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Detection method for opioid active substances based on cell dopamine release effect and detection kit for opioid active substance

A technology for detection kits and active substances, which is applied in the field of detection methods and detection kits for opioid active substances, can solve the problems of low component content, undetectable, impossible immunoassay, etc., and achieve rapid detection, high The effect of sensitive detection

Inactive Publication Date: 2019-06-07
浙江诺迦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fentanyl metabolizes quickly in the human body, resulting in the extremely low content of the target component in the test sample, which cannot be detected
On the other hand, the proliferation of new synthetic fentanyls with diverse molecular structures makes antibody-based immunoassays nearly impossible, and even regulatory detection by GC / LC / MS is difficult

Method used

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  • Detection method for opioid active substances based on cell dopamine release effect and detection kit for opioid active substance
  • Detection method for opioid active substances based on cell dopamine release effect and detection kit for opioid active substance
  • Detection method for opioid active substances based on cell dopamine release effect and detection kit for opioid active substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of pCMV-MOR1 plasmid

[0037] Ligase site EcoR I and not 1, the human mu type opioid receptor MOR1 gene is cloned under the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-Neo, and the eukaryotic expression plasmid pCMV-MOR1 (such as figure 1 shown).

Embodiment 2

[0038] Example 2 Establishment of MOR1 / SK-N-SH stable cell line and blank control Neo / SK-N-SH stable cell line

[0039] The pCMV-MOR1 plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into lentiviral packaging line cell 293V to prepare MOR1 lentivirus, and transfected into SK-N-SH cells. G418 screening and cloning established MOR1 / SK-N-SH stable cells. transfected cell lines. Co-transfect the pCDH-CMV-MCS-EF1-Neo plasmid, pH1 plasmid, and pH2 plasmid into the lentiviral packaging line cell 293V to prepare empty vector lentivirus, and transfect SK-N-SH cells, G418 screening, clone establishment blank Control Neo / SK-N-SH stably transfected cell line. Specific steps are as follows:

[0040] 1) Preparation of packaging line cells: One day before transfection, use DMEM-H complete culture medium (containing 10% FBS and 100U / ml penicillin, 100μg / ml streptomycin double antibody) to make lentiviral packaging line cells 293V into 1 ×10 6 Inoculate a D19cm cell culture dish a...

Embodiment 3

[0049] Example 3 Application of Universal Detection Kit for Opioid Active Substances

[0050] The universal detection kit for opioid active substances developed based on the technical solution of the present invention can be applied to the detection of various samples containing opioid active substances. In this embodiment, we take the hair of a person taking morphine as an example. Specific steps are as follows:

[0051] 1) Hair sample processing: Take 5 hair samples of morphine addicts and 5 hair samples of normal people without smoking history, and the numbers are shown in Table 1.

[0052]

[0053] Cut the hair sample within 3cm of the hair root at 20mg / part, cut it into pieces, put it into a 5ml EP tube, add 2ml of HBSS buffer solution (pH7.4) containing 1% keratinase, add a small amount of zirconium beads and quartz sand, and crush it Shake and pulverize for 1 minute to obtain corresponding sample liquids.

[0054] 2) Cell preparation: mix 5×10 cells one day in adv...

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Abstract

The invention discloses a detection method for opioid active substances based on a cell dopamine release effect and a detection kit for the opioid active substances. The detection kit comprises two components of a detection group kit and a control group kit, wherein the detection group kit comprises a monoclonal cell line MOR1 / SK-N-SH for stably expressing a human mu-type opioid receptor MOR1 geneand an ELISA detection kit; and the control group kit comprises a blank control cell line Neo / SK-N-SH and an ELISA detection kit. According to the detection method and the detection kit, accurate andhigh-sensitivity rapid detection of the opioid active substances, whether natural opioid products or synthetic opioid molecules comprising endless novel synthetic opioid active substances such as fentanyl and various derivatives thereof, can be realized, and a problem of difficulty in supervision of novel synthetic opioid psychoactive substances can be solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting opioid active substances based on cell dopamine release effect and a detection kit thereof. Background technique [0002] The detection and supervision of drug addicts taking new opioid psychoactive substances, such as fentanyl, is a difficult problem for the regulatory authorities of various countries. Because the current detection methods mainly rely on antibody (such as anti-fentanyl antibody) immunoassays, including chromatography, ELISA, etc.; and large-scale instrumental analysis such as GC-MS and LC-MS. However, fentanyl metabolizes quickly in the human body, resulting in the extremely low content of the target detection component in the test sample, which cannot be detected. On the other hand, new synthetic fentanyls emerge in endlessly, and their molecular structures are diverse, making antibody-based immunoassays almost impossible, and ev...

Claims

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Application Information

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IPC IPC(8): G01N33/558
Inventor 范春雷程向荣
Owner 浙江诺迦生物科技有限公司
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