CN107462723A - A kind of THC based on biomembrane interference technique（Hemp）Detection method - Google Patents
A kind of THC based on biomembrane interference technique（Hemp）Detection method Download PDFInfo
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Abstract
The present invention discloses a kind of THC (hemp) detection method based on biomembrane interference technique.The method of the invention detection time is short, and whole detection process only needs 5min or so, and detection sensitivity is minimum can reach 10ng/mL.The method of the invention can carry out the detection of batch sample simultaneously, at most can once carry out the detection of 80 samples, and the whole time only has 30min, realizes high flux sample quick detection.Fibre optical sensor in the method for the invention can be reused by regenerative process, the cost of the detection substantially reduced.
Description
Technical field
The invention belongs to illicit drugs inspection field, especially batch pattern detection field, is related to a kind of based on biomembrane interference
THC (hemp) detection method of technology.
Background technology
Hemp is earliest in known drugs, in terms of being once used for medicine, religion, amusement for a long time.Can aspirate, orally, nozzle
Chew, general 7 milligrams of suction can cause floaty euphoria.It is long-term use of may cause insomnia, anorexia, edgy, easy hair
Anger, to vomit, tremble, hallucinating, making the comprehension and memory loss of people, immunity degradation is easy to obtain various diseases, so as to
Make in poor health, become thin.But it will not typically cause death.The main component of hemp is THC (THC), and it is to maincenter
The most strong psychotropic activity composition of action of nervous system.
The detection method of THC is mainly colloidal gold method and chromatography at present.Although colloidal gold strip detection side
Just, but it is using drug addict's urine as detection object, and its sensitivity only has hundreds of ng/ml, and each test strips can only be examined every time
Survey a people.Although chromatography high sensitivity, the pretreatment process time is longer, it is necessary to dedicated technician and extensive costliness
Instrument.And this technology can be achieved to carry out the saliva of personnel to be tested illicit drugs inspection, so as to avoid because taking urine pair with its high sensitivity
The infringement of object privacy, and the high flux detection for carrying out 80 samples was may be implemented in a few minutes.
Biomembrane interference technique biomembrane interference technique (Bio-1ayer interferometry, BLI) be it is a kind of in real time,
Without the Fast Detection Technique of mark, its principle is to form one layer of biological membranous layer when biomolecule is attached to sensor surface, should
Biological membranous layer causes interference to the waveform of the light through sensor.Interference is detected in a manner of phase shift, can
The change of sensor molecule quantity is attached to detection.BLI technologies have been successfully applied to protein molecule interphase interaction
Detection, also has further research in small molecule detection field.
The content of the invention
It is an object of the invention to provide THC content in a kind of quick, high-throughout detection personnel to be tested's saliva
Technology.The detection method sampling is convenient, and simple to operate, detection is quick, high sensitivity, has weight in batch sample detection occasion
The application value wanted.
The purpose of the present invention is realized by following technical scheme：
A kind of tetrahydrocannabinoldetection detection method based on biomembrane interference technique, comprises the following steps：
Step (1)：The preparation of collaurum.Take the gold chloride (HAuCl of certain volume4) aqueous solution is placed in beaker, presses
100:2 volume ratio adds the sodium water solution of citric acid three, and 15-30min is boiled in heating, until solution takes on a red color, after cooling, adds
The solution of potassium carbonate of 0.5% volume mixes, using the absorbance at spectrophotometer detection 510-525nm；
The mass fraction of described chlorauric acid solution is 0.01%, and the mass fraction of the sodium water solution of citric acid three is 1%；Carbon
The concentration of sour potassium solution is 0.2mol/L；Described said process is all lucifuge；The particle diameter of collaurum described in this method is
15-20nm, there is maximum absorbance value under 518nm wavelength；
Step (2)：The preparation of gold labeling antibody.Take the colloidal gold solution of certain volume, with solution of potassium carbonate adjust PH to
