is the most suitable, really
It is 25 μ g/mL to determine the antibody concentration in follow-up test,；
(6) NaOH solution of 10 μ L 50mM is passed through after the completion of each example reaction with the flow velocity of 10 μ L/min, resists antigen
Body dissociates, and the antibody of next concentration is added after completing chip regeneration, as shown in Fig. 2 initial baseline is 2301RU, after regeneration
Baseline returns to 2297RU, and CV (coefficient of variation) is only 0.12%, shows that chip regeneration effect is preferable；
(7) it is passed through the 30 μ L of antibody totally 5 times of 25 μ g/mL with the flow velocity of 10 μ L/min, the stability of validation instrument, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 948.4RU, and 960.3RU, 946.8RU, 975.4RU, 981.6RU, CV are only
1.63%, show that the stability tested is preferable；
(8) by a series of cocaine standard substance of 30 μ L of variable concentrations (2000ng/mL, 1000ng/mL, 500ng/mL,
250ng/mL, 125ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/
ML, 1.95ng/mL, 0ng/mL) arrive according to being passed through 40 μ L successively with the flow velocity of 10 μ L/min after mixing with 25 μ g/mL antibody equal-volumes
SPR chips obtain SPR response signals, set up standard curve, as a result as shown in Fig. 4 (a) and Fig. 4 (b), in 7.81～1000ng/
In the range of ml, the concentration of cocaine is linear with SPR response signals, and minimal detectable concentration is in 7.81-15.63ng/ml models
Enclose, when cocaine concentration more than 1000ng/ml or less than 7.81ng/ml when, the concentration of cocaine is no longer linear with spr signal
Relation.
(9) basic condition of people's (second) to be detected：Sex man, at the age 34, its urine Jing colloid gold test paper testing result is
Cocaine is positive.
After the saliva 12000pm centrifugation 10min of second, the body such as concentration antibody that the sample and step (5) for taking 30 μ L determines
After product mixing, 30 μ L to SPR chips are passed through with the flow velocity of 10 μ L/min, recording responses signal substitutes into standard curve, tries to achieve saliva
Middle cocaine content 231.39ng/mL.
The verity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and combine that to measure cocaine content in second saliva be 234.82ng/mL, it is consistent with the result that the present invention is measured.
Embodiment 3
(1) SPR instruments are opened, unlocks SPR chip pockets, SPR chips are placed in slot, chip pocket is locked；SPR
Instrument temperature is set as 30 DEG C；
(2) it is passed through 150 μ L0.2M EDCs and 150 μ L0.2M NHSs toward SPR chip surfaces with the flow velocity of about 30 μ L/min
Mixed liquor, activates the carboxylic group of the chip surface；
(3) 2mg/mL BSA- are passed through with the flow velocity of 30 μ L/min on the SPR chips after step (2) activating surface carboxyl
Cocaine solution (pH value is 5.5) so as to be coupled on the carboxyl for activating, it is desirable to which target response value (RU) reaches about 3000；
(4) 100 μ L 2M are passed through with the flow velocity of 30 μ L/min on the SPR chips after step (3) BSA-Cocaine couplings
Ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site for activating；
(5) 6.25 μ g/mL, 12.5 μ g/mL, the 25 μ g/mL of 50 μ L, 50 μ g/mL, 100 μ are passed through with the flow velocity of 30 μ L/min
The cocaine antibody of g/mL gradient concentrations, as a result as shown in figure 1, as a result showing the antibody concentration of 6.25 μ g/mL and 12.5 μ g/mL
Under RU values it is too small, mark song gradient is narrower, reduce sensitivity；RU values under the antibody concentration of 50 μ g/mL and 100 μ g/mL are higher,
But can expend excessive antibody, be unfavorable for cost control, under Integrated comparative, the antibody concentration of 25 μ g/mL is the most suitable, really
