CN113138271A

CN113138271A - Rapid detection method for various drugs based on reading of glucometer signals

Info

Publication number: CN113138271A
Application number: CN202110236140.9A
Authority: CN
Inventors: 胡骏杰; 上官国强; 马文俊; 彭帅
Original assignee: JINING MEDICAL UNIVERSITY
Current assignee: JINING MEDICAL UNIVERSITY
Priority date: 2021-03-03
Filing date: 2021-03-03
Publication date: 2021-07-20

Abstract

The invention discloses a rapid detection method of various drugs based on reading of glucometer signals, which relates to the technical field of drug detection, and comprises the steps of coating a drug-bovine serum albumin compound at the bottom of a 96-hole immune substrate to prepare a porous immune substrate for specific drug detection, when a sample and a corresponding antibody are added, corresponding drug molecules in the sample compete with immunogen fixed in the 96-hole immune substrate for antigen binding sites on a biotinylated antibody together, so that the biotinylated antibody captured by a pore plate is in negative correlation with the drug content in the sample, then the sample is washed by buffer solution softly twice, streptavidin and invertase @ Biotin-liposome beacon are continuously added into the system, after a surfactant is added, the invertase is released by a liposome cavity to catalyze sucrose substrate to be decomposed into glucose, and the detection result is read by a household glucometer, the detection method can rapidly acquire qualitative and quantitative information of drugs in a sample or corresponding abused drug components in a simple device and output mode.

Description

Rapid detection method for various drugs based on reading of glucometer signals

Technical Field

The invention relates to the technical field of drug detection, in particular to a rapid detection method for various drugs based on reading of glucometer signals.

Background

Methamphetamine (Amphetamine), morphine (morphine), ketamine (ketamine) and other substances are collectively called drugs, and abuse of the drugs can bring hidden troubles such as distortion of human thought, behavior and body state and cause serious consequences. The on-site rapid detection of the substances and metabolites thereof in human body fluid samples (particularly urine and saliva) is one of the most direct ways for determining drug abuse behaviors (particularly in the fields of competitive sports, criminal law, dangerous driving and the like), and has important significance for the management and control of related substances and the maintenance of social security.

To date, methods for drug detection in body fluid samples have mainly included gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and enzyme-linked immunosorbent assay (ELISA). In recent years, electrochemical sensors and point of care testing (POCT) have also found application in this area. In all methods, Mass Spectrometry (MS) is still used as a "gold standard" for detecting related substances due to its high accuracy and sensitivity. However, mass spectrometry techniques typically involve complex instrument use and process development, and thus their application is often limited to central laboratories or hospitals where equipment is sophisticated. ELISA kits and electrochemical sensor principles are relatively simple and easy to use, but they still rely on specific instruments (e.g. fluorescence quantitative PCR instrument, electrochemical workstation) for signal readout and data processing. On-site collection and detection devices, such as colloidal gold test strips, integrate on-site collection and rapid analysis of body fluid samples (mainly saliva), and related departments (SAMHSA) have clearly specified that such devices can be used for detection of related substances in body fluids. However, it should be noted that the commercial field collection and detection device has limited analysis sensitivity and flux, and most of them can only provide qualitative detection results, but cannot completely meet the requirements of fast, accurate and quantitative field analysis. The performance of the rapid identification and detection method of related substances still needs to be improved.

Currently, the home blood glucose monitor (PGM) is the most classical real-time collection and diagnosis instrument capable of providing quantitative analysis results, and has the advantages of portability, low price, credible detection results and the like. In conventional wisdom, blood glucose meters are used only for the detection of glucose levels in blood. To take full advantage of the advantages, the home-use glucometer can also be used to detect non-glucose targets, such as biological factors, disease markers, heavy metal ions, and the like. Therefore, the reaction of catalyzing substrate sucrose by sucrose invertase to generate glucose is creatively introduced into the signal output process of drug detection by combining a household blood glucose meter quantitative platform, and a universal method is established for rapidly detecting various drugs in a body fluid sample. The signal output mode of the established platform is simplified, so that the detection efficiency is greatly improved. In the subsequent reaction, more invertase is filled in an internal cavity of a biotinylated liposome (biotin-liposome), the biotinylated liposome is released by adding a surfactant, and the signal is amplified by catalyzing substrate conversion to generate more glucose, so that a more sensitive and accurate detection result is obtained.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a method for rapidly detecting various drugs based on reading of glucometer signals, which uses sucrose invertase as a signal conversion group of immunoreaction to catalyze sucrose to be decomposed into glucose, so that the detection of a target substance is converted into the detection of the content of the glucose, and further converted into a read value of a portable household glucometer; the method realizes signal amplification by coating sucrose signal molecules with liposome so as to realize high specificity, high sensitivity and rapid identification and analysis of a target object.