7.2-7.4 10-20min is stirred at room temperature；By 100:1-2 volume ratio adds 1mg/mL THC monoclonal antibody, room
Temperature stirring 20-30min；Add mass fraction be 10% BSA solution make its final concentration of 1%；20-30min is stirred at room temperature；4-
8 DEG C, 12000rpm centrifugations 20-30min；Supernatant is abandoned, stays precipitation；Precipitation is dissolved with isometric PBS, obtains tetrahydrochysene
Cannabinol gold labeling antibody solution；
Described colloidal gold solution is that step (1) inspection is qualified；Solution of potassium carbonate concentration is 0.2M；THC list
Clonal antibody is the mouse resource monoclonal antibody of purchase；The uncombined position of BSA (bovine serum albumin(BSA)) solution blocking antibody；PBS
The pH of buffer solution is 7.4；
Step (3)：The preparation of optical fiber biosensor.It is molten that THC-BSA is submerged into APS fibre optical sensors end
In liquid, 20-30min is stored at room temperature；Optical fiber biosensor is submerged again in 10% BSA solution, be stored at room temperature 20-30min；
Optical fiber biosensor is submerged in sucrose solution again, is stored at room temperature 20-30min；Room temperature is dried, and is placed in 2~8 DEG C and is dried guarantor
Deposit；
Described APS fibre optical sensors are the 0.5M configured with absolute ethyl alcohol APS (Aminopropyls-ilane, ammonia
Base propyl silane) immersion fibre-optical probe 12 hours and obtained after drying, the fibre-optical probe surface energy after processing passes through albumen
Amino ankyrin molecule；THC-BSA is THC comlete antigen, i.e., has been coupled tetrahydrochysene hemp on BSA surfaces
Phenol molecule, is commercially available；THC-BSA solution concentrations are 50-100 μ g/mL, and sucrose solution concentration is 15%；
Step (4)：Testing sample prepares.After the saliva in personnel to be tested's mandible is picked with cotton swab, cotton swab insertion is contained
Have in the EP pipes of 0.5-1mL solution and discard cotton swab after rotating mixing；Solution for later use in EP pipes；
Described cotton swab is disposable medical cotton swab, and the solution in EP pipes is THC gold mark made from step (2)
Antibody-solutions, sample of the solution as subsequent detection in EP pipes；
Step (5)：The making of standard curve.Using ForteBio Octet Red bio-molecular interaction instrument to gradient
Standard solution is detected, and draws standard curve；Detailed process submerges PBS for the fibre optical sensor that 1. prepared by 8 steps (3)
30-100s is balanced in buffer solution, 2. balance after 8 fibre optical sensors submerge respectively 0ng/mL, 1ng/mL, 10ng/mL,
60-100s enters in 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL THC standard solution
The detection of row gradient standard items, according to testing result, standard curve is drawn, then 10 logarithms for being bottom are done to standard concentration and are turned
Change, draw the functional relation between standard concentration and detection signal, 3. 8 fibre optical sensors after the detection of gradient standard items do not have
Enter 30-100s in glycine-HCI buffer solution to be regenerated, the fibre optical sensor after 4. regenerating submerges 30- in PBS again
100s is balanced；
Described detailed process can be automatically brought into operation progress by instrument after operating software design patterns；Glycine-HCI buffer solution
PH be 1.5-2；Fibre optical sensor is reusable after regeneration；
Step (6)：Sample detection.Using ForteBio Octet Red bio-molecular interactions instrument to step (4)
Sample is detected；Detection process submerges 60-100s in the samples of step (4) for the fibre optical sensor after 1. step (5) balance
Detected, the fibre optical sensor after 2. detecting submerges 30-100s in glycine-HCI buffer solution and regenerated, after 3. regenerating
Fibre optical sensor submerge 30-100s in PBS again and be balanced；Testing result is substituted into the function of step (5), asked
Obtain the concentration of hemp in sample.
Beneficial effects of the present invention：
(1) the method for the invention sampling is convenient, avoids inconvenience of the testing staff in urine sampling and to personnel to be measured
The infringement of privacy.
(2) the method for the invention detection time is short, and whole detection process only needs 5min or so, and detection sensitivity is minimum
It can reach 10ng/mL.
(3) the method for the invention can carry out the detection of batch sample simultaneously, at most can once carry out the inspection of 80 samples
Survey, and the whole time only has 30min, realizes high flux sample quick detection.
(4) fibre optical sensor in the method for the invention can be reused by regenerative process, the detection substantially reduced
Cost.