It is 25 μ g/mL to determine the antibody concentration in follow-up test,；
(6) NaOH solution of 10 μ L 50mM is passed through after the completion of each example reaction with the flow velocity of 30 μ L/min, resists antigen
Body dissociates, and the antibody of next concentration is added after completing chip regeneration, as shown in Fig. 2 initial baseline is 2301RU, after regeneration
Baseline returns to 2297RU, and CV (coefficient of variation) is only 0.12%, shows that chip regeneration effect is preferable；
(7) it is passed through the 50 μ L of antibody totally 5 times of 25 μ g/mL with the flow velocity of 30 μ L/min, the stability of validation instrument, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 948.4RU, and 960.3RU, 946.8RU, 975.4RU, 981.6RU, CV are only
1.63%, show that the stability tested is preferable；
(8) by a series of cocaine standard substance of 50 μ L of variable concentrations (2000ng/mL, 1000ng/mL, 500ng/mL,
250ng/mL, 125ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/
ML, 1.95ng/mL, 0ng/mL) arrive according to being passed through 40 μ L successively with the flow velocity of 30 μ L/min after mixing with 25 μ g/mL antibody equal-volumes
SPR chips obtain SPR response signals, set up standard curve, as a result as shown in Fig. 4 (a) and Fig. 4 (b), in 7.81～1000ng/
In the range of ml, the concentration of cocaine is linear with SPR response signals, and minimal detectable concentration is in 7.81-15.63ng/ml models
Enclose, when cocaine concentration more than 1000ng/ml or less than 7.81ng/ml when, the concentration of cocaine is no longer linear with spr signal
Relation.
(9) basic condition of people (third) to be detected：Sex female, at the age 24, its urine Jing colloid gold test paper testing result is
Cocaine is negative.
After the saliva 12000pm centrifugation 10min of second, the body such as concentration antibody that the sample and step (5) for taking 50 μ L determines
After product mixing, 50 μ L to SPR chips are passed through with the flow velocity of 30 μ L/min, recording responses signal substitutes into standard curve, tries to achieve saliva
Middle cocaine content 48.21ng/mL.
The verity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and combine that to measure cocaine content in the third saliva be 46.38ng/mL, it is consistent with the result that the present invention is measured.
Embodiment 4：
The basic condition of people's (fourth) to be detected：Sex man, age 22, its urine Jing colloid gold test paper testing result are cocker
Because of the positive.
Take 40 μ L fourths urine sample mix with isopyknic antibody after, 30 μ L are passed through successively with the flow velocity of 20 μ L/min and are arrived
SPR chips, recording responses signal substitute into standard curve, and in trying to achieve urine, cocaine content is 954.54ng/mL.
The verity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and combine that to measure cocaine content in fourth urine be 961.26ng/mL, it is consistent with the result that the present invention is measured.
Embodiment 5
The basic condition of people's (penta) to be detected：Sex man, age 31, its urine Jing colloid gold test paper testing result are cocker
Because of the positive.
Take 30 μ L penta blood sample mix with isopyknic antibody after, 30 μ L to SPR are passed through with the flow velocity of 10 μ L/min
Chip, recording responses signal substitute into standard curve, and in trying to achieve blood, cocaine content is 673.76ng/mL.
The verity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and combine that to measure cocaine content in penta blood be 670.27ng/mL, it is consistent with the result that the present invention is measured.
Above-described embodiment is not that the present invention is not limited only to above-described embodiment, as long as meeting for the restriction of the present invention
Application claims, belong to protection scope of the present invention.
