CN106896086A - A kind of morphine detection method based on SPR technique - Google Patents
A kind of morphine detection method based on SPR technique Download PDFInfo
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Abstract
The present invention discloses a kind of morphine detection method based on SPR technique.The method is as sensing chip after the amino on the carboxylic group coupling BSA after SPR chips are activated through EDC/NHS, when testing sample passes through censorchip surface, detect saliva to be detected with the light intensity change of molecular recognition sensitive chip interface total reflection light using surface plasma body resonant vibration detector directly to determine the morphine content in saliva.The inventive method detection sensitivity is high, substantially reduces the loss of drug addict's detection, operating efficiency is improved, with important application value.
Description
Technical field
The invention belongs to illicit drugs inspection field, it is related to a kind of morphine detection method based on SPR technique.
Background technology
Morphine is formed by opium purification, white crystals, and content 10% or so in opium, are colourless or white crystalline powder
Shape, with analgesia, hypnosis, cough-relieving, antidiarrheal, suppresses breathing and enterocinesia effect, is mainly used in alleviating acute sharp pain and cardiogenic
Asthma.Floaty euphoria can be produced after sucking, habituation easier than opium.Long-term use can cause insane, delirium and illusion, excessive
Using respiratory failure can be caused and dead.It was once used as psychotropic substances and gave up opium in history, but because its side effect is excessive,
Finally it is decided to be drugs.
Although methods such as existing chemical method, chromatography, spectroscopic methodology, immunization, capillary electrophoresis and ion mobility spectrometries
It is applied in illicit drugs inspection, but chemical method sensitivity is low, chromatography, capillary electrophoresis and ion mobility spectrometry pretreatment process
Time is more long, it is necessary to dedicated technician and extensive costliness instrument, spectroscopic methodology sensitivity is low and testing sample purity is wanted
Ask higher, the immunization operating time is long.It is current to be applied to during drug abuse is detected be most widely colloid gold test paper, but it is with drug abuse
Personnel's urine is detection object, and its sensitivity only has 300-1000ng/ml.It is achievable right and this technology is with its high sensitivity
Drugs are detected in the saliva of drug addict, so as to avoid the infringement to object privacy, and substantially increase operating efficiency.
Surface plasma body resonant vibration (Surface Plasmon Resonance, SPR) technology is based on physical optics phenomenon
A kind of analytical technology.When polarised light is impinged upon on metal and dielectric interface with resonance angle incidence, it is totally reflected, it is incident
The part energy of light is coupled with surface plasma wave, causes surface plasma body resonant vibration, produces light intensity drastically to reduce
Reflected light.When being determined using optical SPR technology, a kind of functional response thing (such as part) is usually fixed on sensing chip table
On the metal film of face, when add testing sample (acceptor) 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 identity high of SPR technique, high sensitivity, fast
Speed, in situ (without mark), 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 analysis, environmental monitoring, agricultural production.
At present, the report that morphine detection is combined together with SPR technique is had no both at home and abroad.
The content of the invention
The detection sensitivity relatively low it is an object of the invention to be directed to current colloid gold test paper, there is provided a kind of based on SPR's
In real time, quick, highly sensitive morphine detection technique, methods described detection sensitivity is high, substantially reduces drug addict's detection
Loss, with important application value.
The method that morphine of the present invention based on SPR technique is detected, is the carboxyl base after SPR chips are activated through EDC/NHS
As sensing chip after amino on group coupling BSA, when testing sample passes through censorchip surface, using surface plasma
Resonance detector detects that saliva to be detected directly determines with the light intensity change of molecular recognition sensitive chip interface total reflection light
Morphine content in saliva, specifically includes 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；After golden film Surface Creation sulfydryl alkanol generation epoxy radicals, epoxy are processed with epoxychloropropane
Being processed with bromoacetic acid after base covalent bond glucan makes glucan carboxylated, so as to obtain the dextran surface of carboxylated；The SPR
Chip can be coupled the amino on BSA through the carboxylic group after EDC/NHS activation；
Step (2)：200~300 μ L EDC/NHS are passed through with the flow velocity of 10~30 μ L/min toward SPR chip surfaces to mix
Liquid, activates the carboxylic group of the chip surface；
Described EDC/NHS mixed liquors are isometric 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)：On SPR chips after step (2) activating surface carboxyl 0.5 is passed through with the flow velocity of 10~30 μ L/min
~2mg/mL BSA-Morphine solution, makes it be coupled on the carboxyl for activating, it is desirable to which target response value (RU) reaches 2000
~3000；
Described BSA-Morphine is artificial synthesized morphine comlete antigen, i.e., on BSA (bovine serum albumin(BSA)) surface
Morphine (morphine) molecule has been coupled, it is available commercially；PH value is 4.5~5.5；
Step (4)：It is logical with the flow velocity of 10~30 μ L/min on SPR chips after step (3) BSA-Morphine couplings
Enter 75~100 μ L ethanolamine hydrochloric salt solution, be used 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 passed through with the flow velocity of 10~30 μ L/min on SPR chips after step (4) treatment
The morphine Abs of concentration, it is determined that suitable AC；
Described morphine Abs are the mouse resource monoclonal antibody to BSA-Morphine with specific recognition.