Brief description of the drawings
Fig. 1 is the absorbance detection result of the collaurum prepared.
Fig. 2 is the testing result of 8 gradient standard items.
Fig. 3 is the standard curve of 8 gradient standard items.
Embodiment
The present invention, but not limited to this are described 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
Embodiment 1：
(1) gold chloride (HAuCl4) aqueous solution that the mass fraction for taking 100mL is 0.01% is placed in beaker, adds 2mL
Mass fraction is 1% sodium water solution of citric acid three, and 15min is boiled in heating, until solution takes on a red color, after cooling, adds 0.5%
The 0.2mol/L of volume solution of potassium carbonate mixes, and is detected using spectrophotometer, absorbance such as Fig. 1 institutes of the collaurum of preparation
Show, its λ max is at 518nm, and the diameter for the collaurum for showing to prepare is in 15nm or so；
(2) colloidal gold solution 100ml is taken, PH to 7.2 is adjusted with solution of potassium carbonate, 10min is stirred at room temperature；Add 1mg tetra-
Hydrogen cannabinol monoclonal antibody, is stirred at room temperature 20min；2ml BSA solution blocking antibodies are added, 20min is stirred at room temperature；4 DEG C,
12000rpm centrifuges 20min；Supernatant is abandoned, stays precipitation；Precipitation 100mL pH=7.4 PBS dissolves, and obtains four
Hydrogen cannabinol gold labeling antibody solution；
(3) dried in the APS solution for the 0.5M for configuring fibre-optical probe immersion with absolute ethyl alcohol after 12 hours and obtain APS light
Fiber sensor；Its end is submerged in 50 μ g/mL THC-BSA solution, is stored at room temperature 20min；Optical fibre bio is passed again
Sensor is submerged in 10% BSA solution, is stored at room temperature 20min；Optical fiber biosensor is submerged in 15% sucrose solution again, room
Temperature stands 20min；Room temperature is dried, and is placed in 4 DEG C of kept dries；
(4) after the saliva in the mandible of 1-8 personnel to be tested is picked with disposable medical cotton swab, cotton swab insertion is contained
Cotton swab is discarded in the 1.5mL of 0.5mL THC gold labeling antibodies EP pipes and after rotating mixing；After solution in EP pipes is used as
The sample of continuous detection；
(5) gradient standard solution is detected using ForteBio Octet Red bio-molecular interactions instrument,
Draw standard curve；Detailed process is put down for above-mentioned 8 fibre optical sensors prepared 1. are submerged into 30s in PBS
Weighing apparatus, 2. balance after 8 fibre optical sensors submerge respectively 0ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL,
100s carries out the detection of gradient standard items in 200ng/mL, 500ng/mL, 1000ng/mL THC standard solution,
Testing result as shown in Fig. 2 draw standard curve as shown in figure 3, standard concentration is done again 10 be bottom Logarithm conversions, draw
Functional relation between standard concentration and detection signal, 8 fibre optical sensors after 3. gradient standard items detect submerge pH=
30s is regenerated in 1.5 glycine-HCI buffer solution, and the PBS that the fibre optical sensor after 4. regenerating submerges pH=7.4 again delays
30s is balanced in fliud flushing；
(6) detected using the above-mentioned testing sample of ForteBio Octet Red bio-molecular interaction instrument；Detection
Process is detected for the fibre optical sensor after above-mentioned balance is 1. submerged into 100s in 1-8 measuring samples respectively, after 2. detecting
Fibre optical sensor submerge 30s in pH=1.5 glycine-HCI buffer solution and regenerated, the fibre optical sensor after 3. regenerating
30s in pH=7.4 PBS is submerged again to be balanced；Testing result is substituted into the function of step (5), try to achieve No. 1-8
The content of THC is respectively in sample>1000ng/mL, 34.31ng/mL, 475.91ng/mL,<10ng/mL,
114.12ng/mL, 581.25ng/mL, 841.58ng/mL, 74.43ng/mL；
(7) authenticity of the present embodiment testing result is verified：With Gas chromatographyMass spectrometry (GC/MS) and solid phase
It is respectively 1861.55ng/mL that abstraction technique (SPE), which is combined the THC content measured in personnel to be tested's 1-8 salivas,
30.45ng/mL, 465.34ng/mL, 3.13ng/mL, 110.45ng/mL, 573.61ng/mL, 838.41ng/mL, 71.84ng/
ML, it is consistent with the result that the present invention measures.