Claims (10)
1. a kind of cocaine detection method based on SPR technique, it is characterised in that the method is comprised the following steps：
Step (1)：SPR chips are placed in SPR instruments as sensing chip,
Step (2)：200～300 μ L EDC/NHS mixed liquors are passed through toward SPR chip surfaces with the flow velocity of 10～30 μ L/min, it is living
Change the carboxylic group of the chip surface；
Step (3)：0.5 is passed through with the flow velocity of 10～30 μ L/min on the SPR chips after step (2) activating surface carboxyl～
2mg/mL BSA-Cocaine solution so as to be coupled on the carboxyl for activating, it is desirable to target response value (RU) reaches 2000～
3000；
Step (4)：75 are passed through with the flow velocity of 10～30 μ L/min on SPR chips after step (3) BSA-Cocaine is coupled～
100 μ L ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site for activating；
Step (5)：30～50 μ L gradient concentrations are being passed through with the flow velocity of 10～30 μ L/min on SPR chips after step (4) process
Cocaine antibody, it is determined that suitable antibody concentration；
Step (6)：The concentration that a series of cocaine standard substance of 30～50 μ L of variable concentrations are determined with step (5) respectively resists
After body mixing, SPR chips are passed through successively with the flow velocity of 10～30 μ L/min, standard curve is set up；Wherein cocaine antigen with step
Suddenly the concentration antibody equal-volume that (5) determine；
Step (7)：After the concentration antibody equal-volume that the detected sample of 30～50 μ L is determined with step (5) is mixed, with 10～
The flow velocity of 30 μ L/min is passed through SPR chips, and recording responses signal substitutes into standard curve, tries to achieve cocaine content in saliva.
2. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that step (1) is described
SPR chips be first to plate the thick layers of chrome of one layer of 2～3nm on the glass substrate, then plate the thick golden films of one layer of 40～50nm；Golden film table
Face generates epoxy radicals with epoxychloropropane process after generating sulfydryl alkanol, is processed with bromoacetic acid after epoxy radicals covalent bond glucosan
Make glucosan carboxylated, so as to obtain carboxylated dextran surface.
3. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that step (1) SPR instrument
Device temperature is set as 20～30 DEG C.
4. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that step (2) is described
EDC/NHS mixed liquors be EDC and NHS equal-volume mixed solution；Wherein EDC is N- (3- dimethyl aminopropyls)-N '-ethyl
Carbodiimide, concentration are 0.2～0.4M；NHS is N-hydroxy-succinamide, and concentration is 0.1～0.2M.
5. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that step (3) is described
BSA-Cocaine for synthetic cocaine complete antigen, i.e., be coupled on BSA (bovine serum albumin) surface
Cocaine (cocaine) molecule；PH value is 4.5～5.5.
6. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that step (4) is described
Ethanolamine hydrochloric salt solution concentration be 1～2M.
7. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that step (5) is described
Cocaine antibody be the Mus resource monoclonal antibody to BSA-Cocaine with specific recognition.
8. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that every in step (5)
NaOH solution is passed through with the flow velocity of 10～30 μ L/min after the completion of individual cocaine antibody response, is made antigen-antibody dissociate, is completed core
The antibody of next concentration is added after piece regeneration；Described sodium hydroxide solution is 50～100mM.
9. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that in step (7)
Testing sample can be saliva, blood, urine.
10. a kind of cocaine detection method based on SPR technique as claimed in claim 1, it is characterised in that cocaine is most
Low detection sensitivity is in 7.81-15.63ng/ml.
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| CN107478613A (en) * | 2017-08-02 | 2017-12-15 | 杭州晶百检测技术有限公司 | A kind of preparation method of a variety of drug testing chips based on SPR |
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| CN101788489A (en) * | 2010-02-10 | 2010-07-28 | 中国政法大学 | Sensitive thin-film material used for detecting drugs efficiently and preparation method thereof |
| CN103323429A (en) * | 2013-05-30 | 2013-09-25 | 中南大学 | Surface plasmon resonance (SPR) sensor chip for detection of melamine content of milk and its preparation method and detection method |
| 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 |
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| CN101216423A (en) * | 2008-01-18 | 2008-07-09 | 天津市先石光学技术有限公司 | Dairy food antibiotic race detection method based on surface plasma resonance |
| CN101788489A (en) * | 2010-02-10 | 2010-07-28 | 中国政法大学 | Sensitive thin-film material used for detecting drugs efficiently and preparation method thereof |
| CN103323429A (en) * | 2013-05-30 | 2013-09-25 | 中南大学 | Surface plasmon resonance (SPR) sensor chip for detection of melamine content of milk and its preparation method and detection method |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107478613A (en) * | 2017-08-02 | 2017-12-15 | 杭州晶百检测技术有限公司 | A kind of preparation method of a variety of drug testing chips based on SPR |
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