In order to achieve the purpose, the invention provides the following technical scheme: the method comprises the following steps:

the method comprises the following steps: firstly, preparing a 96-hole immune substrate for detecting specific drugs, coupling standard substances such as methamphetamine, morphine, ketamine and the like with Bovine Serum Albumin (BSA) through a chemical synthesis method to form a drug-BSA compound, and respectively fixing the drug-BSA compound as immunogens at the hole bottoms of the 96-hole immune substrate;

step two: corresponding drug components in the body fluid sample and immunogen fixed in a 96-hole immune substrate compete together for antigen binding sites on the corresponding biotinylated monoclonal antibody, so that the biotinylated antibody captured by the hole plate is inversely related to the concentration of the drugs in the sample;

step three: after the glucose sensor is flushed, streptavidin and biotinylation invertase are continuously added into the system, the invertase can catalyze the streptavidin and the biotinylation invertase to be decomposed into glucose after substrates are added, and the detection result can be read by a household glucometer.

As a further scheme of the invention: in the third step, liposome elements are introduced, wherein the internal volume of the liposome is larger, sucrose invertase molecules are encapsulated in an aqueous cavity, and simultaneously, as the liposome can carry a large number of enzyme molecules and is released under a controllable condition by adding a surfactant, more invertase reacts with a substrate in a free state to generate glucose in the process, so that signals are effectively amplified.

As a further scheme of the invention: the operation steps of the rapid drug detection are as follows:

(1) synthesis of Drug molecule-bovine serum albumin complex (Drug-BSA): firstly, mixing and dissolving 20mg of drug compounds (methamphetamine and morphine succinate), 15mg of EDC and 30mg of BSA in 1.0mL of Tris buffer solution, stirring l h in a dark place at 0-4 ℃, then adding 5.0mg of EDC, stirring and reacting for 12h at room temperature, standing overnight, and dialyzing by using the Tris buffer solution to remove unreacted small molecular substances to obtain a conjugate of drug molecules and BSA;

(2) coating of 96-well immune substrates: irradiating a porous plate with ultraviolet ray at an intensity of 30W for 12h at a distance of 75cm from the ultraviolet ray in a 96-well plate to increase the adsorption performance, dissolving the conjugate of the drug molecule and BSA obtained in step (1) in sodium bicarbonate buffer solution with pH of 9.6, and making into 100ng mL solution^-1－20μg mL^-1The solution of (4) was prepared by adding a coating solution in a volume of 50. mu.L to each well of a 96-well immunological substrate, standing overnight in a refrigerator at 4 ℃ and, after removing the coating solution, gently rinsing the well plate twice with a buffer solution and then rinsing it with N₂Drying to obtain a composite coated plate;

(3) synthesis of invertase @ Biotin (Biotin) -liposome beacon: containing DHPE (1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) and 1.0mg biotin-DHPE was first dissolved in 0.1mL chloroform in a 1.5mL centrifuge tube at a biotin-DHPE molar ratio of 19:1, evaporated to dryness naturally to form a lipid membrane, dissolved in a phosphate buffer containing invertase at a pH of 7.4 to form liposomes, incubated further in a 45 ℃ water bath for 20min, passed through a 0.4 μm polycarbonate membrane to produce a uniform size liposome suspension, excess invertase was removed with a 300kDa dialysis bag, stirred at 4 ℃ for 48 h;

(4) acquiring a detection standard curve of the glucometer: immunizing blank target antigens to be detected with different concentrations and a certain amount of antibodies, adding the immunized target antigens into detection holes of a 96-hole immune substrate, washing, gradually adding streptavidin and biotin labeled invertase, adding sucrose, dripping the reaction solution onto blood glucose detection test paper, inserting a blood glucose meter to obtain a reading, drawing the concentration of the antigens by the reading, and performing regression analysis to obtain a standard curve.