Embodiment 2：
(1) gold chloride (HAuCl4) aqueous solution that the mass fraction for taking 100mL is 0.01% is placed in beaker, adds 2mL
Mass fraction is 1% sodium water solution of citric acid three, and 30min is boiled in heating, until solution takes on a red color, after cooling, adds 0.5%
The 0.2mol/L of volume solution of potassium carbonate mixes, and is detected using spectrophotometer, absorbance such as Fig. 1 institutes of the collaurum of preparation
Show, its λ max is at 518nm, and the diameter for the collaurum for showing to prepare is in 15nm or so；
(2) colloidal gold solution 100ml is taken, PH to 7.4 is adjusted with solution of potassium carbonate, 10min is stirred at room temperature；Add 2mg tetra-
Hydrogen cannabinol monoclonal antibody, is stirred at room temperature 30min；2ml BSA solution blocking antibodies are added, 30min is stirred at room temperature；4 DEG C,
12000rpm centrifuges 30min；Supernatant is abandoned, stays precipitation；Precipitation 100mL pH=7.4 PBS dissolves, and obtains four
Hydrogen cannabinol gold labeling antibody solution；
(3) dried in the APS solution for the 0.5M for configuring fibre-optical probe immersion with absolute ethyl alcohol after 12 hours and obtain APS light
Fiber sensor；Its end is submerged in 100 μ g/mL THC-BSA solution, is stored at room temperature 30min；Again by optical fibre bio
Sensor is submerged in 10% BSA solution, is stored at room temperature 30min；Optical fiber biosensor is submerged in 15% sucrose solution again,
It is stored at room temperature 30min；Room temperature is dried, and is placed in 4 DEG C of kept dries；
(4) after the saliva in the mandible of 1-8 personnel to be tested is picked with disposable medical cotton swab, cotton swab insertion is contained
Cotton swab is discarded in the 1.5mL of 0.5mL THC gold labeling antibodies EP pipes and after rotating mixing；After solution in EP pipes is used as
The sample of continuous detection；
(5) gradient standard solution is detected using ForteBio Octet Red bio-molecular interactions instrument,
Draw standard curve；Detailed process is put down for above-mentioned 8 fibre optical sensors prepared 1. are submerged into 60s in PBS
Weighing apparatus, 2. balance after 8 fibre optical sensors submerge respectively 0ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL,
60s carries out the detection of gradient standard items, inspection in 200ng/mL, 500ng/mL, 1000ng/mL THC standard solution
Result is surveyed as shown in Fig. 2 drawing standard curve as shown in figure 3, doing 10 Logarithm conversions for being bottom to standard concentration again, obtains bid
Functional relation between quasi- product concentration and detection signal, 8 fibre optical sensors after 3. gradient standard items detect submerge pH=1.5
Glycine-HCI buffer solution in 60s regenerated, the fibre optical sensor after 4. regenerating submerges pH=7.4 PBS again
Middle 30s is balanced；
(6) detected using the above-mentioned testing sample of ForteBio Octet Red bio-molecular interaction instrument；Detection
Process is detected for the fibre optical sensor after above-mentioned balance is 1. submerged into 60s in 1-8 measuring samples respectively, after 2. detecting
Fibre optical sensor submerges 60s in pH=1.5 glycine-HCI buffer solution and regenerated, and the fibre optical sensor after 3. regenerating is again
60s in pH=7.4 PBS is submerged to be balanced；Testing result is substituted into the function of step (5), try to achieve 1-8 samples
The content of THC is respectively in product>1000ng/mL, 33.52ng/mL, 501.45ng/mL,<10ng/mL,
115.61ng/mL, 603.48ng/mL, 841.53ng/mL, 78.47ng/mL；
(7) authenticity of the present embodiment testing result is verified：With Gas chromatographyMass spectrometry (GC/MS) and solid phase
It is respectively 1861.55ng/mL that abstraction technique (SPE), which is combined the THC content measured in personnel to be tested's 1-8 salivas,
30.45ng/mL, 465.34ng/mL, 3.13ng/mL, 110.45ng/mL, 573.61ng/mL, 838.41ng/mL, 71.84ng/
ML, it is consistent with the result that the present invention measures.