Step (6)：The concentration that a series of morphine standard items of 30~50 μ L of various concentrations are determined with step (5) respectively
After antibody mixing, SPR chips are passed through with the flow velocity of 10~30 μ L/min successively, set up standard curve；Wherein morphine antigen with step
Suddenly the concentration antibody that (5) determine is isometric；
Step (7)：After the concentration antibody that the detected sample of 30~50 μ L and step (5) are determined mixes in equal volume, 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 morphine content in saliva.
Wherein, SPR is the abbreviation of surface plasma resonance.
Further, sweet ammonia is passed through with the flow velocity of 10~30 μ L/min after the completion of the reaction of each morphine Abs in step (5)
Acid solution, makes antigen-antibody dissociate, and completes to add the antibody of next concentration after chip regenerates；Described glycine solution ph
=1.5.
Further, before step (6) is entered, the most suitable concentration antibody of multiple injection step (5) determination 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, need to only mix antigen-antibody and be passed through chip, just can be real
When observed result.
(2) chip of the present invention prepares scheme maturation, has been commercialized, and purchase is convenient, and reusable up to a hundred
It is secondary, it is a kind of very economical detection method.
(3) sensitivity that the inventive method is reached is higher, and the time is shorter, is that other any technologies cannot all compare.
(4) because 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.
Brief description of the drawings
Fig. 1 is the relation between various concentrations antibody and SPR response signals：AC is followed successively by 3.13 μ g/ml, 6.25 μ
g/ml、12.5μg/ml、25μg/ml、50μg/ml、100μg/ml。
Fig. 2 is the regeneration schematic diagram of SPR chips, injects spr signal rapid drawdown after glycine, and be gradually brought to initial baseline
State.
Fig. 3 is to inject 5 SPR response diagrams of constant density antibody respectively.
A is the standard curve of morphine detection in Fig. 4, 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 that following examples are used are produced for GE companies, the surface of the chip is carboxy methylation
Amino on glucan, easily coupling 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, SPR chip pockets are unlocked, SPR chips is placed into slot, lock chip pocket；SPR
Instrument temperature is set as 25 DEG C；
(2) EDC and NHS that inject 250 μ L are passed through in equal volume than mixing toward in SPR chip surfaces with the flow velocity of 20 μ L/min
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
Morphine solution, makes it be coupled on the carboxyl for activating, it is desirable to which target response value (RU) reaches 3000；
(4) monoethanolamine of the 1M of 100 μ L is passed through with the flow velocity of 20 μ L/min on the SPR chips of coupling BSA-Morphine
HCI solution, is used to close the unreacted carboxyl site for activating；
(5) 3.13 μ g/mL, 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 of 40 μ L are passed through with the flow velocity of 20 μ L/min
The morphine Abs of μ g/mL, 100 μ g/mL gradient concentrations, as a result as shown in figure 1, as a result show 3.13 μ g/mL, 6.25 μ g/mL,
RU values under the AC of 12.5 μ g/mL and 25 μ g/mL are too small, and mark song gradient is narrower, reduce sensitivity；100 μ g/mL's is anti-
RU values under bulk concentration are higher, but can expend excessive antibody, are unfavorable for cost control, under Integrated comparative, 50 μ g/mL's
AC is the most suitable, determines the AC in follow-up test for 50 μ g/mL,；
(6) 10 μ L glycine solutions are passed through with the flow velocity of 20 μ L/min after the completion of each example reaction, make antigen-antibody solution
From completion chip adds the antibody of next concentration after regenerating, as shown in Fig. 2 initial baseline is 1748RU, baseline after regeneration
1751RU is returned to, CV (coefficient of variation) is only 0.12%, show that chip regeneration effect is preferable；
(7) it is passed through the μ L of antibody 40 totally 5 times of 50 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 715.4RU, 725.0RU, 729.0RU, 726.0RU, 735.8RU, and CV is only