As a further scheme of the invention: the ketamine in the drug compound in the operation step (1) and BSA are prepared by a glutaraldehyde crosslinking method.

As a still further scheme of the invention: in the operation step (4), streptavidin and biotin labeled invertase are replaced by invertase @ biotin-liposome and a surfactant.

Compared with the prior art, the invention has the beneficial effects that:

(1) the existing rapid drug detection test paper based on gold nanoparticle labeling immunoassay can only provide qualitative analysis results, and the quantitative analysis mostly needs large-scale laboratory instruments. The method takes the glucometer as a signal reading terminal, and provides an accurate quantitative analysis result on the premise of not depending on a laboratory field and instruments on the basis of keeping the characteristics of portability and quickness.

(2) The method skillfully uses liposome-encapsulated enzyme molecules, adds a surfactant to initiate the release of the liposome-encapsulated enzyme molecules, catalyzes a substrate to react to generate a detection signal, and can effectively improve the detection sensitivity of the method and reduce the detection limit, so that the method is more suitable for detecting trace drugs in actual samples.

(3) The method can carry out personalized free combination on the detection objects according to the actual detection requirements so as to realize the simultaneous detection and analysis of single or multiple drugs, single or multiple drugs in the sample.

(4) The method realizes signal output through the glucometer, has low detection cost and simple equipment and operation, is suitable for basic level and is very suitable for popularization and application.

Drawings

FIG. 1 is a schematic diagram of drug detection according to the present invention.

FIG. 2 is a schematic diagram of the liposome principle of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "sleeved/connected," "connected," and the like are to be construed broadly, e.g., "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

Referring to fig. 1-2, in an embodiment of the present invention, a method for rapidly detecting multiple drugs based on a blood glucose meter signal reading includes the following steps:

And simultaneously, as the liposome can carry a large amount of enzyme molecules and is released under a controllable condition by adding a surfactant, more invertase reacts with a substrate in a free state to generate glucose in the process, so that signals are effectively amplified, and a more sensitive detection result is obtained.

The rapid drug detection method comprises the following operation steps:

(2) coating of 96-well immune substrates: irradiating a porous plate with ultraviolet ray at an intensity of 30W for 12h at a distance of 75cm from the ultraviolet ray in a 96-well plate to increase the adsorption performance, dissolving the conjugate of the drug molecule and BSA obtained in step (1) in sodium bicarbonate buffer solution with pH of 9.6, and making into 100ng mL solution^-1－20μg m^L-1Adding 50 mu L of coating solution into each hole of a 96-hole immune substrate, standing overnight in a refrigerator at 4 ℃, then absorbing the coating solution, washing the hole plate twice by using buffer solution softly, and drying by using N2 to obtain a composite coated plate;

Preferably, the ketamine in the drug compound of operation step (1) and BSA are prepared by a glutaraldehyde crosslinking method.

Preferably, the invertase @ biotin-liposome and the surfactant are used in the operation step (4) to replace streptavidin and biotin labeled invertase, so that the detection result is more sensitive.

According to the above preferred embodiment provided by the present invention, taking morphine drug detection as an example, the specific operation steps of the detection are as follows:

the method comprises the following steps: respectively mixing and dissolving 20mg of morphine succinate, 15mg of EDC and 30mg of BSA in 1.0mL of Tris-HCl buffer solution, stirring and stirring l h at 0-4 ℃ in a dark place, adding 5.0mg of EDC, stirring and reacting for 12h at room temperature, standing overnight, and dialyzing by using Tris buffer solution to remove unreacted micromolecule substances to obtain a conjugate of morphine and BSA;

step two: irradiating 96-well immune substrate with ultraviolet light at intensity of 30W, irradiating 96-well plate with ultraviolet light at a distance of 75cm for 12 hr, dissolving the obtained conjugate of morphine and BSA with sodium bicarbonate buffer solution at pH of 9.6 to obtain a solution with concentration of 10ug mL^-1Adding 50 mu L of coating solution into each well of a 96-well immune plate, standing in a refrigerator at 4 ℃ overnight, removing the coating solution, gently washing the well plate twice with buffer solution, and adding N₂Drying to obtain a composite coated plate;

step three: adding 50 mu L of sample solution to be detected with different concentrations and corresponding biotinylated antibody into a detection hole corresponding to a 96-hole immune plate, incubating for 30min at 37 ℃, and washing for 2 times by using buffer solution;

step four: adding streptavidin, incubating for 30min for washing, then adding invertase @ Biotin-liposome beacon, adding 10 mu L of surfactant and sucrose solution after washing is fully combined, and measuring the glucose content of the reaction solution by using blood glucose immunity test paper and a portable glucometer to obtain the working curve of morphine and the content of corresponding substances in a sample to be measured.