Embodiment 3：
(1) gold chloride (HAuCl4) aqueous solution that the mass fraction for taking 100mL is 0.01% is placed in beaker, adds 2mL
Mass fraction is 1% sodium water solution of citric acid three, and 25min is boiled in heating, until solution takes on a red color, after cooling, adds 0.5%
The 0.2mol/L of volume solution of potassium carbonate mixes, and is detected using spectrophotometer, absorbance such as Fig. 1 institutes of the collaurum of preparation
Show, its λ max is at 518nm, and the diameter for the collaurum for showing to prepare is in 15nm or so；
(2) colloidal gold solution 100ml is taken, PH to 7.4 is adjusted with solution of potassium carbonate, 15min is stirred at room temperature；Add 1.5mg
THC monoclonal antibody, is stirred at room temperature 25min；1.5ml BSA solution blocking antibodies are added, 25min is stirred at room temperature；4
DEG C, 12000rpm centrifugations 25min；Supernatant is abandoned, stays precipitation；Precipitation 100mL pH=7.4 PBS dissolves, and obtains
THC gold labeling antibody solution；
(3) dried in the APS solution for the 0.5M for configuring fibre-optical probe immersion with absolute ethyl alcohol after 12 hours and obtain APS light
Fiber sensor；Its end is submerged in 75 μ g/mL THC-BSA solution, is stored at room temperature 25min；Optical fibre bio is passed again
Sensor is submerged in 10% BSA solution, is stored at room temperature 25min；Optical fiber biosensor is submerged in 15% sucrose solution again, room
Temperature stands 25min；Room temperature is dried, and is placed in 4 DEG C of kept dries；
(4) after the saliva in the mandible of 1-8 personnel to be tested is picked with disposable medical cotton swab, cotton swab insertion is contained
Cotton swab is discarded in the 1.5mL of 0.5mL THC gold labeling antibodies EP pipes and after rotating mixing；After solution in EP pipes is used as
The sample of continuous detection；
(5) gradient standard solution is detected using ForteBio Octet Red bio-molecular interactions instrument,
Draw standard curve；Detailed process is put down for above-mentioned 8 fibre optical sensors prepared 1. are submerged into 100s in PBS
Weighing apparatus, 2. balance after 8 fibre optical sensors submerge respectively 0ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL,
100s carries out the detection of gradient standard items in 200ng/mL, 500ng/mL, 1000ng/mL THC standard solution,
Testing result as shown in Fig. 2 draw standard curve as shown in figure 3, standard concentration is done again 10 be bottom Logarithm conversions, draw
Functional relation between standard concentration and detection signal, 8 fibre optical sensors after 3. gradient standard items detect submerge pH=
100s is regenerated in 1.5 glycine-HCI buffer solution, and the PBS that the fibre optical sensor after 4. regenerating submerges pH=7.4 again delays
100s is balanced in fliud flushing；
(6) detected using the above-mentioned testing sample of ForteBio Octet Red bio-molecular interaction instrument；Detection
Process is detected for the fibre optical sensor after above-mentioned balance is 1. submerged into 100s in 1-8 measuring samples respectively, after 2. detecting
Fibre optical sensor submerge 100s in pH=1.5 glycine-HCI buffer solution and regenerated, the fibre optical sensor after 3. regenerating
100s in pH=7.4 PBS is submerged again to be balanced；Testing result is substituted into the function of step (5), try to achieve 1-8
The content of THC is respectively in number sample>1000ng/mL, 27.42ng/mL, 471.35ng/mL,<10ng/mL,
105.56ng/mL, 583.14ng/mL, 836.41ng/mL, 70.56ng/mL；
(7) authenticity of the present embodiment testing result is verified：With Gas chromatographyMass spectrometry (GC/MS) and solid phase
It is respectively 1861.55ng/mL that abstraction technique (SPE), which is combined the THC content measured in personnel to be tested's 1-8 salivas,
30.45ng/mL, 465.34ng/mL, 3.13ng/mL, 110.45ng/mL, 573.61ng/mL, 838.41ng/mL, 71.84ng/
ML, it is consistent with the result that the present invention measures.