1.02%, show the repeatability of experiment preferably；
(8) by a series of morphine standard items of 40 μ L of various concentrations (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、
0.98ng/mL, 0.49ng/mL, 0ng/mL) led to successively with the flow velocity of 20 μ L/min according to after mixing in equal volume with 50 μ g/mL antibody
Enter 40 μ L to SPR chips and obtain SPR response signals, set up standard curve, as a result as shown in Fig. 4 (a) and Fig. 4 (b), 3.91~
In the range of 125ng/ml, the concentration of morphine is linear with SPR response signals, and minimal detectable concentration is in 3.91-7.81ng/ml
Scope, when morphine concentration is more than 125ng/ml or less than 3.91ng/ml, concentration and the no longer linear pass of spr signal of morphine
System.
(9) basic condition of people's (first) to be detected：Sex man, at the age 27, its urine is through colloid gold test paper testing result
Morphine is positive.
After by the saliva 12000pm of first centrifugation 10min, take 40 μ L sample mix with isometric 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, and morphine content is in trying to achieve saliva
Beyond mark song, more than 125ng/mL.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and be combined that to measure morphine content in first saliva be 641.86ng/mL, it is consistent with the result that the present invention is measured.
Embodiment 2
(1) SPR instruments are opened, SPR chip pockets are unlocked, SPR chips is placed into slot, lock chip pocket；SPR
Instrument temperature is set as 25 DEG C；
(2) it is passed through 100 μ L0.2M EDC's and 100 μ L0.1M NHS 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-Morphine solution (pH value is 4.5), makes it be coupled on the carboxyl for activating, it is desirable to which target response value (RU) reaches about
2000；
(4) 75 μ L 2M are passed through with the flow velocity of 10 μ L/min on the SPR chips after step (3) BSA-Morphine couplings
Ethanolamine hydrochloric salt solution, is used to close the unreacted carboxyl site for activating；
(5) 3.13 μ g/mL, 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 of 30 μ L are passed through with the flow velocity of 10 μ L/min
The morphine Abs of μ g/mL, 100 μ g/mL gradient concentrations, as a result as shown in figure 1, as a result show 3.13 μ g/mL, 6.25 μ g/mL,
RU values under the AC of 12.5 μ g/mL and 25 μ g/mL are too small, and mark song gradient is narrower, reduce sensitivity；100 μ g/mL's is anti-
RU values under bulk concentration are higher, but can expend excessive antibody, are unfavorable for cost control, under Integrated comparative, 50 μ g/mL's
AC is the most suitable, determines that the AC in follow-up test is 50 μ g/mL；
(6) 10 μ L glycine solutions are passed through with the flow velocity of 10 μ L/min after the completion of each example reaction, make antigen-antibody solution
From completion chip adds the antibody of next concentration after regenerating, as shown in Fig. 2 initial baseline is 1748RU, baseline after regeneration
1751RU is returned to, CV (coefficient of variation) is only 0.12%, show that chip regeneration effect is preferable；
(7) it is passed through the μ L of antibody 30 totally 5 times of 50 μ g/mL with the flow velocity of 10 μ L/min, the repeatability of confirmatory experiment, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 715.4RU, 725.0RU, 729.0RU, 726.0RU, 735.8RU, and CV is only
1.02%, show the repeatability of experiment preferably；
(8) by a series of morphine standard items of 30 μ L of various concentrations (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、
0.98ng/mL, 0.49ng/mL, 0ng/mL) led to successively with the flow velocity of 10 μ L/min according to after mixing in equal volume with 50 μ g/mL antibody
Enter 30 μ L to SPR chips and obtain SPR response signals, set up standard curve, as a result as shown in Fig. 4 (a) and Fig. 4 (b), 3.91~
In the range of 125ng/ml, the concentration of morphine is linear with SPR response signals, and minimal detectable concentration is in 3.91-7.81ng/ml
Scope, when morphine concentration is more than 125ng/ml or less than 3.91ng/ml, concentration and the no longer linear pass of spr signal of morphine
System.