It should be noted that the working principle of the portable blood glucose meter is the prior art, which is common knowledge of those skilled in the art, and thus, the details are not described herein.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention without departing from the spirit and scope of the invention.

Claims

1. A method for rapidly detecting various drugs based on reading of glucometer signals is characterized by comprising the following steps: the method comprises the following steps:

the method comprises the following steps: firstly, preparing a 96-hole immune substrate for detecting specific drugs, coupling standard substances such as methamphetamine, morphine, ketamine and the like with bovine serum albumin through a chemical synthesis method to form a drug-BSA (bovine serum albumin) compound, and respectively fixing the drug-BSA compound as immunogens at the bottom of a 96-hole immune substrate hole;

2. The method for rapidly detecting multiple drugs based on signal readout of a blood glucose meter according to claim 1, wherein: in the third step, liposome elements are introduced, wherein the internal volume of the liposome is larger, sucrose invertase molecules are encapsulated in an aqueous cavity, and simultaneously, as the liposome can carry a large number of enzyme molecules and is released under a controllable condition by adding a surfactant, more invertase reacts with a substrate in a free state to generate glucose in the process, so that signals are effectively amplified.

3. The method for rapidly detecting multiple drugs based on signal readout of a blood glucose meter according to claim 2, wherein: the operation steps of the rapid drug detection are as follows:

(1) synthesis of drug molecule-bovine serum albumin complex: firstly, mixing and dissolving 20mg of drug compound, 15mg of EDC and 30mg of BSA in 1.0mL of Tris buffer solution, stirring l h at 0-4 ℃ in a dark place, then adding 5.0mg of EDC, stirring and reacting for 12h at room temperature, standing overnight, and dialyzing by using Tris buffer solution to remove unreacted micromolecule substances to obtain a conjugate of drug molecules and BSA;

(2) coating of 96-well immune substrates: irradiating a porous plate with ultraviolet ray at an intensity of 30W for 12h at a distance of 75cm from the ultraviolet ray in a 96-well plate to increase the adsorption performance, dissolving the conjugate of the drug molecule and BSA obtained in step (1) in sodium bicarbonate buffer solution with pH of 9.6, and making into 100ng mL solution^-1－20μg mL^-1The solution of (4 ℃ C.) was prepared by adding 50. mu.L of the coating solution to each well of a 96-well immunoplate at 4 ℃After overnight storage in a refrigerator, the coating was aspirated and the well plates were rinsed gently twice with buffer and N₂Drying to obtain a composite coated plate;

(3) synthesis of invertase @ Biotin-liposome Beacon: dissolving DHPE and 1.0mg biotin-DHPE in 0.1mL chloroform in a 1.5mL centrifuge tube, wherein the molar ratio of biotin-DHPE is 19:1, naturally evaporating to dryness to form a lipid membrane, dissolving the lipid membrane in a phosphate buffer solution containing invertase, wherein the pH of the phosphate buffer solution is 7.4, forming liposomes, continuing to incubate in a water bath at 45 ℃ for 20min, passing through a 0.4 μm polycarbonate membrane to produce a liposome suspension of uniform size, removing excess invertase with a 300kDa dialysis bag, and stirring at 4 ℃ for 48 h;

4. The method for rapidly detecting multiple drugs based on signal readout of a blood glucose meter according to claim 3, wherein: the ketamine in the drug compound in the operation step (1) and BSA are prepared by a glutaraldehyde crosslinking method.

5. The method for rapidly detecting multiple drugs based on signal readout of a blood glucose meter according to claim 3, wherein: in the operation step (4), streptavidin and biotin labeled invertase are replaced by invertase @ biotin-liposome and a surfactant.