Claims (5)
- A kind of 1. THC (hemp) detection method based on biomembrane interference technique, it is characterised in that this method include with Lower step：The preparation of step (1), gold labeling antibodyColloidal gold solution is adjusted into PH to 7.2-7.4,10-20min is stirred at room temperature；By 100:1-2 volume ratio adds 1mg/mL THC monoclonal antibody, 20-30min is stirred at room temperature；Adding the BSA solution that mass fraction is 10% makes its final concentration For 1%, 20-30min is stirred at room temperature；It is subsequently placed at 4-8 DEG C and centrifuges, precipitation is dissolved with isometric PBS, obtains four Hydrogen cannabinol gold labeling antibody solution；Described collaurum particle diameter is 15-20nm, there is maximum absorbance value under 518nm wavelength；Step (2), testing sample prepareAfter the saliva in personnel to be tested's mandible is picked with cotton swab, it is placed in 0.5-1mL THC gold labeling antibody solution, revolves Turn to obtain required testing sample solution after mixing；Step (3), testing sample detectionStep (2) testing sample is detected using ForteBio Octet Red bio-molecular interactions instrument, detected Journey is as follows：1. the test side for regenerating the APS optical fiber biosensors after balancing is immersed in 60- in step (2) testing sample solution 100s is detected；2. the test side of the APS optical fiber biosensors after detection re-starts regeneration balance；3. testing result is substituted into THC standard curve, the concentration of hemp in testing sample is obtained.
- 2. a kind of THC (hemp) detection method based on biomembrane interference technique as claimed in claim 1, it is special Sign is described THC standard curve using ForteBio Octet Red bio-molecular interaction instrument to gradient Standard solution is detected, and draws standard curve, and detailed process is as follows：1. the test side of multiple APS optical fiber biosensors is immersed in 30-100s in PBS and is balanced；2. balance after multiple fibre optical sensors test side be immersed in respectively 0ng/mL, 1ng/mL, 10ng/mL, 50ng/mL, 60-100s carries out gradient mark in 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL THC standard solution The detection of quasi- product, according to testing result, standard curve is drawn, then 10 Logarithm conversions for being bottom are done to standard concentration, obtain bid Functional relation between quasi- product concentration and detection signal, i.e. THC standard curve.
- 3. a kind of THC (hemp) detection method based on biomembrane interference technique as claimed in claim 1, it is special Sign is that APS fibre optical sensors regeneration equilibrium process is that the test side of APS optical fiber biosensors is immersed in into glycine-HCI 30-100s is regenerated in buffer solution；The test side of fibre optical sensor after regeneration is immersed in 30-100s in PBS and entered Row balance.
- A kind of 4. THC (hemp) detection side based on biomembrane interference technique as described in claim 1 or 2 or 3 Method, it is characterised in that the preparation process of described APS optical fiber biosensors is as follows：1) using the common blank of 0.5M APS alcohol solution dippings optical fiber biosensor test side 12 hours and dry；Wherein The amino ankyrin molecule that the test side surface energy of sensor passes through albumen after processing；2) test side of sensor after above-mentioned processing is immersed in THC-BSA solution, is stored at room temperature 20-30min；3) test side of sensor after above-mentioned processing is immersed in the BSA solution that mass content is 10%, is stored at room temperature 20- 30min；4) test side of sensor after above-mentioned processing is immersed in sucrose solution, is stored at room temperature 20-30min；5) room temperature is dried, and is placed in 2~8 DEG C of kept dries；THC-BSA is THC comlete antigen, i.e., has been coupled THC molecule on BSA surfaces.
- 5. a kind of THC (hemp) detection method based on biomembrane interference technique as claimed in claim 4, it is special Sign is that THC-BSA solution concentrations are 50-100 μ g/mL.
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| CN101580544A (en) * | 2008-02-21 | 2009-11-18 | 曾立波 | Plate for detecting immunity of cannabis and tetrahydrocannabinol monoclonal antibody through collaurum tag |
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