(9) basic condition of people's (second) to be detected：Sex man, at the age 30, its urine is through colloid gold test paper testing result
Morphine is positive.
After by the saliva 12000pm centrifugations 10min of second, the bodies such as the concentration antibody of sample and step (5) determination of 30 μ L are taken
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 morphine content exceeds mark song, more than 125ng/ml.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and be combined that to measure morphine content in second saliva be 418.19ng/mL, it is consistent with the result that the present invention is measured.
Embodiment 3
(1) SPR instruments are opened, SPR chip pockets are unlocked, SPR chips is placed into slot, lock chip pocket；SPR
Instrument temperature is set as 30 DEG C；
(2) it is passed through 150 μ L0.2M EDC's and 150 μ L0.2M NHS 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
Morphine solution (pH value is 5.5), makes it 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-Morphine couplings
Ethanolamine hydrochloric salt solution, is used to close the unreacted carboxyl site for activating；
(5) 3.13 μ g/mL, 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 of 50 μ L are passed through with the flow velocity of 30 μ L/min
The morphine Abs of μ g/mL, 100 μ g/mL gradient concentrations, as a result as shown in figure 1, as a result show 3.13 μ g/mL, 6.25 μ g/mL,
RU values under the AC of 12.5 μ g/mL and 25 μ g/mL are too small, and mark song gradient is narrower, reduce sensitivity；100 μ g/mL's is anti-
RU values under bulk concentration are higher, but can expend excessive antibody, are unfavorable for cost control, under Integrated comparative, 50 μ g/mL's
AC is the most suitable, determines that the AC in follow-up test is 50 μ g/mL；
(6) 10 μ L glycine solutions are passed through with the flow velocity of 30 μ L/min after the completion of each example reaction, make antigen-antibody solution
From completion chip adds the antibody of next concentration after regenerating, as shown in Fig. 2 initial baseline is 1748RU, baseline after regeneration
1751RU is returned to, CV (coefficient of variation) is only 0.12%, show that chip regeneration effect is preferable；
(7) it is passed through the μ L of antibody 50 totally 5 times of 50 μ g/mL with the flow velocity of 30 μ L/min, the repeatability of confirmatory experiment, as a result such as
Shown in Fig. 3；The RU of 5 repetitions is respectively 715.4RU, 725.0RU, 729.0RU, 726.0RU, 735.8RU, and CV is only
1.02%, show the repeatability of experiment preferably；
(8) by a series of morphine standard items of 30 μ L of various concentrations (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、
0.98ng/mL, 0.49ng/mL, 0ng/mL) led to successively with the flow velocity of 10 μ L/min according to after mixing in equal volume with 50 μ g/mL antibody
Enter 50 μ L to SPR chips and obtain SPR response signals, set up standard curve, as a result as shown in Fig. 4 (a) and Fig. 4 (b), 3.91~
In the range of 125ng/ml, the concentration of morphine is linear with SPR response signals, and minimal detectable concentration is in 3.91-7.81ng/ml
Scope, when morphine concentration is more than 125ng/ml or less than 3.91ng/ml, concentration and the no longer linear pass of spr signal of morphine
System.
(9) basic condition of people (third) to be detected：Sex man, at the age 20, its urine is through colloid gold test paper testing result
Morphine is negative.
After by the saliva 12000pm centrifugations 10min of second, the bodies such as the concentration antibody of sample and step (5) determination of 50 μ L are taken
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 morphine content 98.31ng/mL.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and be combined that to measure morphine content in the third saliva be 96.26ng/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 37, its urine is morphine through colloid gold test paper testing result
It is positive.
Take the urine of 40 μ L fourths sample mix with isometric antibody after, 30 μ L are passed through with the flow velocity of 20 μ L/min successively
To SPR chips, recording responses signal substitutes into standard curve, Morphine in Urine content is tried to achieve beyond mark song, more than 125ng/mL.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and be combined and measure fourth Morphine in Urine content for 542.18ng/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 35, its urine is morphine through colloid gold test paper testing result
It is positive.
Take the blood of 30 μ L penta sample mix with isometric antibody after, 30 μ L are passed through with the flow velocity of 10 μ L/min and are arrived
SPR chips, recording responses signal substitutes into standard curve, Morphine in Blood content is tried to achieve beyond mark song, more than 125ng/mL.
The authenticity of the lower the present embodiment testing result of checking：Extracted with Gas chromatographyMass spectrometry (GC/MS) and solid phase
Take technology (SPE) and be combined and measure penta Morphine in Blood content for 467.29ng/mL, it is consistent with the result that the present invention is measured.
Above-described embodiment is not that, for limitation of the invention, the present invention is not limited only to above-described embodiment, as long as meeting
Application claims, belong to protection scope of the present invention.
Claims (10)
1. a kind of morphine detection method based on SPR technique, it is characterised in that the method is comprised the following steps：
Step (1)：SPR chips are placed into SPR instruments as sensing chip,
Step (2)：200~300 μ LEDC/NHS mixed liquors are passed through toward SPR chip surfaces with the flow velocity of 10~30 μ L/min, are activated
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 SPR chips after step (2) activating surface carboxyl~
2mg/mL BSA-Morphine solution, makes it be coupled on the carboxyl for activating, it is desirable to and target response value (RU) reaches 2000~
3000；
Step (4)：On SPR chips after step (3) BSA-Morphine couplings 75 are passed through with the flow velocity of 10~30 μ L/min
~100 μ L ethanolamine hydrochloric salt solution, are used to close the unreacted carboxyl site for activating；
Step (5)：30~50 μ L gradient concentrations are passed through with the flow velocity of 10~30 μ L/min on SPR chips after step (4) treatment
Morphine Abs, it is determined that suitable AC；
Step (6)：The concentration antibody that a series of morphine standard items of 30~50 μ L of various concentrations are determined with step (5) respectively
After mixing, SPR chips are passed through with the flow velocity of 10~30 μ L/min successively, set up standard curve；Wherein morphine antigen and step (5)
The concentration antibody of determination is isometric；
Step (7)：Concentration antibody that the detected sample of 30~50 μ L and step (5) are determined is isometric mix after, 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 morphine content in saliva.
2. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that described in step (1)
SPR chips are 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 surface
Generation epoxy radicals is processed with epoxychloropropane after generation sulfydryl alkanol, being processed with bromoacetic acid after epoxy radicals covalent bond glucan makes
Glucan carboxylated, so as to obtain the dextran surface of carboxylated.
3. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that step (1) SPR instruments
Temperature is set as 20~30 DEG C.
4. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that described in step (2)
EDC/NHS mixed liquors are isometric mixed solution of EDC and NHS；Wherein EDC is N- (3- dimethyl aminopropyls)-N '-ethyl carbon
Diimine, concentration is 0.2~0.4M；NHS is N-hydroxy-succinamide, and concentration is 0.1~0.2M.
5. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that described in step (3)
BSA-Morphine is artificial synthesized morphine comlete antigen, i.e., be coupled Morphine on BSA (bovine serum albumin(BSA)) surface
(morphine) molecule；PH value is 4.5~5.5.
6. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that described in step (4)
The concentration of ethanolamine hydrochloric salt solution is 1~2M.
7. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that described in step (5)
Morphine Abs are the mouse resource monoclonal antibody to BSA-Morphine with specific recognition.
8. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that in step (5) each
Glycine solution is passed through with the flow velocity of 10~30 μ L/min after the completion of morphine Abs reaction, antigen-antibody is dissociated, complete chip
The antibody of next concentration is added after regeneration；Described glycine solution is ph=1.5.
9. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that treating in step (7)
Test sample product can be saliva, blood, urine.
10. a kind of morphine detection method based on SPR technique as claimed in claim 1, it is characterised in that the minimum inspection of morphine
Sensitivity is surveyed in 3.91-7.81ng/ml.
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| CN201610876967.5A Pending CN106896086A (en) | 2016-10-08 | 2016-10-08 | A kind of morphine detection method based on SPR technique |
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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 |
| CN110716058A (en) * | 2019-11-19 | 2020-01-21 | 杭州纽蓝科技有限公司 | Chip for detecting hypersensitive C reactive protein |
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